Fiona Young

Effects of blue-green algal toxin cylindrospermopsin (CYN) on human granulosa cells in vitro.

The blue-green algal toxin cylindrospermopsin (CYN) occurs in public water supplies. CYN was hepatotoxic when administered orally to mice, and cytotoxic and genotoxic to human cell lines. To determine the effects of CYN on primary human IVF-derived granulosa cells, 0-1 microg/ml CYN was added to cells for 2, 4 or 6h+/-hCG (n=6), or for 24, 48 and 72 h (n=6). Cytotoxicity was evaluated by MTT assay, and secreted progesterone or estrogen quantified by radioimmunoassay. 24h exposure to 1 microg/ml CYN was cytotoxic (p<0.05), whereas 0.0625 microg/ml CYN did not cause cytotoxicity or affect estrogen production, but did inhibit basal progesterone production (p<0.01). Similarly, 6h exposure to 1 microg/ml CYN did not affect cytotoxicity or hCG-stimulated estrogen production, but did inhibit hCG-stimulated progesterone production (p<0.01). In this in vitro assay, CYN inhibited progesterone production and therefore has the potential to be an endocrine disrupter by changing the progesterone:estrogen ratio in women.

Marine compounds selectively induce apoptosis in female reproductive cancer cells but not in primary-derived human reproductive granulosa cells.

Anticancer properties of tyrindoleninone and 6-bromoisatin from Dicathais orbita were tested against physiologically normal primary human granulosa cells (HGC) and reproductive cancer cell lines. Tyrindoleninone reduced cancer cell viability with IC50 values of 39 µM (KGN; a tumour-derived granulosa cell line), 39 μM (JAr), and 156 μM (OVCAR-3), compared to 3516 μM in HGC. Apoptosis in HGC’s occurred after 4 h at 391 µM tyrindoleninone compared to 20 µM in KGN cells. Differences in apoptosis between HGC and KGN cells were confirmed by TUNEL, with 66 and 31% apoptotic nuclei at 4 h in KGN and HGC, respectively. These marine compounds therefore have potential for development as treatments for female reproductive cancers.

The physiology of the ovary: Maturation of ovarian granulosa cells and a novel role for antioxidants in the corpus luteum

During folliculogenesis the granulosa cells divide whilst in contact with each other, and so exhibit some of the characteristics of stem cells. In vitro we have shown that bovine granulosa cells from 3-7 mm follicles, like stem cells, divide without the need for a substratum, and produce colonies of cells. Growth factors, bFGF and IGFs, stimulate their division. These cells secrete and assemble a basal lamina, suggesting that the follicular basal lamina is produced by the granulosa cells. They have the morphological characteristics of follicular granulosa cells. Thus this system is ideal for studying the functions of immature granulosa cells because the cells do not spontaneously differentiate or luteinize into luteal cells, as occurs in culture on a substratum. On differentiation into luteal cells in vivo the cells express the steroidogenic enzymes for progesterone production and accumulate beta-carotene. During culture of bovine luteal cells we observed that a proportion of the steroidogenic enzyme cholesterol side-chain cleavage cytochrome P450 enzyme became chemically cross-linked to its electron donor, adrenodoxin. P450 enzymes produce oxygen free radicals and oxygen free radicals can cause cross-linking between proteins in close proximity. Cell protect against this damage by the use of antioxidant vitamins. Repleting the cultured luteal cells with beta-carotene reduced the amount of cross-linking. We conclude that the high levels of beta-carotene in corpora lutea are to protect against damage due to oxygen free radicals generated in the course of progesterone synthesis.

Are the Traditional Medical Uses of Muricidae Molluscs Substantiated by Their Pharmacological Properties and Bioactive Compounds

Marine molluscs from the family Muricidae hold great potential for development as a source of therapeutically useful compounds. Traditionally known for the production of the ancient dye Tyrian purple, these molluscs also form the basis of some rare traditional medicines that have been used for thousands of years. Whilst these traditional and alternative medicines have not been chemically analysed or tested for efficacy in controlled clinical trials, a significant amount of independent research has documented the biological activity of extracts and compounds from these snails. In particular, Muricidae produce a suite of brominated indoles with anti-inflammatory, anti-cancer and steroidogenic activity, as well as choline esters with muscle-relaxing and pain relieving properties. These compounds could explain some of the traditional uses in wound healing, stomach pain and menstrual problems. However, the principle source of bioactive compounds is from the hypobranchial gland, whilst the shell and operculum are the main source used in most traditional remedies. Thus further research is required to understand this discrepancy and to optimise a quality controlled natural medicine from Muricidae.

Development of an in vitro reproductive screening assay for novel pharmaceutical compounds

An in vitro reproductive cell‐based toxicity assay was developed using MLTC‐1 (murine Leydig tumour cell line) in order to examine the reproductive toxicity of two novel nanopharmaceutical compounds, namely ethylene glycol mono allyl ether and poly(ethylene glycol) octa‐functionalized polyhedral oligomeric silsesquioxane. Three commonly used cytotoxicity assays, namely the MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H ‐tetrazolium bromide], MTS [3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H ‐tetrazolium] and Crystal Violet assays, were compared, and the MTT assay proved to be the most accurate and reproducible for the MLTC‐1 cell line. The doubling rate of the MLTC‐1 cells was 30±3.5 h and the optimal seeding density for the MTT assay was 20000 cells per well, and the optimized MTT assay utilized a 4 h cell adherence followed by incubation with 0.5 mg/ml MTT for 1 h. The intra‐ and inter‐assay CV (coefficient of variation) values were 12.3 and 11% respectively. MLTC‐1 cells only produce the reproductive hormone progesterone in response to hCG (human chorionic gonadotropin), which stimulated progesterone production dose‐dependently from 0 to 100 m.i.u. (milliinternational units)/ml (2706±1118 ng/ml). H2O2 as a negative control killed 100% of cells at 1000 μg/ml. The two nanopharmaceutical compounds were cytotoxic at concentrations ≥0.1 μg/ml, but hCG decreased cytotoxicity to ≥1000 μg/ml (P <0.001). hCG‐stimulated progesterone synthesis afforded some protection against the cytotoxic effects of the two novel nanotechnology compounds; therefore doses ≤100 μg/ml and an exposure period of 1 h would be recommended for testing in in vivo animal reproductive assays.

Cylindrospermopsin, a blue-green algal toxin, inhibited human luteinised granulosa cell protein synthesis in vitro.

The blue-green algal toxin cylindrospermopsin (CYN) inhibits protein synthesis, and CYP450 enzymes metabolise CYN to cytotoxic endproducts. Human chorionic gonadotrophin (hCG) stimulates the de novo synthesis of StAR and CYP450 aromatase. Human IVF-derived granulosa cells (GC) (n=7) were exposed to 0-5μM CYN±1IU/ml hCG for 2-24h. After 24h pre-culture GC responded to hCG by increasing estradiol 17β (E(2)) and progesterone (P(4)) synthesis. Three micromolar of CYN±1IU/ml hCG for 24h was not cytotoxic and did not affect basal or hCG-stimulated E(2) or P(4) production, but did inhibit protein synthesis (p<0.05, n=4). hCG-stimulated steroidogenesis was not reduced by CYN, suggesting a lack of effect on StAR or CYP450 aromatase protein synthesis. hCG enhanced the effects of CYN on GC protein synthesis. Twenty four hours exposure to 0.1μM CYN did not affect GC, supporting the establishment of a 0.0024μM Guideline level for CYN in public water supplies.

Pathogen and Particle Associations in Wastewater: Significance and Implications for Treatment and Disinfection Processes

Disinfection guidelines exist for pathogen inactivation in potable water and recycled water, but wastewater with high numbers of particles can be more difficult to disinfect, making compliance with the guidelines problematic. Disinfection guidelines specify that drinking water with turbidity ≥1 Nephelometric Turbidity Units (NTU) is not suitable for disinfection and therefore not fit for purpose. Treated wastewater typically has higher concentrations of particles (1–10 NTU for secondary treated effluent). Two processes widely used for disinfecting wastewater are chlorination and ultraviolet radiation. In both cases, particles in wastewater can interfere with disinfection and can significantly increase treatment costs by increasing operational expenditure (chemical demand, power consumption) or infrastructure costs by requiring additional treatment processes to achieve the required levels of pathogen inactivation. Many microorganisms (viruses, bacteria, protozoans) associate with particles, which can allow them to survive disinfection processes and cause a health hazard. Improved understanding of this association will enable development of cost-effective treatment, which will become increasingly important as indirect and direct potable reuse of wastewater becomes more widespread in both developed and developing countries. This review provides an overview of wastewater and associated treatment processes, the pathogens in wastewater, the nature of particles in wastewater and how they interact with pathogens, and how particles can impact disinfection processes.

Undifferentiated murine embryonic stem cells used to model the effects of the blue–green algal toxin cylindrospermopsin on preimplantation embryonic cell proliferation

Undifferentiated mouse embryonic stem cell (mES) proliferation in vitro resembles aspects of in vivo pre-implantation embryonic development. mES were used to assess the embryo-toxicity of cylindrospermopsin (CYN), a water contaminant with an Australian Drinking Water Guideline (ADWG) of 1 μg/L. mES exposed to 0-1 μg/mL CYN for 24-168 h were subjected to an optimised crystal violet viability assay. mES exposed to retinoic acid ± 1 μg/L CYN differentiated into neural-like cells confirmed by morphological examination and RT-PCR for Oct4, Brachyury and Nestin. The CYN No Observed Effect Concentration (OEC) was 0.5 μg/mL, the Lowest OEC was 1 μg/mL (p < 0.001, n = 3), and the IC50 was 0.86 μg/mL after 24 h. The ADWG 1 μg/L CYN did not affect differentiation or proliferation after 72 h, but decreased proliferation after 168 h (p < 0.05). We conclude that higher algal bloom-associated CYN concentrations have the potential to impair in vivo pre-implantation development, and the mES crystal violet assay has broad application to screening environmental toxins.

An in vitro high-throughput assay for screening reproductive and toxic effects of anticancer compounds

An in vitro assay was developed that simultaneously tested the effects of anticancer drug candidates on cytotoxicity, hormone synthesis, and gonadotrophin responsiveness using the choriocarcinoma JAr cell line. JAr culture conditions were optimized and then cells were exposed to a marine mollusc extract in the presence and absence of hCG. The intra‐ and interassay coefficients of variation of the optimized 1 H thiazolyl blue tetrazolium bromide assay were 11.3% and 10.9%, respectively. hCG (1,000 mIU/mL) increased progesterone (P4) synthesis after 24 H (P < 0.05). The mollusc extract significantly decreased cell viability, with the IC50 affected by incubation time, but not hCG. P4 synthesis was inhibited at low concentrations of the anticancer extract, but stimulated at the highest concentration, and complex interactions of P4 were also found with hCG. In conclusion, the optimized assay is useful to characterize the effects of novel drugs on cytotoxicity, basal, and gonadotrophin‐stimulated P4 synthesis in vitro, and can be used to inform subsequent in vivo studies.