Bioanalytical strategies for the quantification of xenobiotics in biological fluids and tissues 8

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 c

Chapter 8 Conclusions

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

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

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

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