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

1.1 Preface to Chapter 1 A concise literature survey emphasizing the xenobiotics absorption, distribution, metabolism and its biotransformation in the organism are dealt in this chapter

Chapter 1 Introduction

1.1 Preface to Chapter 1

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

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 man-made 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

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

eating-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 do 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]

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 do not normally participate in the biochemical

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

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

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

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

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