Bioanalytical strategies for the quantification of xenobiotics in biological fluids and tissues 1

Chapter Chapter Introduction Chapter 1.1 Preface to Chapter A concise literature survey emphasizing the xenobiotics absorption, distribution, metabolism and its biotransformation in the organism are dealt in this chapter in detail, which may help to understand adverse health effects represented by these substances. This is followed by a brief literature survey on the micro extraction and quantification techniques and their significance in the disease diagnostics. The aim and scope of the present study are presented at the end of this chapter. Chapter 1.2 Xenobiotics A xenobiotic is defined as a chemical that is not usually found at significant concentration or expected to reside for long periods in organisms. In addition to manmade chemicals, natural products that are present in much higher concentration than are usual could also be of interest if they have potent biological properties, special medicinal properties. Any compound in the environment that poses a risk of exposure to a given organism also called xenobiotics [1]. Xenobiotic chemicals can be classified based on their exposure medium as they enter the body via the environment, diet and medication. During the past 50 or so years, vast quantities of diverse synthetic chemicals (xenobiotics) have entered the environment because of efforts to increase agricultural productivity and because of modern industrial processes. Chemicals which are exposed to the environment as a result of industrial effluent, fertilizers usage, natural disaster and accidents are accumulated in the air, water and soil. Environmental xenobiotics include chemical carcinogens, herbicides, insecticides, fungicides, styrene, polychlorinated biphenyls, nitrosamines, aromatic hydrocarbons, biphenyls, halogenated hydrocarbons and chemicals from building and constructing environments such as flame retardants, plasticizers, UV-blockers and biocides [2]. The contribution from industrial point sources, for instance, incineration industries (e.g. coal, tar, steel and gas production) are polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and persistent organic pollutants (POPs). These pollutants are generally chemically stable over long periods of time. Hence, many xenobiotics are recalcitrant and persist in the environment and increase in Chapter concentration with time. For example, burning of plastics and certain fertilizers forms dioxin, a xenobiotic toxin, so pervasive and it can be found in nearly every human being [3]. Many inorganic species may also be of particular concern if they are recalcitrant during most processes of wastewater treatment. These may include salts of common ions such as sodium, potassium, calcium, chloride and bromide, as well as trace heavy metals [4, 5]. Similarly, when xenobiotic compounds such as agrochemicals and industrial chemicals are utilized, they eventually reach the soil environment. Such chemicals in soil are totally available to microorganisms, plant roots via direct, contact exposure; subsequently these organisms are consumed as part of food web processes and bioaccumulation may occur, increasing exposures to higher organisms up the food chain [6, 7]. Persistent pesticides, chemical solvents and others tend to slowly invade the environment, bioaccumulate in the food chain, and have long half-lives in animals and humans [8]. Ingestion of contaminated fruits and vegetables is a potential pathway of human exposure to xenobiotics. Fruits and vegetables may become contaminated by several different pathways. Ambient air pollutants may be deposited on or absorbed by plants, or dissolved in rainfall or irrigation waters that contact the plants. Plant roots may also absorb pollutants from contaminated soil and groundwater. The addition of pesticides, soil additives, and fertilizers may also result in food contamination [9]. Another potential pathway of xenobiotics towards the human body is via foods from animal origin. The major exposure route both for humans and animals is by ingestion of endocrine disrupting chemicals (EDCs) via food intake, which leads to bioaccumulation and bio-magnification, especially towards species at the top level of food chain. For example, in the case of fish eating4 Chapter birds and marine mammals, it has been found that these mammals may contain concentrations of POPs many times higher than those found in the fish on which they feed [9-11]. In the same way, a large number pharmaceuticals and personal care products (PPCPs) are continuously released into the environment. Antibiotics, vitamins, supplements, and sexual enhancement drugs are contained in this group. "Personal care products" may include cosmetics, fragrances, menstrual care products, lotions, shampoos, soaps, toothpastes, and sunscreen [12]. Effluents from sewage treatment plants are well known to be the major source for introduction of PPCPs into the aquatic system. PPCPs enter into the environment through individual human activity and as residues from manufacturing, agribusiness, veterinary use, and hospital and community use. Individuals may add PPCPs to the environment through waste excretion and bathing as well as by directly disposing of unused medications to septic tanks, sewers, or rubbish vegetables. Because PPCPs tend to dissolve relatively easily and not evaporate at normal temperatures, they often end up in soil and water bodies. Illicit drugs such as methamphetamine and cocaine are another type of PPCP. The manufacturers of these products may accidentally spill or purposefully dump harmful by-products directly into the environment [13, 14]. The major routes by which the aforementioned environmental toxicants enter the body are through the skin, the lungs, and the gastrointestinal tract and they biotransformed within the body by the process called xenobiotic metabolism. It is noteworthy to comprehend the xenobiotic metabolism for the understanding of their effects on an organism [15]. Chapter 1.3 Xenobiotic metabolism Directly or following some conversions, xenobiotics absorbed by the human body can circulate throughout the organism with physiological fluids. The disposition of a xenobiotic in the organism consists of absorption, distribution, biotransformation and excretion [16] (Figure 1.1). Through absorption, they can undergo accumulation in various tissues and organs or they can be excreted from the organism unchanged or as polar metabolites. Some xenobiotics can act directly on the exterior surface of the plasma membrane; they bind to a specialized protein (receptor) in the membrane. Reaction with that membrane receptor can cause an endogenous compound to move from the plasma membrane to other compartments in the cell, such as the nucleus, to effect a biological response [6]. After entering the blood by absorption or intravenous administration, xenobiotics are available for distribution throughout the body. Heart, liver, kidney, brain and other well perfused organs receive most of lipohilic xenobiotics within the first few minutes after absorption. Patterns of xenobiotic distribution reflect certain physiological properties of the organism and physicochemical properties of the xenobiotics [16]. Uptake of xenobiotics into organs or tissues may occur either by passive diffusion or by unique transport processes. Within tissues, binding storage, or biotransformation can occur. During biotransformation, xenobiotic compounds, often lipophilic in nature, are converted to more polar compounds in order to be excreted from the body. Xenobiotics are species which not normally participate in the biochemical Chapter pathways of an organism [17, 18]. The same enzymes which are responsible for the metabolic activation of a safe, effective pharmaceutical may also transform inert chemicals into dangerous reactive species. These reactive species may (i) interact with the cellular environment to provoke chemical changes that assist in healing, (ii) have no effect at all, or (iii) react with cellular environment and lead to lethal effects such as cell death or cancer. The process may deplete beneficial substances and/or may generate undesirable reactive species from essential substances such as oxygen [19, 20]. Figure 1.1 The disposition of a xenobiotic in the organism. 1.4 Xenobiotic bioactivation In the past, the concept of biotransformation often implied detoxification. In recent years it has become apparent that this is not always the case. In certain Chapter instances, biotransformation enzymes, through a process called ‘bioactivation’, may give rise to stable or unstable metabolic products which are more toxic than the parent compounds. The general purpose of biotransformation reactions is detoxication, since xenobiotics should be transformed to metabolites, which are more readily excreted. However, depending on the structure of the chemical and the enzyme catalyzing the biotransformation reaction, metabolites with a higher potential for toxicity than the parent compound are often formed (Figure 1.2). This process is termed bioactivation and is the basis for the toxicity and carcinogenicity of many xenobiotics with a low chemical reactivity [22]. The interaction of the toxic metabolite initiates actions that eventually may result in cell death, cancer, organ failure and other manifestation of toxicity. Formation of reactive and more toxic metabolites is more frequently associated with phase I reactions; however, phase II reactions may also be involved in toxication as well as combinations of phase I and phase II reactions. Thus, biotransformation does not always imply detoxification, in certain instances metabolites will be produced that are capable of reacting with tissue macromolecules or obtaining toxic properties greater than those of the parent molecule [23]. Fast rates of absorption into the blood and slow rates of excretion from the body can also lead to high concentrations of xenobiotics in the body. Chapter Figure 1.2 General mechanisms for activation of a xenobiotic. 1.5 Xenobiotic toxicity All living organisms depend upon a large and complex array of chemical signalling systems to guide biological development and control cell and organ activity. The presence of a xenobiotic in the environment affects that natural scheme and always represents a risk for living organisms. Xenobiotic toxins are capable of disrupting body chemistry in many ways. Possible consequence is any imaginable symptom or disease. Several xenobiotics have the potential to disrupt reproductive, developmental, and neurological processes and some agents in common use have carcinogenic, epigenetic, endocrine-disrupting, and immune-altering action [24]. Some toxicants appear to have biological effect at miniscule levels and certain chemical compounds are persistent and bioaccumulative within the human body. The Chapter biological effects initiated by a xenobiotic are not related simply to the innate toxic properties of the xenobiotic such as the initiation, intensity, and duration of a toxic response. It was suggested that chemically inert drugs may be activated in vivo to metabolic products that are capable of forming covalent bonds with proteins, nucleic acids, and other endogenous substances, and the adducts are capable of inducing carcinogenesis or tissue (and cellular) necrosis [25]. When reactive metabolites are formed during bioactivation of xenobiotic, the target of their toxic action(s) is dependent on their stability. Short-lived intermediates generally exert their toxicity in the tissue(s) where they are produced, whereas stable ones may be formed in one tissue (usually liver) and released into the bloodstream and then affect other tissues [24-25]. 1.6 Role of xenobiotics in ovarian tumor Ovarian tumor is the most lethal gynecological malignancy among women. Despite advances in medicine and technology, the survival rate of women diagnosed with ovarian cancer has remained rather unchanged over the past 30 years [26]. The five year survival rate for early stage ovarian cancer is approximately 92%, but it is difficult to detect ovarian cancer in an early stage due to indefinite clinical symptoms. Unfortunately, most patients will be diagnosed with advanced stage disease, in which the five year survival rate is only 30% [27]. Early detection implies the screening of cancer at an early stage in its development. The screening approaches that facilitate early cancer detection must be capable of detecting small tumors at a stage when they can be cured, thus improving patient mortality. Further, an effective cancer screening approach must be cost-effective, acceptable to patients, and associated with limited 10 Chapter morbidity. However, the molecularly heterogeneous nature of cancer poses a challenge for the early detection which needs to identify an array of biomarkers to confirm its occurrence. Further, ovarian cancer cells with various histological types may express tumor markers differently; hence it is important to use multiple tumor markers to detect all ovarian cancers. In the last two decades, intensive efforts have been made to find new biomarkers for the early diagnosis of ovarian cancer [28].With advanced technology, a large number of biomarkers have been found to be associated with ovarian cancer, especially biomarkers that reflect both chemical exposure and the subsequent biological effect. Hence, information on xenobiotic chemicals plays an important role in biomarker discovery and by means, the early detection of ovarian cancer. Epidemiologic evidence on the relationship between xenobiotic chemicals and the development of cancer has been investigated several years. Generally, cancer is believed to arise from a single cell which has become “initiated” by mutation of a few crucial genes, caused by random errors in DNA replication or a reaction of the DNA with chemical species of exogenous or endogenous origin [29]. The mutations are directly related to malignant transformation of already existing benign tumor as well. Conceivably, the mutations may be the result of a local collapse in the intercellular processes which are responsible for stability of genotype, and thereby trigger a cascade of mutations [30]. This series of changes may be due to mutations of many different genes in many cells as well as to other factors affecting the integrity of tissues. This mutagenic origin of cancer may be owing to exposition to different carcinogens present in our environment. Therefore these mutations are not caused by a single chemical, however a higher rate of mutations occurs in toxic environment 11 Chapter containing one or more carcinogens [31, 32]. Hence, the information on the body burden of carcinogens provides evidence for malignant transformation. 1.7 Importance of xenobiotic quantification in body fluids and tissues Xenobiotic quantification can be of great value for associating adverse health effects with exposure to environmental chemicals, but several problems arise in attempts to understand the association. Most toxicological concerns are related to chronic diseases that can take decades to develop (e.g., cancer, dementias, neurologic disorders, osteoporosis, and arthritis) [32, 33]. Given the complexity of chronic disease processes, it is sometimes difficult to associate the onset and development of a disease with a specific chemical. The association of some chemicals with a disease process is well accepted (e.g., appearance of specific proteins in familial Alzheimer’s disease) and in some cases the association is not definite (e.g., DNA adducts). In some cases, exposure to some chemicals that not definitely produce disease (or not produce it through an understood mechanism) might nevertheless lead to the appearance of some effect. Such an effect is commonly referred as biological marker [34]. This insidious effect of some xenobiotics is mediated by their ability to mimic natural hormones in the body. One of the best known is those that mimic the steroid hormone estrogens. These agents, often referred as xenoestrogens, include pesticides and many common industrial chemicals (e.g., organochlorine pesticides (OCPs), PCBs, bisphenol A). Such a chemical biomarker is a parameter that can be used to measure the progress of disease or the effects of treatment. 12 Chapter However, the final effect of xenobiotic exposure is not related to the action of a single chemical, but to the combined effect of hundreds of different chemicals present in our body fluids and tissues. Hence, quantification of a wide range of potential chemical compounds in biological fluids and tissues may lead to proper understanding of the origin and progress of a disease. For detection and quantification of xenobiotics in complex biological samples, many sophisticated laboratory techniques, developed during the last decade, can now detect exposures to pollutants at very low concentrations, and can assess their behaviour, fate, and effect at the cellular or molecular level. 1.8 Analytical strategies for xenobiotics in complex biological samples The analysis of samples of human physiological fluids presents a formidable challenge to the analyst. In order to determine steroids and a variety of organic compounds, those samples have to be subjected to tedious and time-consuming sample preparation operations. Samples of physiological fluids are characterized by a very complex matrix, which essentially precludes direct determination of the analytes using common analytical procedures and techniques. The biological samples such as human bio-fluids (serum, plasma, urine and milk) and bio-solid samples such as tissue are complex fatty matrices, with presence of large amounts of co-extracted lipids. Thus post-clean-up of the extract is required to remove interferences. Traditional methods were time consuming and multistep approaches. For these reasons, in recent years, there are many innovations can be 13 Chapter found in sample preparation techniques that can be applied to complex biological fluids and biological solid samples. 1.8.1 Sample preparation techniques The main analytical challenges for biological samples are (i) sample volumes are limited; (ii) contaminants are present at trace levels and (iii) complexity of the sample. Due to these limitations, multistep analytical methods are not suitable. As a result, simple microextraction methods such as solid-phase microextraction (SPME) [35], stir bar sorptive extraction (SBSE) [36], liquid-phase microextraction (LPME) [37] and electromembrane extraction (EME) [38] have been reported in the literature. Likewise, exhaustive extraction such as solid-phase extraction (SPE) [39] and extraction by molecularly imprinted polymer (MIP) [40] also have been reported biofluid samples. In case of analysis of volatile and semi-volatile organic compounds, a singlestep isolation and/or enrichment process is the best solution. This requirement is met by the techniques enabling both analyte isolation and enrichment in a single stage. The most often used techniques of analyte isolation and enrichment from body fluids samples are liquid-liquid extraction (LLE), LPME, SPE, and SPME [41, 42]. Sample preparation methods for solid, semisolid and highly viscous biological matrices are more challenging than the methods for liquid samples, because of the diversity of solid samples. Classical extraction techniques such as liquid–liquid and solid phase extractions can be applied for solid samples. However they require tedious steps including mincing, shredding, grinding, pulverizing and pressurizing, to render the sample and its components into a non-viscous, particulate-free and relatively 14 Chapter homogeneous liquid state. Therefore, microextraction is a not viable approach for these samples. To eliminate these complications in dealing with complex bio-solid and semi-solid samples, modern extraction techniques such as pressurized liquid extraction [43], together with supercritical fluid extraction [44], subcritical water extraction, microwave assisted extraction [45], and matrix solid-phase dispersion [46], have demonstrated great potential. The main advantages of the modern techniques, methods could be tailor for simultaneous extraction and cleanup. 1.8.2 Quantification techniques The accurate quantification of hazardous environmental contaminants along with biologically relevant molecules is an essential prerequisite for monitoring and understanding biological processes. Precise quantification of xenobiotics in biosamples (e.g. serum, plasma, cyst fluids, urine, saliva, sweat, hair, tissues) is great challenges in analytical toxicology. Gas chromatography (GC), high performance liquid chromatography (HPLC), capillary electrophoresis (CE) or immunoassays (IA) can be used to solve particular analytical problems. Gas chromatography-mass spectroscopy (GC-MS) is the most sensitive, specific and universal analytical method for low mass xenobiotics. Since large numbers of samples are generally analyzed in studies of diseases, GC-MS which allows the study of a wide variety of compounds is desirable. Precise quantification can be performed using the selected ion mode (SIM) and use of internal standards. Different components in the mixtures are separated due to the different absorptive interaction between the components in the gas stream and the stationary phase. For most GC detectors, identification is based solely on retention time on the column. 15 Chapter GC-MS provides quantitative and qualitative information on the amounts and chemical structure of each compound. Screening of the unknown target can be achieved by using mass spectral library searching which is essential for monitoring studies [47]. The compatibility of GC-MS with analytical and database software is also an added advantage for high through-put analysis. The time consuming derivatization step can be done effortlessly with injection port derivatization. HPLC has also been widely used for the analysis of the pollutants. It is routinely used not only in the analysis of thermally labile, non-volatile ionic compounds but for all types of molecules from the smallest ions to even large biological molecules. It is also a powerful analytical tool in environmental monitoring. However, there are problems associated with HPLC analysis: (i) The limit of detection is usually above the very low sample concentration of organic pollutants in the sample; (ii) The environmental sample cannot be analyzed directly due to the complex matrix. Hence for the isolation and pre-concentration of target organic contaminants from the sample prior to HPLC analysis, efficient pre-concentration steps have been developed. Analysis of trace metals in body fluids plays an important role in the disease diagnostics, since the non-biodegradable nature of most metals makes them of the most insidious pollutants. The monitoring of trace metals in humans is therefore very important to assess their impact on chronic diseases. Hence, sensitive analytical techniques are utilized for this purpose, because most heavy metals are present in human body fluids at concentrations ranging from the sub nano-molar level to the micro-molar level. Several spectrometric techniques, such as hydride generation 16 Chapter atomic absorption spectroscopy, inductively coupled plasma-mass spectrometry (ICPMS), graphite furnace atomic absorption spectroscopy, capillary electrophoresis, have been investigated for metal determination. ICP-emission spectroscopy analysis (ICPOES) is a cost effective and an important tool for trace element quantification in biological and clinical samples. 1.9 Objective of the current study As mentioned in the xenobiotics section, accumulation of xenobiotics in human tissues and fluids has been reported to cause irreversible damage to vital tissues and organs which eventually leading to severe illness like ovarian cancer, which incidentally is the fourth, most common cancer in Singapore. For the better understanding of the role of xenobiotics in the progression of ovarian cancer, it is necessary to monitor the levels of these xenobiotics and their metabolites in biological fluids. Accurate estimation of these chemicals poses real challenge because of the complexity of biological matrix. Hence, there is a need for the development of simple and rapid analytical techniques for the analysis of complex biological matrix. The work described in this thesis mainly focused on development of analytical procedure with suitable sample preparation (microextraction) techniques to analyze bio-fluids such as ovarian tumour cyst fluid, blood plasma and serum. Using these techniques, we explored ovarian tumor cyst fluids, one of the less explored body fluids in terms of composition of xenobiotics and their impact on the severity of the cancer. Wide range of potential carcinogens including estrogens, OCPs, PCBs, heterocyclic amines, aromatic amines, organic acids, polybrominated diphenyl ethers (PBDEs), nitrosamine compounds and inorganic trace elements were quantified in 17 Chapter both malignant and benign ovarian tumor cyst fluids, using simple and cost effective micro-extraction techniques such as micro-solid phase extraction (µ-SPE) and hollow fibre protected liquid-phase microextraction (HF-LPME). Furthermore, apart from impact of xenobiotic on human tissues, we investigated the effect of most common steroid xenobiotics (estrogens) on zebra fish which is known to be a very good modal for the human genome. In addition, we developed a novel analytical procedure for early prognosis of ovarian cancer based on the existence of natural cancer biomarker protein, endorepellin. Conventional proteomics coupled with AFM imaging was used to distinguish cancer samples from non-cancer samples. 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A 1095 (2005) 203. 20 [...]... leading to severe illness like ovarian cancer, which incidentally is the fourth, most common cancer in Singapore For the better understanding of the role of xenobiotics in the progression of ovarian cancer, it is necessary to monitor the levels of these xenobiotics and their metabolites in biological fluids Accurate estimation of these chemicals poses real challenge because of the complexity of biological. .. affecting the integrity of tissues This mutagenic origin of cancer may be owing to exposition to different carcinogens present in our environment Therefore these mutations are not caused by a single chemical, however a higher rate of mutations occurs in toxic environment 11 Chapter 1 containing one or more carcinogens [ 31, 32] Hence, the information on the body burden of carcinogens provides evidence for. .. different chemicals present in our body fluids and tissues Hence, quantification of a wide range of potential chemical compounds in biological fluids and tissues may lead to proper understanding of the origin and progress of a disease For detection and quantification of xenobiotics in complex biological samples, many sophisticated laboratory techniques, developed during the last decade, can now detect... cyst fluids, one of the less explored body fluids in terms of composition of xenobiotics and their impact on the severity of the cancer Wide range of potential carcinogens including estrogens, OCPs, PCBs, heterocyclic amines, aromatic amines, organic acids, polybrominated diphenyl ethers (PBDEs), nitrosamine compounds and inorganic trace elements were quantified in 17 Chapter 1 both malignant and benign... 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For the better understanding of the role of xenobiotics in the progression of ovarian cancer, it is necessary to monitor the levels of these xenobiotics. directly into the environment [13 , 14 ]. The major routes by which the aforementioned environmental toxicants enter the body are through the skin, the lungs, and the gastrointestinal tract and they. understanding of the origin and progress of a disease. For detection and quantification of xenobiotics in complex biological samples, many sophisticated laboratory techniques, developed during the