Evidence-based hormonal, mutational, and endocrine-disrupting chemical-induced zebrafish as an alternative model to study PCOS condition similar to mammalian PCOS model.

Polycystic ovarian syndrome (PCOS) causes swollen ovaries in women at reproductive age due to hormonal disorder with small cysts on the outer edges. The cause of the disorder is still yet to be found. Multiple factors have increased PCOS prevalence, hyperandrogenism, oxidative stress, inflammation, and insulin resistance. Various animal PCOS models have been developed to imitate the pathophysiology of PCOS in humans. Zebrafish is one of the most versatile animal experimental models because of the transparency of the embryos, small size, and rapid growth. The zebrafish similarity to higher vertebrates made it a useful non-mammalian model for PCOS drug testing and screening. This review provides an insight into the usage of zebrafish, a non-mammalian model for PCOS, as an opportunity for evaluating future initiatives in such a research domain.

Determination of Median Lethal Concentration (LC50) for Endosulfan, Heptachlor and Dieldrin Pesticides to African Catfish, Clarias gariepinus and Their Impact on Its Behavioral Patterns and Histopathological Responses.

Pesticides such as endosulfan, heptachlor and dieldrin persist in aquatic environments as a result of their resistance to biodegradation. However, there is no adequate information about the toxicity of endosulfan, heptachlor and dieldrin to the aquatic organism, African catfish (Clarias gariepinus)-a high valued widely distributed commercially interesting species. The current experiment was performed with the aim to determine the median lethal concentration (LC50) of endosulfan, heptachlor and dieldrin to African catfish (Clarias gariepinus); their behavioral abnormalities and histopathological alterations in several vital organs. A total of 324 juvenile fish were exposed for 96 h to six concentrations of endosulfan and dieldrin at 0, 0.001, 0.002, 0.004, 0.008 and 0.016 ppm, and to heptachlor at concentrations of 0, 0.02, 0.04, 0.08, 0.16 and 0.32 ppm for dose-response tests. The study demonstrated that the species is highly susceptible to those contaminants showing a number of behavioral abnormalities and histopathological changes in gill, liver and muscle. The 96-h LC50 value of endosulfan, dieldrin and heptachlor for the African catfish was found as 0.004 (0.001-0.01) mg/L, 0.006 mg/L and 0.056 (0.006-0.144) mg/L, respectively. Abnormal behaviors such as erratic jerky swimming, frequent surfacing movement with gulping of air, secretion of mucus on the body and gills were observed in response to the increasing exposure concentrations. Histopathological alterations of liver, gill and muscle tissues were demonstrated as vacuolization in hepatocytes, congestion of red blood cells (RBCs) in hepatic portal vein; deformed secondary lamellae and disintegrated myotomes with disintegrated epidermis, respectively. These findings are important to monitor and responsibly manage pesticide use in and around C. gariepinus aquacultural areas.

GR15 peptide of S-adenosylmethionine synthase (SAMe) from Arthrospira platensis demonstrated antioxidant mechanism against H2O2 induced oxidative stress in in-vitro MDCK cells and in-vivo zebrafish larvae model.

GR15 is a short molecule or peptide composed of aliphatic amino acids and possesses to have antioxidant properties. The GR15, 1GGGAFSGKDPTKVDR15 was identified from the protein S-adenosylmethionine synthase (SAMe) expressed during the sulfur departed state of Arthrospira platensis (spirulina or cyanobacteria). The in-silico assessment and the structural features of GR15 showed its antioxidant potency. Real-time PCR analysis found the up-regulation of ApSAMe expression on day 15 against oxidative stress due to 10 mM H2O2 treatment in A. platensis (Ap). The antioxidant activity of GR15 was accessed by the cell-free antioxidant assays such as ABTS, SARS, HRAS and NO; the results showed dose-dependent antioxidant activity. The toxicity assay was performed in both in vitro and in vivo models, in which peptide does not exhibit any toxicity in MDCK cell and zebrafish embryos. The intercellular ROS reduction potential of GR15 peptide was also investigated in both in vitro and in vivo models including LDH assay, antioxidant enzymes (SOD and CAT), and fluorescent staining assay (DCFDA, Hochest and Acridine orange sting) was performed; the results showed that the GR15 peptide was effectively reduced the ROS level. Further, RT-PCR demonstrated that GR15 enhanced the antioxidant property and also up-regulated the antioxidant gene, thus reduced the ROS level in both in vitro and in vivo models. Based on the results obtained from this study, we propose that GR15 has the potential antioxidant ability; hence further research can be directed towards the therapeutic product or drug development against disease caused by oxidative stress.

Tryptophan-tagged peptide from serine threonine-protein kinase of Channa striatus improves antioxidant defence in L6 myotubes and attenuates caspase 3-dependent apoptotic response in zebrafish larvae.

This study reports the antioxidant property and molecular mechanism of a tryptophan-tagged peptide derived from a teleost fish Channa striatus of serine threonine-protein kinase (STPK). The peptide was tagged with tryptophan to enhance the antioxidant property of STPK and named as IW13. The antioxidant activity of IW13 peptide was investigated using in vitro methods such as DPPH, ABTS, superoxide anion radical scavenging and hydrogen peroxide scavenging assay. Furthermore, to investigate the toxicity and dose response of IW13 peptide on antioxidant defence in vitro, L6 myotubes were induced with generic oxidative stress due to exposure of hydrogen peroxide (H2O2). IW13 peptide exposure was found to be non-cytotoxic to L6 cells in the tested concentration (10, 20, 30, 40 and 50 µM). Also, the pre-treatment of IW13 peptide decreased the lipid peroxidation level and increased glutathione enzyme activity. IW13 peptide treatment upregulated the antioxidant enzyme genes: GPx (glutathione peroxidase), GST (glutathione S transferase) and GCS (glutamine cysteine synthase), in vitro in L6 myotubes and in vivo in zebrafish larvae against the H2O2-induced oxidative stress. The results demonstrated that IW13 renders protection against the H2O2-induced oxidative stress through a cellular antioxidant defence mechanism by upregulating the gene expression, thus enhancing the antioxidant activity in the cellular or organismal level. The findings exhibited that the tryptophan-tagged IW13 peptide from STPK of C. striatus could be a promising candidate for the treatment of oxidative stress-associated diseases.

Intracellular ROS scavenging and antioxidant regulation of WL15 from cysteine and glycine-rich protein 2 demonstrated in zebrafish in vivo model.

Antioxidant peptides are naturally present in food, especially in fishes, and are considered to contain rich source of various bioactive compounds that are structurally heterogeneous. This study aims to identify and characterize the antioxidant property of the WL15 peptide, derived from Cysteine and glycine-rich protein 2 (CSRP2) identified from the transcriptome of a freshwater food fish, Channa striatus. C. striatus is already studied to contain high levels of amino acids and fatty acids, besides traditionally known for its pharmacological benefits in the Southeast Asian region. In our study, in vitro analysis of WL15 peptide exhibited strong free radical scavenging activity in 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), superoxide anion radical and hydrogen peroxide (H2O2) scavenging assay. Further, to evaluate the cytotoxicity and dose-response, the Human dermal fibroblast (HDF) cells were used. Results showed that the treatment of HDF cells with varying concentrations (10, 20, 30, 40 and 50 µM) of WL15 peptide was not cytotoxic. However, the treatment concentrations showed enhanced antioxidant properties by significantly inhibiting the levels of free radicals. For in vivo assessment, we have used zebrafish larvae for evaluating the developmental toxicity and for determining the antioxidant property of the WL15 peptide. Zebrafish embryos were treated with the WL15 peptide from 4 h of post-fertilization (hpf) to 96 hpf covering the embryo-larval developmental period. At the end of the exposure period, the larvae were exposed to H2O2 (1 mM) for inducing generic oxidative stress. The exposure of WL15 peptide during the embryo-larval period showed no developmental toxicity even in higher concentrations of the peptide. Besides, the WL15 peptide considerably decreased the intracellular reactive oxygen species (ROS) levels induced by H2O2 exposure. WL15 peptide also inhibited the H2O2-induced caspase 3-dependent apoptotic response in zebrafish larvae was observed using the whole-mount immunofluorescence staining. Overall results from our study showed that the pre-treatment of WL15 (50 µM) in the H2O2-exposed zebrafish larvae, attenuated the expression of activated caspase 3 expressions, reduced Malondialdehyde (MDA) levels, and enhanced antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT). The gene expression of antioxidant enzymes such as glutathione S-transferase (GST), glutathione peroxide (GPx) and γ-glutamyl cysteine synthetase (GCS) was found to be upregulated. In conclusion, it can be conceived that pre-treatment with WL15 could mitigate H2O2-induced oxidative injury by elevating the activity and expression of antioxidant enzymes, thereby decreasing MDA levels and cellular apoptosis by enhancing the antioxidant response, demonstrated by the in vitro and in vivo experiments.

Molecular mechanism of down-regulating adipogenic transcription factors in 3T3-L1 adipocyte cells by bioactive anti-adipogenic compounds.

Obesity is growing at an alarming rate, which is characterized by increased adipose tissue. It increases the probability of many health complications, such as diabetes, arthritis, cardiac disease, and cancer. In modern society, with a growing population of obese patients, several individuals have increased insulin resistance. Herbal medicines are known as the oldest method of health care treatment for obesity-related secondary health issues. Several traditional medicinal plants and their effective phytoconstituents have shown anti-diabetic and anti-adipogenic activity. Adipose tissue is a major site for lipid accumulation as well as the whole-body insulin sensitivity region. 3T3-L1 cell line model can achieve adipogenesis. Adipocyte characteristics features such as expression of adipocyte markers and aggregation of lipids are chemically induced in the 3T3-L1 fibroblast cell line. Differentiation of 3T3-L1 is an efficient and convenient way to obtain adipocyte like cells in experimental studies. Peroxisome proliferation activated receptor γ (PPARγ) and Cytosine-Cytosine-Adenosine-Adenosine-Thymidine/Enhancer-binding protein α (CCAAT/Enhancer-binding protein α or C/EBPα) are considered to be regulating adipogenesis at the early stage, while adiponectin and fatty acid synthase (FAS) is responsible for the mature adipocyte formation. Excess accumulation of these adipose tissues and lipids leads to obesity. Thus, investigating adipose tissue development and the underlying molecular mechanism is important in the therapeutical approach. This review describes the cellular mechanism of 3T3-L1 fibroblast cells on potential anti-adipogenic herbal bioactive compounds.

TL15 of Arthrospira platensis sulfite reductase scavenges free radicals demonstrated in oxidant induced larval zebrafish (Danio rerio) model.

The antioxidant role of sulfite reductase (SiR) derived from Arthrospira platensis (Ap) was identified through a short peptide, TL15. The study showed that the expression of ApSiR was highly expressed on day ten due to sulfur deprived stress in Ap culture. TL15 peptide exhibited strong antioxidant activity when evaluated using antioxidant assays in a concentration ranging from 7.8 and 125 µM. Further, the cytotoxicity of TL15 peptide was investigated, even at the higher concentration (250 µM), TL15 did not exhibit any toxicity, when tested in vitro using human leucocytes. Moreover, a potential reduction in reactive oxygen species (ROS) production was observed due to the treatment of TL15 peptide (>15.6 µM) to H2O2 exposed leucocytes. For the in vivo assessment of TL15 toxicity and antioxidant ability, experiments were performed in zebrafish (Danio rerio) larvae to analyse the developmental toxicity of TL15 peptide. Results showed that, exposure to TL15 peptide in tested concentrations ranging from 10, 20, 40, and 80 µM, did not affect the development and physiological parameters of the zebrafish embryo/larvae such as morphology, survival, hatching and heart rate. Fluorescent assay was performed using DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) to examine the production of intracellular reactive oxygen species (ROS) in zebrafish treated with TL15 peptide during the embryo-larval stages. Fluorescent images showed that pre-treatment with TL15 peptide to attenuate the H2O2 induced ROS levels in the zebrafish larvae in a dose-dependent manner. Further to uncover the underlying biochemical and antioxidant mechanism, the enzyme activity of superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (LPO) levels were studied in zebrafish larvae. TL15 pre-treated groups showed enhanced antioxidant enzyme activity, while the hydrogen peroxide (H2O2) exposed larvae showed significantly diminished activity. Overall results from the study revealed that, TL15 act as a potential antioxidant molecule with dose-specific antioxidant property. Thus, TL15 peptide could be an effective and promising source for biopharmaceutical applications.

Pharmacologic downregulation of protein arginine methyltransferase1 expression by adenosine dialdehyde increases cell senescence in breast cancer.

We investigated the role of protein arginine methylation (PAM) in estrogen receptor (ER)-positive breast cancer cells through pharmacological intervention. Tamoxifen (TAM) or adenosine dialdehyde (ADOX), independently, triggered cell cycle arrest and down-regulated PAM, as reduced protein arginine methyltransferase1 (PRMT1) mRNA and asymmetric dimethylarginine (ADMA) levels. Synergistic effect of these compounds elicited potent anti-cancer effect. However, reduction in ADMA was not proportionate with the compound-induced down-regulation of PRMT1 mRNA. We hypothesized that the disproportionate effect is due to the influence of the compounds on other methyltransferases, which catalyze the arginine dimethylation reaction and the diversity in the degree of drug-protein interaction among these methyltransferases. In silico analyses revealed that independently, ADOX or TAM, binds with phosphatidylethanolamine-methyltransferase (PEMT) or betaine homocysteine-methyl transferase (BHMT); and that the binding affinity of ADOX with PEMT or BHMT is prominent than TAM. These observations suggest that in breast cancer, synergistic effect of ADOX + TAM elicits impressive protective function by regulating PAM; and plausibly, restoration of normal enzyme activities of methyltransferases catalyzing arginine dimethylation could have clinical benefits.

Peroxiredoxin of Arthrospira platensis derived short molecule YT12 influences antioxidant and anticancer activity.

This study demonstrates both the antioxidant and anticancer potential of the novel short molecule YT12 derived from peroxiredoxin (Prx) of spirulina, Arthrospira platensis (Ap). ApPrx showed significant reduction in reactive oxygen species (ROS) against hydrogen peroxide (H2 O2 ) stress. The complementary DNA sequence of ApPrx contained 706 nucleotides and its coding region possessed 546 nucleotides between position 115 and 660. Real-time quantitative reverse transcription polymerase chain reaction analysis confirmed the messenger RNA expression of ApPrx due to H2 O2 exposure in spirulina cells at regular intervals, in which the highest expression was noticed on Day 20. Cytotoxicity assay was performed using human peripheral blood mononuclear cells, and revealed that at 10 µM, the YT12 did not exhibit any notable toxicity. Furthermore, ROS scavenging activity of YT12 was performed using DCF-DA assay, in which YT12 scavenged a significant amount of ROS at 25 µM in H2 O2 -treated blood leukocytes. The intracellular ROS in human colon adenocarcinoma cells (HT-29) was regulated by oxidative stress, where the YT12 scavenges ROS in HT-29 cells at 12.5 µM. Findings show that YT12 peptide has anticancer activity, when treated against HT-29 cells. Through the MTT assay, YT12 showed vital cytotoxicity against HT-29 cells. These finding suggested that YT12 is a potent antioxidant molecule which defends ROS against oxidative stress and plays a role in redox balance.

Arthrospira platensis transglutaminase derived antioxidant peptide-packed electrospun chitosan/ poly (vinyl alcohol) nanofibrous mat accelerates wound healing, in vitro, via inducing mouse embryonic fibroblast proliferation.

In this present study, we have carried out the antioxidant function of transglutaminase (TG) identified from Arthrospira platensis (Ap) transcriptome. The antioxidant peptide ML11 (MLRSIGIPARL) has been predicted from the transglutaminase core domain and the peptide's free radical scavenging potential was evaluated and it shows that it functions on a dose dependent manner. The ML11 peptide cell toxicity was analysed in the human blood leucocytes which resulted no cytotoxic activity in any of the cell population. Moreover, the nanofibre mat encapsulated with antioxidant peptide ML11 was prepared by electrospinning technique. The antioxidant peptide ML11 encapsulated mat showed increase in fibre diameter compared to the chitosan polyvinyl alcohol blended mat. The change in the crystalline behaviour of both chitosan and polyvinyl alcohol polymer to the amorphous nature was determined by X-ray diffraction at the broad band between 20 and 30° (2θ°). FTIR revealed the functional groups which present in the polymer as well as the interaction between their components of chitosan (CS) and polyvinyl alcohol (PVA). The fibre retains the antioxidant activity due to the peptide encapsulated by scavenging the intracellular ROS that was confirmed by flowcytometry and fluorescence microscopy. The ML11 peptide encapsulated mat showed no cytotoxicity in the NIH-3T3 mouse embryonic fibroblast cells. Also, ML11 peptide encapsulated fibre showed potential wound healing activity in NIH-3T3 cells. Taken altogether, the study indicates that the wound healing potential of the ML11 peptide encapsulated nano fibre mat may be used as biopharmaceutical drug.

In-silico analysis and mRNA modulation of detoxification enzymes GST delta and kappa against various biotic and abiotic oxidative stressors.

This study reports the comprehensive comparative information of two different detoxification enzymes such as glutathione S-transferases (GSTs) delta and kappa from freshwater giant prawn Macrobrachium rosenbergii (designated as MrGSTD and MrGSTK) by investigating their in-silico characters and mRNA modulation against various biotic and abiotic oxidative stressors. The physico-chemical properties of these cDNA and their polypeptide structure were analyzed using various bioinformatics program. The analysis indicated the variation in size of the polypeptides, presence or absence of domains and motifs and structure. Homology and phylogenetic analysis revealed that MrGSTD shared maximum identity (83%) with crustaceans GST delta, whereas MrGSTK fell in arthropods GST kappa. It is interesting to note that MrGSTD and MrGSTK shared only 21% identity; it indicated their structural difference. Structural analysis indicated that MrGSTD to be canonical dimer like shape and MrGSTK appeared to be butterfly dimer like shape, in spite of four ß-sheets being conserved in both GSTs. Tissue specific gene expression analysis showed that both MrGSTD and MrGSTK are highly expressed in immune organs such as haemocyte and hepatopancreas, respectively. To understand the role of mRNA modulation of MrGSTD and MrGSTK, the prawns were inducted with oxidative stressors such as bacteria (Vibrio harveyi), virus [white spot syndrome virus (WSSV)] and heavy metal, cadmium (Cd). The analysis revealed an interesting fact that both MrGSTD and MrGSTK showed higher (P < 0.05) up-regulation at 48 h post-challenge, except MrGSTD stressed with bacteria, where it showed up-regulation at 24 h post-challenge. Overall, the results suggested that GSTs are diverse in their structure and possibly conferring their potential involvement in immune protection in crustaceans. However, further study is necessary to focus their functional differences at proteomic level.

Effects of dietary peppermint (Mentha piperita) essential oil on survival, growth, cannibalism and hepatopancreatic histopathology of Portunus pelagicus juveniles.

A two-part experiment was performed to determine whether dietary peppermint oil could improve the growth and/or decrease aggression among blue swimmer crab, Portunus pelagicus early juveniles. A total of five isonitrogenous diets were made that contained increasing peppermint oil levels of 0.00, 0.05, 0.10, 0.50 or 1.00%.? These diets were fed to 45 replicate crabs in each treatment (total of 225 crabs) for 12 days, the final sizes and weights were measured, and then placed in 3 replicate containers (30 in total/treatment) to allow the opportunity for cannibalism over 10 days.? After 10 days, the remaining crabs were examined for any histopathological changes in gills or hepatopancreas.? Results showed dietary peppermint oil, at the tested levels, had no effect on the growth or cannibalism, in either experiments (p > 0.05).? However, there were substantial changes in the hepatopancreatic histopathology that included thinner tubules and significantly less B- and R-cells from 0.10% dietary peppermint oil and above.? The unaffected growth or cannibalism indicate that the levels of dietary peppermint oil used were insufficient and further investigations are required, particularly on the implications to the hepatopancreatic changes. ?