Bioanalytical strategies for the quantification of xenobiotics in biological fluids and tissues 5

Chapter 5 Determination of inorganic trace elements in ovarian tumor cyst fluid samples by inductively coupled plasma optical emission spectrometry... 5.1 Preface to Chapter 5 Inductivel

Chapter 5 Determination of inorganic trace elements in

ovarian tumor cyst fluid samples by inductively coupled plasma optical emission spectrometry

5.1 Preface to Chapter 5

Inductively coupled plasma-optical emission spectroscopy was used to study the variations in trace elemental concentrations between benign and malignant human ovarian tumor cyst fluid specimens and to understand the effects of these elements in the progression of ovarian tumor The samples were prepared by microwave assisted acid digestion prior to ICP-OES analysis Many of the trace elements were identified and their concentrations were estimated in 30 cyst fluids (15 benign and 15 malignant) samples The elements Cr, Cd, Cu, Fe, Mn, Pb, Zn and Ni were found to

be present in all samples in significant concentration Among them Fe, Cr, Pb, and Ni were highly elevated in malignant samples compared with benign cyst fluids The excess levels of trace elements observed in the malignant tumor samples could either

be a cause or a consequence of ovarian cancer Regarding their role in the initiation or promotion of ovarian cancer, one possible interpretation is that the elevated levels of

Fe, Cr, Pb and Ni could have led to the formation of free radicals that adversely affect

DNA thereby causing ovarian tumor

5.2 Introduction

Trace elements play a vital role in all biological systems They take part in all metabolic and physiological processes, being components of different enzymes and catalysts for chemical interactions in living cells [1-3] Trace elements deficiency or excess amount of trace elements with respect to the human physiological level has been found in patients with certain diseases, including cancer Study on the trace metals for human health has been attracting much of attention in recent decades Several elements are usually determined in body fluids and tissues for the diagnosis and monitoring of various disorders, nutritional deficiencies, and occupational or environmental exposure [1-3].The number of elements of interest has increased in the recent decade, not only the trace metals but the heavy metals as well

The study of the role of the trace elements has been emphasized in the search for the possible causes of cancer The International Agency for Research on Cancer has proposed trace elements for their carcinogenesis [4, 5] Some of them or their compounds are now recognized as carcinogenic to humans Those elements include:

Be, Cr, Co, Ni, As, Cd, Sb, Pb, Hg, and Pt The carcinogenesis of Mn, Fe, Cu, Zn, Se and Sr has not been completely revealed yet, however assessment of their xenobiotic effects associated with disease progress is necessary [6] Numerous investigations have been carried out in studying the role of trace elements in the development or inhibition of cancer To understand the cause for metal induced tumors, it is important

to understand the mechanism of metal carcinogenicity

Significant differences in the concentration of various trace metals in the blood and tissues of the cancerous patients compared with the healthy personnel have

been reported [7, 8] Ovarian cancer is one of the common types of cancer worldwide and it is mainly associated with environmental factors rather than genetics The role of environmental pollutants thus becomes important in the prognosis and screening of the disease

Several studies have been reported in the recent years regarding the trace metal evaluation in the body liquids Blood is a transport medium for trace metals to and from the tissues and it provides rapid and reliable information about the trace metal metabolism in human body [3, 6] Consequently, whole blood, serum and plasma have been used in biological research for the determination of trace metal status of individuals and groups [9-11] Nonetheless, in the case of ovarian tumor, both benign and malignant ovarian cyst fluids are good source of information about tumor marker and other compounds which are associated with the origin and prognosis of tumor Thus, ovarian cyst fluid analysis will give more reliable information about the metal induced carcinogenicity of trace elements rather than any other body fluids

Determination of metal ions at trace level is difficult for the biological samples, owing to trace amount of these metal ions and complex contents of the matrices Hence, preconcentration and separation technologies have to be employed to extract analytes from the complex matrix so as to obtain suitable concentrations of the analytes for accurate determination Sample preparation is

an important step for metal analysis of biological samples The choice of sample preparation depends on the type of sample and the selection of instrument Commonly used preparation methods are acid digestion, microwave-assisted digestion Similarly,

several spectrometric techniques, such as FAAS [12], hydride generation AAS [13], inductively coupled plasma-mass spectrometry [14], graphite furnace AAS [15], capillary electrophoresis [16], atomic fluorescence spectrometry [17] have been investigated for metal determination

ICP-OES is one of the powerful tools for the determination of metals in a variety of different sample matrices The technique is based upon the spontaneous emission of photons from atoms and ions that have been excited in a RF discharge Liquid and gas samples may be injected directly into the instrument, while solid samples require extraction or acid digestion so that the analytes will be present in a solution The sample solution is converted to an aerosol and directed into the central channel of the plasma At its core the ICP sustains a temperature of approximately 10

000 K, so the aerosol is quickly vaporized Analyte elements are liberated as free atoms in the gaseous state Further collisional excitation within the plasma imparts additional energy to the atoms, promoting them to excited states Sufficient energy is often available to convert the atoms to ions and subsequently promote the ions to excited states Both the atomic and ionic excited state species may then relax to the ground state via the emission of a photon These photons have characteristic energies that are determined by the quantized energy level structure for the atoms or ions Thus the wavelength of the photons can be used to identify the elements from which they originated The total number of photons is directly proportional to the concentration

of the originating element in the sample

In this current study, benign and malignant cyst fluids samples of ovarian tumor patients were studied for the estimation of metal concentration by microwave

assisted acid digestion followed by ICP-OES technique Moreover, the trace metals are responsible for many complex biological interactions and such relationships are quite complicated, hence reliable prediction of a certain disease based on trace metal analysis requires suitable mathematical or statistical model to distinguish healthy and diseased subjects

5.3 Materials and methods

5.3.1 Reagents and samples

Analytical-reagent grade nitric acid (Merck), Hydrofluoric and hydrochloric acids (Merck), Distilled, de-ionized water (DDW) and ultrapure were used for sample preparation

A total of 30 cyst fluids (15 benign and 15 malignant) samples were analyzed

in this study The collection and storage of the cyst fluid was done as described in chapter 2 100 µL of each cyst fluid was diluted (10 -fold) to 1 mL with ultrapure prior to the acid digestion.

5.3.2 Microwave-assisted acid digestion

The samples are digested prior to analysis with nitric acid in a closed microwave digestion system (Milestone Inc., CT, USA) An aliquot of sample (1 mL) was digested with 1.5 mL of HNO3 diluted with DDW (1+2) for 30 min at 300W

power After cooling to room temperature, the digest was poured into poly(propylene) autosampler tubes and diluted to 10 mL with DDW Prior to use, these tubes were thoroughly cleaned in a sequence with detergent, water, a mixture of HNO3 (1.4 mol

L-1) and HCl (1.1 mol L-1) followed by soaking in HNO3 (0.7 mol L-1) and a final

rinse with Milli-Q water All pipette tips used for blood, plasma, cyst fluid, water and were rinsed in a sequence with HNO3 and DDW

5.3.3 ICP-OES analysis

A Perkin-Elmer dual-view optima 5030 DV ICP-OES system was used for measuring the concentrations of elements The mode of time scan was chosen to measure the concentration of metal ions due to its low consumption of sample The operational conditions of the ICP-OES system are given in Table 5.1

Table 5.1

Operational parameters of ICP-OES

5.4 Results and discussion

In the present work, trace metal analysis was carried out in the benign and malignant cyst fluid samples The metal elements were identified and their concentrations were estimated Most of the metals were present in all 30 samples and the concentration ranges from ng g-1 to µg g-1 levels Basic statistical distribution

parameters of essential trace metals (Cd, Se, Cu, Fe, Mn, Cr, Ni, Pb and Zn) in the

malignant and benign cyst fluids are shown in Table 5.2, whereas, the average metal concentrations are shown in Figure 5.1, for comparative evaluation

Figure 5.1 Comparison of average trace metals in the benign and malignant cyst fluid

samples

Large variations were observed among minimum and maximum concentrations of trace metals in all the groups In the malignant samples, Cu (1.835

µg g-1), Fe (2.252 µg g-1), Pb (1.904 µg g-1), Cr (2.309 µg g-1), Zn (1.059 µg g-1) and

Ni (1.352 µg g-1) emerged as major contributors on the average, while; slightly lower

mean concentrations were observed for Cd, Se and Mn Most of the trace metals revealed random distribution in both samples, as shown by large standard deviation (SD) values Mean concentrations of Fe, Cr, Pb and Ni in the malignant samples were significantly higher than those observed in the benign samples; however, the average concentrations of Cd, Se, Cu and Mn were found to be slightly differing in the malignant cyst fluids compared with the benign samples

Table 5.2

Distribution of trace elements in benign and malignant ovarian cyst fluid samples

Elements

In the present work, significantly elevated levels (p<0.002) of Cr were observed in the malignant samples when compared to the benign samples Despite its metabolic essentiality, chromium is a recognized carcinogen Chromium toxicity is primarily associated with exposure to hexavalent chromium compounds rather than to trivalent chromium compounds, which have relatively low toxicity [18, 19] Once it enters into the cell, chromium (VI) is partially reduced to the trivalent state, which produces genotoxic effects [20, 21] The observed elevated levels of chromium in the malignant samples of ovarian tumor, in the present work, support this mechanism of chromium carcinogenicity Similar trend was reported in the trace elemental analysis

in cancerous and normal breast tissues, chromium levels were observed to be high in cancerous tissues [22]

Iron is essential for the normal physiological functions in humans, since it is

an integral part of many proteins and enzymes It plays a vital role in the regulation of cell growth and in differentiation [23, 24] Physiological maintenance of relatively constant levels of iron is very crucial, since both iron deficiency and iron overload are harmful, and can contribute to disease development in a number of ways Iron overload is associated with several chronic diseases, such as cancer [25] Iron can both cause and promote cancer It causes tissue damage by acting as a catalyst in cell reactions The excess accumulation of iron in humans is associated with an increased risk of cancer in some previous studies [26] In the present study, higher levels (p<0.01) of iron are observed in the malignant samples relative to the benign samples Though this observation is well supported by most of the earlier works [27, 28] contradictory results have also been reported [29, 30]

From the table, the concentration of copper in the malignant samples is observed to be higher (p< 0.01) than that in the benign samples Copper is found in all living cells Though it is essential for a wide range of biochemical processes, it is also

a potentially toxic substance Moreover, being an important cofactor for angiogenesis, copper can affect carcinogenesis [31, 32] Without copper, the mediators of angiogenesis cannot function, thereby stopping the growth of new blood vessels The excess copper levels observed in the benign possibly promote the malignant transformation of ovarian tumor The present findings in agreement with the results of earlier studies on tumor, irrespective of the nature of the sample [33, 34]

Similarly, Pb is a toxic metal and its neurotoxic, and hematologic effects are well known [8].The majority of lead intoxication is related to glutathione metabolism Lead exposure in animal models alters the reduced glutathione level Glutathione is

an important substrate, which impacts the action of several drugs and toxins via its conjugation in the liver An increase in the incidence of hypertension has been observed following lead exposure in humans [8] In the present study, elevated levels

of Pb ((p<0.01) is observed in malignant samples

Nickel and certain nickel compounds have been listed by the National Toxicology Program (NTP) as being reasonably anticipated to be carcinogens Nikel compounds enhance cell proliferation by inhibiting apoptotic processes and allow for cell adaptation to metal-toxicity Nickel alters normal growth control by distinct epigenetic mechanisms [35] Nickel compounds have been observed to inhibit histone acetylation a process which facilitates the accessibility of transcription factors to DNA and this inhibition apparently contributes to tumor growth The current study shows the elevated levels of Ni (p<0.012) in malignant samples over benign samples