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

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Chapter 8 Conclusions













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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
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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
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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.
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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.