Chapter Chapter Conclusions 163 Chapter 8.1 Conclusions The current research work described in this thesis focused on the application of various analytical methodologies for the determination of environmental xenobiotics in biological fluids and tissues. The extraction techniques as well as analysis strategies, efficient for biological fluids are explained. A simple porous membrane protected µ-SPE technique in conjunction with GC-MS, proved to be a more efficient method for the extraction of a wide range of various environmental compounds (endocrine disrupters) as well as biological xenobiotics (i.e. estrogens) from complex biological fluids and tissues. Similarly, hollow fibre protected liquid phase micro-extraction together with GC-EI-MS has been successfully demonstrated to be an efficient analytical technique for determining the amount of estrogens in zebra fish liver, and the accumulation pattern of estrogens were well studied by the tank experiments. In a biomonitoring study, using a very simple µ-SPE technique, a total of 60 compounds were analyzed from the 20 tumor cyst fluids, and provided a data set which gave an insight on the chemical concentration both in benign and malignant cyst fluids. The extraction procedures employed for those analysis, were fine-tuned in terms of sorbent material and other extraction conditions. For all procedures, quality assurance was evaluated to ensure the reliability of the results. For most of the analysis GC-MS was used for the detection of analytes. GC-MS method parameters (i.e. column temperature) and derivatization of analytes were optimized for better identification. Since the aim of the study was to explore the biologically important tumor fluids and tissues, the choice of analytes should be pertinent to the tumor, such as 164 Chapter carcinogens and EDCs. Most of the analytes chosen are abundant in the environment, and in one way or another, available to human exposure. It includes the well known pollutants such as OCPs, PCBs, nitrosamines, heterocyclic amines and biological metabolites such as organic acids and estrogenic compounds. Further, the type of sample also is an important criterion to achieve the aim of the study. Ovarian tumor cyst fluids were analyzed for derive information on the xenobiotic biomarkers that might influence or directly associated with the progression of the disease. Compared to other body fluids, such as blood plasma and serum, ovarian cyst fluids are more relevant to the objective of the study. Further, the cyst fluids were studied for their and inorganic trace element levels by applying a combination of high throughput analytical strategies. The accumulation behavior of the biologically significant environmental compounds, estrogens, on an organism was observed by taking zebrafish as a model for this study. The accumulation pattern over time was observed and similar accumulation can be expected on humans. The influence of endorepellin, an important biomarker for tumor, has been successfully evaluated using an interesting analytical procedure and significant results which were more consistent with the earlier findings were obtained. The levels of organic compounds and inorganic trace elements in cyst fluids studied were, as expected, slightly lowered, yet comparable with other body fluids and tissues. However, for the first time, the concentration trend between benign and malignant stage tumor samples was well documented in this study. In the case of estrogens, among the four estrogens studied, the synthetic estrogen, diethylstilbestrol is higher in malignant cyst fluids. The other three estrogens which are naturally occur in human body exhibit a different trend in that, their levels significantly were higher 165 Chapter in benign than malignant cyst fluids. This could be understandable that the synthetic estrogen level is influenced by environmental factors, whereas the natural estrogens levels are influenced by the internal factors such as metabolic changes. In the case of ovarian tumor, the role of estrogens was well reported earlier. The trend obtained in this study shed a light on their role in disease progress. Considering other xenobiotics, the levels of each compound was quantified in benign and malignant of cyst fluids. The results showed that, although, most of the compounds were present in both groups of samples, they were relatively mere highly correlated in the malignant group than in the benign groups. With the aid of PCA, their trend in the two groups of samples is compared and individual and compound group trend analysis was also explored in detail. In the case of PCBs, the results showed that their levels were significantly higher in malignant samples compared to benign group. Two hundred and nine individual congeners were screened, among them 87 congeners were detected in both groups of samples. Their levels and trend in cyst fluids were estimated with the compound composer database software. In the case of trace elements no significant trend was observed since their presence was not consistent in the samples studied. Nevertheless, some metals were found to have higher levels in malignant than benign cyst fluids and their possible mechanism towards carcinogenicity was discussed. The study on factors associated with the malignant transformation of benign cyst fluids is an emerging research interest in ovarian cancer research. The present study explored the environmental factors in detail and other internal factors such as estrogens and endorepellin protein to distinguish benign and malignant stage ovarian tumor. 166 Chapter The following conclusions can be drawn from the present study; 1) Complex biological matrices i.e. ovarian cyst fluids, were successfully analysed for wide range of environmental xenobiotic levels by the simple and rapid microextration techniques. 2) For the first time, the malignant and benign stage ovarian tumor cyst fluids were effectively compared in terms of concentration levels of compounds which give some clear insights of the influence of xenobiotics in the disease progression. 3) The complete profile of environmental organic, inorganic and steroid chemicals in cyst fluids helpful to understand their synergic effect on the malignant transformation and etiologic research of cancer. Based on the conclusions drawn from the current work, the following implications are made for further studies: 1) For profiling studies, the numbers of samples studied influence the results to some extent. In this work, the number of cyst fluid samples was largely restricted by their availability, cost and biological nature. Inclusion of more samples would enhance the reliability of the results. 2) The association of chemical levels on the disease progress is greatly influenced by the characteristics of the sample, i.e. patient’s information such as age, ethnic group, smoking and eating habits and pathology. If the sample groups were sorted by those data, more precise results would be possible. 167 . Since the aim of the study was to explore the biologically important tumor fluids and tissues, the choice of analytes should be pertinent to the tumor, such as Chapter 8 165 carcinogens. plasma and serum, ovarian cyst fluids are more relevant to the objective of the study. Further, the cyst fluids were studied for their and inorganic trace element levels by applying a combination. The trend obtained in this study shed a light on their role in disease progress. Considering other xenobiotics, the levels of each compound was quantified in benign and malignant of cyst fluids.