American Journal of Advanced Drug Delivery

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Review Article Xenobiotics: An Essential Precursor for Living System

Dhaval K. Patel* and Dr. Dhrubo Jyoti Sen

Department of Pharmaceutical Chemistry, Shri Sarvajanik Pharmacy College, Gujarat Technological University, Arvind Baug, Mehsana-384001, Gujarat, India

Date of Receipt- 20/07/2013 ABSTRACT Date of Revision- 22/07/2013 Date of Acceptance- 24/07/2013 A xenobiotic is a chemical which is found in an organism but which is not normally produced or expected to be present in it. It can also cover substances which are present in much higher concentrations than are usual. Specifically, drugs such as antibiotics are xenobiotics in humans because the human body does not produce them itself, nor are they part of a normal diet. Natural compounds can also become xenobiotics if they are taken up by another organism, such as the uptake of natural human hormones by fish found downstream of sewage treatment plant outfalls, or the chemical defenses produced by some organisms as protection against predators. Xenobiotic metabolism is the set of metabolic pathways that modify the chemical structure of xenobiotics. In general, drugs are metabolized more Address for slowly in fetal, neonatal and elderly humans and animals than in Correspondence adults. The body removes xenobiotics by xenobiotic metabolism. Department of This consists of the deactivation and the excretion of xenobiotics and Pharmaceutical happens mostly in the liver. Excretion routes are urine, feces, breath, Chemistry, Shri and sweat. Hepatic enzymes are responsible for the metabolism of Sarvajanik Pharmacy xenobiotics by first activating them (oxidation, reduction, hydrolysis College, Gujarat and/or hydration of the xenobiotic) and then conjugating the active Technological secondary metabolite with glucuronic or sulphuric acid, or University, Arvind glutathione followed by excretion in bile or urine. Environmental Baug, Mehsana- xenobiotics are xenobiotic substances with a biological activity that 384001, Gujarat, are found as pollutants in the natural environment. India E-mail: dkpatel3742 Keywords: Xenobiotic metabolism, Metabolic pathways, Oxidation, @gmail.com Reduction, Hydrolysis, Secondary metabolite, Environmental xenobiotics. INTRODUCTION

However, the term xenobiotics is such as dioxins and polychlorinated very often used in the context of pollutants biphenyls and their effect on the biota,

American Journal of Advanced Drug Delivery www.ajadd.co.uk Patel et al______ISSN 2321-547X because xenobiotics are understood as XENOBIOTIC METABOLISM/DRUG substances foreign to an entire biological METABOLISM system, i.e. artificial substances, which did not exist in nature before their synthesis by Drug metabolism also known as humans. The term xenobiotic is derived xenobiotic metabolism is the biochemical from the Greek words ξένος (xenos) = modification of pharmaceutical substances foreigner, stranger and βίος (bios, vios) = or xenobiotics respectively by living life, plus the Greek suffix for adjectives - organisms, usually through specialized τικός, -ή, -ό (tic). Xenobiotics are any enzymatic systems. Drug metabolism often chemical compounds that are found in a converts lipophilic chemical compounds into living organism, but which are foreign to more readily excreted hydrophilic products. that organism, in the sense that it does not The rate of metabolism determines the normally produce the compound or consume duration and intensity of a drug's it as part of its diet. For example, in humans, pharmacological action. most drugs are part of this category, since The body removes xenobiotics by people don't produce them naturally, or xenobiotic metabolism. This consists of the consume them under normal circumstances. deactivation and the excretion of xenobiotics Xenobiotics can also be defined as and happens mostly in the liver. Excretion substances that are present in higher-than- routes are urine, feces, breath and sweat. normal concentrations, or ones that are Hepatic enzymes are responsible for the entirely artificial and did not exist before metabolism of xenobiotics by first activating they were produced synthetically by them (oxidation, reduction, hydrolysis humans. Drug metabolism is divided into and/or hydration of the xenobiotic) and then three Phases. In Phase I, enzymes such as conjugating the active secondary metabolite cytochrome P450 oxidases introduce with glucuronic or sulphuric acid or reactive or polar groups into xenobiotics. glutathione, followed by excretion in bile or These modified compounds are then urine. An example of a group of enzymes conjugated to polar compounds in Phase II involved in xenobiotic metabolism is hepatic reactions. These reactions are catalysed by microsomal cytochrome P450. These transferase enzymes such as glutathione S- enzymes that metabolize xenobiotics are transferase. Finally, in Phase III, the very important for the pharmaceutical conjugated xenobiotics may be further industry, because they are responsible for processed, before being recognized by efflux the breakdown of medications. transporters and pumped out of cells. Organisms can also evolve to A compound that is normal to one tolerate xenobiotics. An example is the co- organism may be a xenobiotic to another. A evolution of the production of tetrodotoxin commonly used example of this is the effect in the rough-skinned newt and the evolution experienced by fish that live downstream of tetrodotoxin resistance in its predator, the from the outlet of a sewage treatment plant. common garter snake. In this predator-prey Hormones produced by humans may be pair, an evolutionary arms race has produced present, even in treated water and these high levels of toxin in the newt and compounds are foreign as far as fish are correspondingly high levels of resistance in concerned1. the snake. This evolutionary response is based on the snake evolving modified forms of the ion channels that the toxin acts upon, so becoming resistant to its effects2.

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conjugated to polar compounds in Phase II reactions. These reactions are catalyzed by transferase enzymes such as glutathione S- transferase. Finally, in Phase III, the conjugated xenobiotics may be further processed, before being recognized by efflux transporters and pumped out of cells4,5. Tetrodotoxin FACTORS AFFECTING DRUG Xenobiotic metabolism (from the METABOLISM Greek xenos "stranger" and biotic "related to living beings") is the set of metabolic The duration and intensity of pathways that modify the chemical structure pharmacological action of most lipophilic of xenobiotics, which are compounds drugs are determined by the rate they are foreign to an organism's normal metabolized to inactive products. The biochemistry, such as drugs and poisons. Cytochrome P450 mono-oxygenase system These pathways are a form of is the most important pathway in this regard. biotransformation present in all major In general, anything that increases the rate of groups of organisms and are considered to metabolism (e.g. enzyme induction) of a be of ancient origin. These reactions often pharmacologically active metabolite will act to detoxify poisonous compounds; decrease the duration and intensity of the however, in some cases, the intermediates in drug action. The opposite is also true (e.g., xenobiotic metabolism can themselves be enzyme inhibition). However, in cases the cause of toxic effects. where an enzyme is responsible for The reactions in these pathways are metabolizing a pro-drug into a drug, enzyme of particular interest in medicine as part of induction can speed up this conversion and drug metabolism and as a factor contributing increase drug levels, potentially causing to multidrug resistance in infectious diseases toxicity. and cancer chemotherapy. The actions of Various physiological and some drugs as substrates or inhibitors of pathological factors can also affect drug enzymes involved in xenobiotic metabolism metabolism. Physiological factors that can are a common reason for hazardous drug influence drug metabolism include age, interactions. These pathways are also individual variation (e.g. pharmacogenetics), important in environmental science, with the enterohepatic circulation, nutrition, xenobiotic metabolism of microorganisms intestinal flora, or sex differences. determining whether a pollutant will be In general, drugs are metabolized broken down during bioremediation, or more slowly in fetal, neonatal and elderly persist in the environment. The enzymes of humans and animals than in adults.Genetic xenobiotic metabolism, particularly the variation (polymorphism) accounts for some glutathione S-transferase are also important of the variability in the effect of drugs. With in agriculture, since they may produce 3 N-acetyltransferases (involved in Phase II resistance to pesticides and herbicides . reactions), individual variation creates a Drug metabolism is divided into group of people who acetylate slowly (slow three Phases. In Phase I, enzymes such as acetylators) and those who acetylate quickly, cytochrome P450 oxidases introduce split roughly 50:50 in the population of reactive or polar groups into xenobiotics. Canada. This variation may have dramatic These modified compounds are then

AJADD[1][3][2013]262-270 Patel et al______ISSN 2321-547X consequences, as the slow acetylators are hydrophobic compounds across these more prone to dose-dependent toxicity. barriers cannot be controlled and organisms, Cytochrome P450 monooxygenase system therefore, cannot exclude lipid-soluble enzymes can also vary across individuals, xenobiotics using membrane barriers. with deficiencies occurring in 1-30% of However, the existence of a people, depending on their ethnic permeability barrier means that organisms background. were able to evolve detoxification systems Pathological factors can also that exploit the hydrophobicity common to influence drug metabolism, including liver, membrane-permeable xenobiotics. These kidney, or heart diseases. In-silico modeling systems therefore solve the specificity and simulation methods allow drug problem by possessing such broad substrate metabolism to be predicted in virtual patient specificities that they metabolize almost any populations prior to performing clinical non-polar compound.7 Useful metabolites studies in human subjects6. This can be used are excluded since they are polar, and in to identify individuals most at risk from general contain one or more charged groups. adverse reaction. The detoxification of the reactive by- products of normal metabolism cannot be BIOTRANSFORMATION OF achieved by the systems outlined above, XENOBIOTICS because these species are derived from normal cellular constituents and usually That the exact compounds an share their polar characteristics. However, organism is exposed to will be largely since these compounds are few in number, unpredictable, and may differ widely over specific enzymes can recognize and remove time, is a major characteristic of xenobiotic them. Examples of these specific toxic stress7. The major challenge faced by detoxification systems are the glyoxalase xenobiotic detoxification systems is that system, which removes the reactive they must be able to remove the almost- aldehyde methylglyoxal and the various limitless number of xenobiotic compounds antioxidant systems that eliminate reactive from the complex mixture of chemicals oxygen species9,10. involved in normal metabolism. The solution that has evolved to address this PHASES OF DETOXIFICATION problem is an elegant combination of physical barriers and low-specificity The metabolism of xenobiotics is enzymatic systems. often divided into three Phases: All organisms use cell membranes as modification, conjugation and excretion. hydrophobic permeability barriers to control These reactions act in concert to detoxify access to their internal environment. Polar xenobiotics and remove them from cells. compounds cannot diffuse across these cell membranes and the uptake of useful PHASE I–MODIFICATION molecules is mediated through transport proteins that specifically select substrates In Phase I, a variety of enzymes acts from the extracellular mixture. This to introduce reactive and polar groups into selective uptake means that most their substrates. One of the most common hydrophilic molecules cannot enter cells, modifications is hydroxylation catalyzed by since they are not recognized by any specific the cytochrome P-450-dependent mixed- transporters8. In contrast, the diffusion of function oxidase system. These enzyme

AJADD[1][3][2013]262-270 Patel et al______ISSN 2321-547X complexes act to incorporate an atom of Phase I metabolism of drug oxygen into nonactivated hydrocarbons, candidates can be simulated in the laboratory which can result in either the introduction of using non-enzyme catalysts13. This example hydroxyl groups or N-, O- and S-dealkylation of a biomimetic reaction tends to give of substrates11. The reaction mechanism of the products that often contain the Phase I P-450 oxidases proceeds through the metabolites. As an example, the major reduction of cytochrome-bound oxygen and metabolite of the pharmaceutical trimebutine, the generation of a highly-reactive oxyferryl desmethyl trimebutine (nor-trimebutine), can species, according to the following scheme12. be efficiently produced by in-vitro oxidation of the commercially available drug. + + NADPH+H +RH ADP +H2O+ROH Hydroxylation of an N-methyl group leads to expulsion of a molecule of formaldehyde, Phase I reactions (also termed while oxidation of the O-methyl groups takes nonsynthetic reactions) may occur by place to a lesser extent. oxidation, reduction, hydrolysis, cyclization, decyclization and addition of oxygen or Oxidation removal of hydrogen, carried out by mixed  Cytochrome P450 mono-oxygenase function oxidases, often in the liver. These system oxidative reactions typically involve a  Flavin-containing mono-oxygenase cytochrome P450 mono-oxygenase (often system abbreviated CYP), NADPH and oxygen. The  Alcohol dehydrogenase and aldehyde classes of pharmaceutical drugs that utilize dehydrogenase this method for their metabolism include  Monoamine oxidase phenothiazines, paracetamol and steroids. If  Co-oxidation by peroxidases the metabolites of Phase I reactions are Reduction sufficiently polar, they may be readily  NADPH-cytochrome P450 reductase excreted at this point. However, many Phase I products are not eliminated rapidly and Cytochrome P450 reductase, also known undergo a subsequent reaction in which an as NADPH:ferrihemoproteinoxidoreductase, endogenous substrate combines with the NADPH:hemoproteinoxidoreductase, newly incorporated functional group to form a NADPH:P450 oxidoreductase, P450 highly polar conjugate. reductase, POR, CPR, CYPOR, is a A common Phase I oxidation involves membrane-bound enzyme required for conversion of a C-H bond to a C-OH. This electron transfer to cytochrome P450 in the reaction sometimes converts a microsome of the eukaryotic cell from a pharmacologically inactive compound (a pro- FAD- and FMN-containing enzyme drug) to a pharmacologically active one. By NADPH:cytochrome P450 reductase The the same token, Phase I can turn a nontoxic general scheme of electron flow in the molecule into a poisonous one (toxification). POR/P450 system is: NADPH → FAD → Simple hydrolysis in the stomach is normally FMN → P450 → O2 an innocuous reaction, however there are  Reduced (ferrous) cytochrome P450 exceptions. For example, Phase I metabolism During reduction reactions, a converts acetonitrile to HOCH2CN, which chemical can enter futile cycling, in which it rapidly dissociates into formaldehyde and gains a free-radical electron, then promptly hydrogen cyanide, both of which are toxic. loses it to oxygen (to form a superoxide anion).

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Hydrolysis Conjugates and their metabolites can  Esterases and Amidase be excreted from cells in Phase III of their  Epoxide hydrolase metabolism, with the anionic groups acting as affinity tags for a variety of membrane PHASE II – CONJUGATION transporters of the multidrug resistance 16 protein (MRP) family . These proteins are In subsequent Phase II reactions, these members of the family of ATP-binding activated xenobiotic metabolites are cassette transporters and can catalyse the conjugated with charged species such as ATP-dependent transport of a huge variety of glutathione (GSH), sulfate, glycine, or hydrophobic anionsand thus act to remove glucuronic acid. Sites on drugs where Phase II products to the extracellular medium, conjugation reactions occur include carboxyl where they may be further metabolized or 17,18 (-COOH), hydroxyl (-OH), amino (-NH2), excreted . and sulfahydryl (-SH) groups. Products of conjugation reactions have increased ENVIRONMENTAL XENOBIOTIC molecular weight and tend to be less active than their substrates, unlike Phase I reactions Environmental xenobiotic are which often produce active metabolites. The xenobiotic substances with a biological addition of large anionic groups (such as activity that are found as pollutants in the GSH) detoxifies reactive electrophiles and natural environment. produces more polar metabolites that cannot diffuse across membranes, and may, Pharmaceuticals therefore, be actively transported.These Pharmaceutical drug is a chemical reactions are catalyzed by a large group of used for the alteration, diagnosis, prevention broad-specificity transferase, which in and treatment of disease, health conditions or combination can metabolize almost any structure/function of the human body. hydrophobic compound that contains Pharmaceutically active compounds (PhACs) nucleophilic or electrophilic groups13. One of are those pharmaceuticals that have by one the most important classes of this group is route or another entered the environment as that of the glutathione S-transferase (GSTs). the parent compound or as pharmacologically active metabolites. Drugs are developed with PHASE III – FURTHER the intention of having a beneficial biological MODIFICATION AND EXCRETION effect on the organism to which they are administered, but many such compounds all After Phase II reactions, the too often pass into the environment where xenobiotic conjugates may be further they may exert an unwanted biological 19 metabolized. A common example is the effect . processing of glutathione conjugates to For many years PhACs have been all Acetyl cysteine (mercapturic acid) but ignored as environmental researchers conjugates15. Here, the γ-glutamate and concentrated on the well-known glycine residues in the glutathione molecule environmentally dangerous chemicals that are removed by Gamma- were/are largely used in agriculture and glutamyltranspeptidase and dipeptidases. In industry. But with increasing technology to the final step, the cystine residue in the help in the separation and identification of conjugate is acetylated. multiple compounds in a mixture, PhACs and their effects have received increasing

AJADD[1][3][2013]262-270 Patel et al______ISSN 2321-547X attention20. PhACs have not (until relatively hisconstant guidance and support for this recently) been seen as potentially toxic work. because regulations associated with pharmaceuticals are typically overseen by REFERENCES human health organizations which have limited experience with environmental 1. Brodie ED, Ridenhour BJ, Brodie ED issues21. (2002). "The evolutionary response of Nearly all categories of predators to dangerous prey: hotspots and pharmaceuticals including pain killers coldspots in the geographic mosaic of (analgesics and anti-inflammatory), coevolution between garter snakes and newts". Evolution 56 (10): 2067–82. antibiotics (antibacterial), anticonvulsant 2. Geffeney S, Brodie ED, Ruben PC, Brodie drugs, β-blockers, blood lipid regulators, X- ED (2002). "Mechanisms of adaptation in a ray contrast media, cytostatic drugs predator-prey arms race: TTX-resistant (Chemotherapy), oral contraceptives, and sodium channels". Science 297 (5585): veterinary pharmaceuticals among many 1336–9. others have been found in the environment22. 3. Boyland E, Chasseaud LF (1969). "The role of glutathione and glutathione S-transferases CONCLUSION in mercapturic acid biosynthesis". Adv. Enzymol. Relat. Areas Mol. Biol. 32: 173– Drugs such as antibiotics are 219. xenobiotics in human because the human 4. Guengerich FP (2001). "Common and uncommon cytochrome P450 reactions body does not produce them itself, nor are related to metabolism and chemical they part of a normal diet. The body removes toxicity". Chem. Res. Toxicol. 14 (6): 611– xenobiotics by xenobiotic metabolism. 50. Xenobiotic metabolism is the set of metabolic 5. Schlichting I, Berendzen J, Chu K, Stock pathways that modify the chemical structure AM, Maves SA, Benson DE, Sweet RM, of xenobiotics. The body removes xenobiotics Ringe D, Petsko GA, Sligar SG (2000). by xenobiotic metabolism. This consists of "The catalytic pathway of cytochrome the deactivation and the excretion of p450cam at atomic resolution". Science 287 xenobiotics, and happens mostly in the liver. (5458): 1615–22. Excretion routes are urine, feces, breath, and 6. Rostami-Hodjegan A, Tucker GT (2007). sweat. Hepatic enzymes are responsible for "Simulation and prediction of in vivo drug metabolism in human populations from in- the metabolism of xenobiotics by first vitro data". Nat Rev Drug Discov 6 (2): 140– activating them (oxidation, reduction, 8. hydrolysis and/or hydration of the 7. Jakoby WB, Ziegler DM (1990). "The xenobiotic), and then conjugating the active enzymes of detoxication". J. Biol. Chem. secondary metabolite with glucuronic or 265 (34): 20715–8. sulphuric acid, or glutathione, followed by 8. Mizuno N, Niwa T, Yotsumoto Y, excretion in bile or urine. Environmental Sugiyama Y (2003). "Impact of drug xenobiotics are xenobiotic substances with a transporter studies on drug discovery and biological activity that are found as pollutants development". Pharmacol. Rev. 55 (3): 425– in the natural environment. 61. 9. Thornalley PJ (1990). "The glyoxalase system: new developments towards ACKNOWLEDGEMENT functional characterization of a metabolic The author Dhaval K. Patel is very pathway fundamental to biological life". much thankful to Dr. Dhrubo Jyoti Sen for Biochem. J. 269 (1): 1–11.

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10. Sies H (1997). "Oxidative stress: oxidants Export pumps for conjugates with and antioxidants". Exp. Physiol. 82 (2): 291– glutathione, glucuronate or sulfate". 5. Biofactors 17 (1-4): 103–14. 11. Guengerich FP (2001). "Common and 17. König J, Nies AT, Cui Y, Leier I, Keppler D uncommon cytochrome P450 reactions (1999). "Conjugate export pumps of the related to metabolism and chemical multidrug resistance protein (MRP) family: toxicity". Chem. Res. Toxicol. 14 (6): 611– localization, substrate specificity, and 50. MRP2-mediated drug resistance". Biochim. 12. Schlichting I, Berendzen J, Chu K, Stock Biophys. Acta 1461 (2): 377–94. AM, Maves SA, Benson DE, Sweet RM, 18. Commandeur JN, Stijntjes GJ, Vermeulen Ringe D, Petsko GA, Sligar SG (2000). NP (1995). "Enzymes and transport systems "The catalytic pathway of cytochrome involved in the formation and disposition of p450cam at atomic resolution". Science 287 glutathione S-conjugates. Role in (5458): 1615–22. bioactivation and detoxication mechanisms 13. Akagah B, Lormier AT, Fournet A, Figadère of xenobiotics". Pharmacol. Rev. 47 (2): B (2008). "Oxidation of antiparasitic 2- 271–330. substituted quinolines using 19. Halling-Sorensen et al. (1998). Occurrence, metalloporphyrin catalysts: scale-up of a fate and effects of pharmaceutical biomimetic reaction for metabolite substances in the environment-A Review. production of drug candidates". Org. Chemosphere 36: 357-393. Biomol. Chem. 6 (24): 4494–7. 20. Daughton and Ternes. (1999). 14. Liston HL, Markowitz JS, DeVane CL Pharmaceuticals and personal care products (2001). "Drug glucuronidation in clinical in the environment: Agents of subtle psychopharmacology". J. Clin. change? Environmental Health Perspectives Psychopharmacol. 21 (5): 500–15. 117: 907-938. 15. Boyland E, Chasseaud LF (1969). "The role 21. Jones et al. (2001). Human pharmaceuticals of glutathione and glutathione S-transferases in the aquatic environment. Environmental in mercapturic acid biosynthesis". Adv. Technology 22: 1383-1394. Enzymol. Relat. Areas Mol. Biol. 32: 173– 22. Heberer. (2002). Occurrence, fate and 219. removal of pharmaceutical residues in the 16. Homolya L, Váradi A, Sarkadi B (2003). aquatic environment-A review of recent "Multidrug resistance-associated proteins: research data. Toxicology Letters 131: 5-17.

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Table 1. Metabolism of xenobiotics

Mechanism Involved enzyme14 Co-factor Location14

Methylation Methyl transferase S-adenosyl-L-methionine Liver. Kidney, lungs, CNS

3'-phosphoadenosine-5'- Sulphation Sulfotransferases Liver, kidney, intestine phosphosulfate N-acetyltransferases liver, lung, spleen, Acetylation bile acid-CoA:amino acid N- acetyl coenzyme A gastric mucosa, RBCs, acyltransferase ymphocytes liver, kidney, intestine, Glucuronidation UDP-glucuronosyltransferase UDP-glucuronic acid lung, skin, prostate, brain

Glutathione conjugation glutathione S-transferase glutathione liver, kidney

Glycine conjugation acetyl Co-enzyme glycine liver, kidney

Figure.1. Cytochrome P450 oxidases are Figure.2. Phases I and II of the metabolism of a important enzymes in xenobiotic metabolism. lipophilic xenobiotic

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