Shamba Chatterjee

Anti-androgenic endocrine disrupting activities of chlorpyrifos and piperophos.

The present work describes the screening and characterization of some common endocrine disrupting chemicals for their (anti)androgenic activities. Various chemicals (mostly pesticides and pharmaceuticals) were screened with the NIH3T3 cell line stably expressing human androgen receptor (hAR) and luciferase reporter gene for their ability to stimulate luciferase activity or inhibit the response that was evoked by 0.4nM testosterone. The most potent anti-androgenic compounds identified in our assay included chlorpyrifos, endosulfan and piperophos. Finally, the chemicals were analyzed for their effects on steriodogenesis in rat Leydig cells. Piperophos and chlorpyrifos showed a significant decrease in testosterone biosynthesis by Leydig cells. RT-PCR studies showed decrease in the expression of key steroidogenic enzymes: cytochrome P450scc, 3beta-HSD and 17beta-HSD and immunoblot analysis demonstrated a decrease in steroidogenic acute regulatory (StAR) protein expression by both these chemicals. Chlorpyrifos also showed a decrease in LH receptor stimulated cAMP production. In conclusion, we demonstrate that commonly used pesticides like chlorpyrifos and piperophos pose serious threat to male reproductive system by interfering at various levels of androgen biosynthesis.

Screening of some anti-progestin endocrine disruptors using a recombinant yeast based in vitro bioassay

The present study was aimed to develop a sensitive, fast and user friendly progesterone receptor transactivation assay using recombinant yeast cells, Saccharomyces cerevisiae, modified to express human progesterone receptor (PR) and progesterone response element (PRE) driving the expression of green fluorescent protein. Stimulation of cells with increasing concentrations of progesterone resulted in significant elevation in fluorescence activity, with the minimum effective dose of progesterone being 0.1 nM. RU486, significantly inhibited progesterone induced transactivation and non-progesterogenic steroids failed to transactivate PR till 10 microM concentrations. About 7 different chemicals (mostly pesticides or their metabolites) like DDT and its metabolites, nonylphenol, endosulfan were screened in this assay system for their role in transactivation and they were all found to be anti-progestative and IC50 values within the range of 3-20 microM. Further, the assay was used to analyze the endocrine disrupting activity of extracted water samples from leather industries known for their high content of various chemicals and it was found to be rich in anti-progestative compounds. It resulted in about 30% reduction in transactivation. In conclusion, we demonstrated that this yeast based bioassay provides a rapid and robust assay for high throughput screening of (anti)progestative compounds from various sources.

Biodegradation of pyridine by the new bacterial isolates S. putrefaciens and B. sphaericus

In this study, two bacterial strains capable of utilizing pyridine as a sole carbon source were isolated from biofilters. Based on the biochemical test, the organisms were identified as Shewanella putrefaciens and Bacillus sphaericus. In liquid cultures, S. putrefaciens and B. sphaericus degraded pyridine quite effectively up to 500 mg L(-1). S. putrefaciens degrades 500 mg L(-1) of pyridine completely within 140 h, whereas the B. sphaericus degrades 500 mg L(-1) of pyridine only nearly 75% and takes a longer duration of 150 h. S. putrefaciens used pyridine as sole carbon and energy source better than B. sphaericus. Monods and Haldanes inhibitory growth models were used to obtain maximum specific growth rate (micro(max)), half saturation (K(s)) and substrate inhibition (K(i)) constant for pyridine by using S. putrefaciens and B. sphaericus. The high value of K(i) for S. putrefaciens than B. sphaericus indicates that the inhibition effect can be observed only in a high concentration range. The S. putrefaciens degrades pyridine with a faster rate than B. sphaericus. S. putrefaciens can be used effectively for the treatment of pyridine bearing wastewater and as an inoculum in a biofilter treating pyridine-laden gas.

Development of a yeast-based assay to determine the (anti)androgenic contaminants from pulp and paper mill effluents in India

We have constructed an efficient and reliable yeast-based detection system to evaluate the androgenic activity of endocrine disruptors from pulp and paper mill effluents (PPME). This system consists of human androgen receptor and androgen response elements driven β-galactosidase genes transformed in yeast, Saccharomyces cerevisiae. The transcriptional activation by known androgens, correlated with androgenic activities as measured by other assay systems. This assay system when applied to evaluate anti-androgenic activities, the known anti-androgens effectively inhibited reporter gene induction by testosterone. The specificity of the assay was tested by incubating the transformed cells with supraphysiological concentrations of non-androgenic steroids and none of them gave a significant response. The extracted PPME from five different mills demonstrated strong androgenic activities (about five- to eight-folds over control). These results suggest that PPME are rich in androgenic chemicals and the employed detection system could be applicable to primary screening for effectors on androgen receptor functions.

Assessment of luteinizing hormone receptor function in an endometrial cancer cell line, Ishikawa cells in response to human chorionic gonadotrophin (hCG).

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) play an important role in the development and maintenance of male and female gonads. Both these hormones act through the same specific receptor LH/hCG receptor (LHR). Recent studies have shown the existence of functional LHR in several non-gonadal tissues. The aim of this study was to confirm the functional existence of LHR in an endometrial adenocarcinoma cell line, Ishikawa cells, which has been used since long as an in vitro uterine endometrium model. Reverse transcriptase-polymerase chain reaction (RT-PCR) data showed the stable expression of LHR in this cell line. However, the receptor failed to activate the PKA pathway in response to hCG, which is the most conventional mode of LH/hCG action in target tissues. When tested for other pathways, hCG failed to activate them either. Nested RT-PCR confirmed the existence of full-length LHR and this was further supported by Western blot. This study demonstrated that although Ishikawa cells do possess a full-length LHR, which was confirmed by RT-PCR, nested RT-PCR, Western blot and DNA sequencing, it failed to activate the conventional LH-mediated downstream signaling. Based on these data we hypothesize that in Ishikawa cells LH/hCG does not utilize its conventional receptor. Whether it acts through some other receptor is a question, which can be answered through future research.

Progesterone receptor agonists and antagonists as anticancer agents.

Progesterone is a major female steroid hormone produced by the ovarian corpus luteum and by the placental syncytiotrophoblast during the second trimester. The biological effects of this steroid hormone are mediated by the ubiquitously expressed progesterone receptor. The exact link between progesterone and female reproductive organ cancer is a controversial issue with various cross-talks. The present review summarizes recent trends in the development of some (anti)progestagen in the cure and management of breast and uterine cancers.

Cytotoxic biphenyl-4-carboxylic acid targets the tubulin–microtubule system and inhibits cellular migration in HeLa cells

Abstract Two structurally similar biphenyl compounds, biphenyl-2-carboxylic acid (B2C) and biphenyl-4-carboxylic acid (B4C), were selected to assess their cellular cytotoxic and antimitotic behaviour in the quest of a potent anticancer compound. The HeLa and MCF-7 cell lines were used to determine the cytotoxic effect of the two biphenyl compounds using the MTT assay. Confocal microscopy was performed to analyze the degree of nuclear condensation and fragmentation associated with depolymerized microtubules that result from inhibiting in vitro tubulin polymerization. Circular dichroism spectroscopy along with DTNB kinetics were conducted to predict alterations in the tubulin secondary structure and global conformational changes in the tertiary structure of the protein, respectively. Finally, a wound healing assay was employed to assess whether cellular migration was inhibited in the treated HeLa cells. B4C imparted more cellular cytotoxicity in the HeLa and MCF-7 cell lines (IC50 ∼ 4 μM). B4C inhibited in vitro tubulin polymerization into microtubules with an IC50 of 50 μM. Confocal microscopy of treated cell indicated presumptive apoptosis, exhibiting fragmented nuclei with significant microtubular disruption, which was confirmed by Western blot analysis. Circular dichroism revealed a significant reduction in the α-helix content of treated tubulin. DTNB kinetics showed that approximately six –SH groups were buried in the structure. The wound healing assay revealed that B4C prevented cellular invasion. B4C had greater cytotoxic and antimitotic effects against HeLa cells than B2C. To delineate the specific mechanism of action of B4C and its derivatives, further research is warranted.

Development of a Multidrug Transporter Deleted Yeast-Based Highly Sensitive Fluorescent Biosensor to Determine the (Anti)Androgenic Endocrine Disruptors from Environment

A competent and consistent androgen receptor transactivation assay has been developed using pleiotropic drug resistance transporters Pdr5, Snq2 and Yor1 deleted yeast strain, Saccharomyces cerevisiae, intended to express the human androgen receptor and androgen response element (probasin promoter) driving the expression of green fluorescent protein to determine endocrine disruptors from pulp and paper mill effluents (PPME). Stimulation of cells by known androgens, correlated with androgenic activities as measured by other reported bioassay systems. This yeast-based assay system when applied to evaluate anti-androgenic activities, the known anti-androgens effectively inhibited fluorescence reporter gene induction by dihydrotestosterone. The specificity of the assay was experienced by incubating the recombinant yeast cells with supraphysiological concentrations of non-androgenic steroidal compounds and none of them yielded considerable response. Further, the assay was used to analyze the extracted PPME from different mills confirmed strong androgenic activities. In conclusion, these results support the earlier report by us that PPME are rich in androgenic compounds and the employed detection system provides novel high throughput fluorescence based biosensor system for successful well sensitive (picogram level) screening of (anti)androgenic chemicals from various environmental sources.

Biofuel from microalgae for sustainable development

The upcoming shortage of fossil fuels and the environmental threat of global climate change has led mankind to search for alternatives concerning its energy supply. Today, enormous efforts are made to maximize the productivity of biomass and identify new species of plants and processes to accomplish the future demand of food, fodder, materials and energy. Biofuels play a crucial role in mitigating CO2 emission, reducing global warming and acting as potential cheap alternative in comparison with the soaring prices per barrel of petroleum. The production of algal biomass for biofuels combined with the production of bulk chemicals, application in wastewater treatment, aquaculture, food and feed ingredients as well as in human health is presently drawing attention in many countries. This paper briefly elucidates the recent status of microalgae utilization for biodiesel production, together with their cultivation, harvesting, and processing.