BIOCHEMICAL CHARACTERIZATION OF TOXIC CONSTITUENTS OF SEED EXTRACT OF AZADIRACHTA INDICA A. JUSS

ABSTRACT

The  toxic  constituents  of  seed  extracts  of  Azadirachta  indica  A.  Juss  were  isolated  and biochemically  characterised  to  elucidate  the  spectrum  of  toxicity.  The  constituents  were isolated using toxicity-guided technique.Acute toxicity test established an oral LD50 >5 g/kg in mice. Chronic oral administration of the extracts and fractions significantly (P<0.05) increased the activities of ALP, AST and ALT. The methanol extract (ME) did not cause any significant change in bilirubin concentration.  Lower doses (100  and 500 mg/kg) of the extract reduced urea levels whereas  higher  doses of both extract  and fractions  caused  significant  (P<0.05) increase  in  urea  levels.  The  crude  extract  and  fractions  lowered  fasting  blood  sugar  and cholesterol concentrations in normal treated rats. The ME caused significant (P<0.05) increase in Na+ concentration and reduction in K+  concentration whereas the concentrations of HCO3- and  Cl-   remained  unchanged  throughout  the  experimental  period.  The  ME  also  reduced neutrophil count and caused significant (P<0.05) increase in Hb concentration, PCV percentage and lymphocyte  count. Lower doses of ME increased  platelet  count  whereas  higher doses caused  a reduction.  With the exception  of TN-1 and TN-2, the  extract and other fractions significantly (P<0.05) increased the body and organ weights  of treated rats. Tissue sections from the liver of ME-treated rats showed uniformly edematous and hepatocyte necrosis with steatosis and portal tract inflammation. Kidney tissues showed edematous necrotic tubules with destruction of basement membrane. Comparison of the magnitude of liver toxicity showed that TN-2 was more toxic than TN-1. TN-1 caused extensive areas of liver cell edema while TN-2 caused hepatic necrosis with extensive severe changes. Structure elucidation revealed TN-1 and TN-2 to be 6-deacetylnimbin and nimbolide respectively.

CHAPTER ONE

INTRODUCTION

Toxicology is  the  study of the  dynamic  interaction of chemicals  with  living system. It is also the science of numerous industries and regulatory agencies from those involved with development and regulation of food additives to those involved with use and the remediation of hazardous chemicals. Toxicological investigations explore the interaction of chemicals with biological systems by focusing on the adverse effects caused by such interaction.

Toxicity defines the degree of damage a substance is able to cause an exposed organism, such as cells (cytotoxicity) or organs (organotoxicity) such as the liver (hepatotoxicty) and  kidney (nephrotoxicity) (UNECEGHS, 2008).  The  potency of a chemical depends on its movement through the body to the target site (toxicokinetics); its ability to interact with the body to cause harm (toxicodynamics); and the dose the body receives (exposure level) which is in turn modified by the toxicokinetics and toxicodynamics of the chemical. Both the kinetics and dynamics depend upon the current biochemical status of the organism e.g. enzyme levels at the time of exposure, nutritional status and stress levels. A variety of substances including chemical, physical and biological factors could cause  varying degrees of damage to  cells  or  tissues which manifest as toxicity.

Generally, three types of toxic agents are recognized: chemical, biological and physical (UNECEGHS, 2008). Chemicals include inorganic substances such as lead, mercury, asbestos, hydroflouric acid, chlorine gas and organic compounds such as methyl alcohol, most medications and poisons from living things. Physical toxic agents include direct blows, sound, vibrations, heat, cold, non-ionizing electromagnetic radiation such as infrared and visible light, and ionizing radiations such as x-rays, , , and  radiations. Biological entity includes those bacteria and viruses that are able to induce  disease in living  organism (UNECEGHS,  2008).  Toxic  reaction  may  differ  depending  on  the duration of exposure. A single exposure or multiple exposures occurring over 1 or 2 days represents acute exposure. Multiple exposures continuing over a longer period represent a chronic  exposure.  Toxicological  investigations  are  also  concerned with  the  possible

harmful effects of contact with small concentration of chemicals over long period of time. This type of chronic situation is called low-level, long –term exposure. The harmful effect resulting from either acute or chronic exposure may be reversible or irreversible. The relative reversibility of the toxic effect depends on the recuperative properties of the affected organ (Liu, 2004).

1.2 Drug-Induced Toxicity

Drug toxicity may occur with overdosage of a medication, accumulation of the drug in the body over time or the inability of the patient’s body to eliminate the drug. Drug-induced liver injury (DILI) is a major health problem that challenges not only health care professionals but also the pharmaceutical industry and drug regulatory agencies. Ostapowicz et al. (2002) stated that DILI accounts for more than 50% of acute liver failure, including hepatotoxicity caused by overdose of acetaminophen and idiosyncratic liver injury triggered by other drugs.

Because of the significant patient morbidity and mortality associated with DILI , the U.S Food and Drug Administration (FDA) has removed several drugs from market including  bromfenac  and  ebrotidine  (Zimmerman,  1999;  Hunter,  1999;  Anonymous

1998). DILI is the most common cause for the withdrawal of drugs from the pharmaceutical market (Lasser et al., 2002).

1.2.1   Hepatotoxicity

Many xenobiotics (drugs and environmental chemicals) are capable of causing some degree of liver injury. Xenobiotic-induced liver toxicity is implicated in 2 – 5% of hospitalizations for jaundice, an estimated 15 – 30% of the cases of fulminant liver failure, and 40% of the acute hepatitis cases in individuals older than 50 years (Bass, 1996).  Most  drug-induced  liver  injuries  resolve  once  the  drug  is  withdrawn,  but morbidity may be severe and prolonged as recovery ensues. A 5% overall mortality rate for drug-induced liver injury has been reported (Werth et al., 1993). The liver is prone to xenobiotic-induced injury because of its central role in xenobiotic metabolism, its portal location within the circulation, and its anatomical and physiological structure (Jones, 1996). The liver is divided into multiple lobules, each centered around a terminal hepatic

venule and surrounded peripherally by six portal tracts. The regional pattern of hepatocellular necrosis with some xenobiotic-induced liver injuries can be understood by dividing the liver into functional subunits referred to as acini (Rapport et al., 1993; Jones

1996). Each liver acinus is divided into three concentric zones of hepatocytes radiating from a portal tract and terminating at one or more adjacent terminal hepatic venules. Hepatocytes closest to the portal tract (Zone one) receive blood most enriched with oxygen and other nutrients and are most resistance to injury. Hepatocytes more distal to the blood supply receive a lower concentration of essential nutrients, making them more susceptible to ischemic or nutritional damage. Most important for xenobiotic-induced hepatic  damage,  the  centrilobular  (zone  three)  hepatocytes are  the  primary  sites  of cytochrome P450 enzymes activity which makes them most susceptible to xenobiotic- induced liver injury (Thurman, 1986).

1.2.1.1 Types of Xenobiotic-Induced Liver Injuries

Xenobiotic-induced  liver  injuries  can  be  broadly  classified  into  cytotoxic (necrotic or steatotic), cholestatic, or mixed (Bass, 1996). The presence of an injury can be established on the basis of clinical and biochemical evidence. However, histological examination  of  a  liver  biopsy  specimen  remains  the  only  means  of  definitively diagnosing the type of injury present (Friendman et al., 1996; Friendman et al., 2003).

i.          Hepatocellular Necrosis

Hepatocellular  necrosis  can  range  in  severity  from  increases  of  amino  transferase enzymes and jaundice to overt hepatic failure (Zimmerman, 1978). With intrinsic hepatotoxins, non specific gastrointestinal symptoms such as nausea or vomiting begin within  few  hours  of  exposure.  These  symptoms  often  resolve  within  48-72  hours followed by a 1-2 day period of relative well-being. Overt liver failure is generally established within 3-5 days, characterized by jaundice, coagulopathies, neurological symptoms, and acute renal failure. The degree of aminotransferase enzyme increases, hyperbilirubinenia, and prolongation of the prothrombin time have prognostic significance, as does the appearance of any manifestation of hepatic encephalopathy (Moutt et al., 1975).

ii. Toxic Hepatitis

In toxic hepatitis, hepatocellular necrosis is a hallmark of the injuries, but the associated symptoms and  histological pattern of injury are  nearly identical to those observed with acute viral hepatitis (Pande et al., 1996; Barnard, 1994). Histologically, these injuries reflect diffuse hepatocellular necrosis, which may be associated with cholestasis. Lobular structure is generally maintained, and even in severe cases, areas of necrosis are surrounded by viable hepatocytes that reveal various degrees of degenerative changes (Zimmerman, 1993). Prominent monocytic or eosinophilic inflammatory infiltrates are common. These injuries are thought to result from bioactivation of toxic metabolite (Zimmerman, 1993). Symptoms of toxic hepatitis range from increases of hepatic aminotransferase enzymes (Serum glutamate-oxalate transaminase, SGOT and Serum glutamate-pyruvate transaminase, SGPT) to signs of overt liver failure.   With drugs such as phenytorn, these injuries often present with onset of fever and nausea, which may be accompanied by diffuse rash (Brown et al., 1986). As with hepatocellular necrosis induced by intrinsic hepatotoxins, clinical and biochemical markers have prognostic value (Mitchell et al., 1976).

iii. Steatosis

Steatosis  results  from the  abnormal accumulation of triglycerides  within  the hepatocyte (Hoyumpa et al., 1975). Macrovesicular steatosis is characterized by a single large cytoplasmic vacuole of triglyceride within the hepatocyte that displaces the nucleus peripherally.  The  etiology  of  macrovesicular  steatosis  is  multifactorial,  including increased mobilization of fatty acids, increased hepatic synthesis of fatty acids, increased synthesis of triglyceride from fatty acids, and deficient removal of triglyceride from the hepatocyte  via  defective  very  low  density  lipoprotein (VLDL) synthesis  (Salaspuro,

2003). Microvesicular steatosis is a less common but more severe variant, resulting from deficient  mitochondrial -oxidation of fatty acid and the presence of multiple  small droplets of triglyceride (Keeffe et al., 2004). The -oxidation of fatty acids (a  process results in the production of acetyl-coenzyme A moieties is the source of ATP  in most cells and  its disruption promotes the esterification of fatty acid in the  cytoplasm to

triglyceride, robbing the cell of energy. Valproic acid is an established cause of microvesicular steratosis, which resembles Reye syndrome and is in fact more likely to occur in young children. Valproic acid-induced liver injury is thought to result from phase I bioactivation (Eadie et al., 1988). Cytochrome P450 enzymes mediate the production of valproic acid, an oxidative metabolite capable of generating coenzyme derivatives. Production and accumulation of these derivatives may inhibit mitochondrial -oxidation via depletion of free co-enzyme concentration (Kesterson et al., 1984).

iv. Cholestasis

Xenobiotic-induced cholestasis results from the disruption of bile production or flow. Hepatocanalicular (hypersensitivity) cholestasis is characterized by prominent monocytic portal inflammation and secondary damage to bile Canaliculi, as seen with chlorpromazine. These drugs metabolites interfere with bile acid secretion via disruption

of canalicular membrane fluidity and Na+/K+-ATPase (Elias and Boyer, 1979). Overt

jaundice is accompanied by extreme increases of alkaline phosphatase and conjugated serum bilirubin. Hepatic aminotransferase enzymes are only mildly increased  in the absence of significant necrosis (Zimmerman et al., 1993).

v. Hepatic Vascular Injury

Veno-occlusive disease is a severe form of drug induced liver injury characterized by thrombosis of efferent hepatic venules, leading to centrilobular necrosis and liver outflow obstruction, which can progress to congestive cirrhosis. The condition presents with  onset  of severe  abdominal pain,  hepatomegaly, and  jaundice,  accompanied  by extreme increases of hepatic aminotransferase and alkaline phosphatase enzyme (McDonald et al., 1993). Oral contraceptive can also produce another type of vascular lesion called peliosis hepatitis, in which weakening of sinusoidal membrane leads to the development of blood-filled sacs within the hepatic parenchyma (McDonald et al., 1993).

vi. Hepatic Tumors

Chronic use of oral contraceptives is associated with the development of hepatic adenomas, benign tumors typically observed only in women of child bearing age. These tumors usually resolve completely with drug withdrawal, and risk of tumors development is  highly correlated  with  the  duration of drug  exposure  (Edmondson et  al.,  1977). Hepatocellular carcinomas have  been associated with the chronic use of androgenic steroid (Henderson et al., 1983).

1.2.1.2      Drug Metabolism in Xenobiotic-Induced Liver Injury

Most drugs are not intrinsically toxic to the liver but can cause injury secondary to the production of hepatotoxic drug metabolite, a process known as bioactivation (Vessey,

1996; Dahm and Jones, 1996). Because gastrointestinal absorption is enhanced by lipid solubility, most xenobiotics are highly lipophilic compounds which are poorly excreted by the kidney (Vessey, 1996). The liver plays a critical role in promoting excretion of these  compounds by transforming them into  metabolites of greater  water  solubility. Metabolic reactions are of two types, phase I and phase II (Vessey, 1996). Phase I (oxidation, reduction, or hydrolysis) reactions typically occur first, and enhance water solubility by generating hydroxyl, carboxy, or epoxide functional groups on the parent compound. These functional groups facilitate phase II reactions (conjugation with glucuronate, sulfate, acetate, or glutathione moieties). Conjugation reactions enhance water solubility and renal excretion (Vessey, 1996). Phase II reactions also play a role in the   prevention  of   xenobiotic-induced   liver   injury   because   most   conjugates  are biologically inactive (Lee, 1995; Reuben, 2004). Disruption of normal phase II processes can lead to accumulation of hepatotoxic phase I metabolites.

Phase I oxidation and reductions are primarily catalyzed by cytochrome P450 enzymes,  a  supergene  family  of haeme-containing, mixed-function oxidase enzymes found in great concentration in the smooth endoplasmic reticulum of centrilobular hepatocytes (Bernhardt, 1995; Jessica et al., 2003). These enzyme reactions have the potential  to  induce  cellular  injury  through  several  mechanisms  of  toxicity.  The cytochrome P450 enzyme-catalyzed oxidation of xenobiotics generates a highly electrophilic  intermediate  capable  of  forming  covalent  adducts  with  critical  circular

macromoles such as thiol-containing membrane proteins that regulate calcium homeostatsis (Bellomo and Orrenius, 1985). This induction of increased intracellular calcium concentrate may be the common pathway leading to cell death. Cytochrome P450 enzyme-mediated reduction of halogenated hydrocarbons can also generate free radical intermediates, which can directly damage cell membrane via lipid peroxidation, or can target nucleophilic DNA residues (Thor Orrenius, 1980; Lynch Price, 2007; Recknagel et  al.,  1989).  Similar  cellular  damage can result  from the  generation of reactive oxygen species such as hydrogen peroxide and hydroxyl free radical during a process known as redox cycling (Abate et al., 1990; Skett et al., 2001). Redox cycling occurs when a reduced substrate reoxidizes in the presence of oxygen, thereby reducing the oxygen molecule (Myers et al., 1977).

1.2.1.3   Determinant of Host Susceptibility to Xenobiotic-Induced liver injury

Xenobiotic-induced liver injuries can be broadly classified as intrinsic or idiosyncratic (Bass, 1996). Intrinsic injuries are predictable, in that a threshold dose exists in all individuals leading to zonal liver necrosis accompanied by little or no signs of inflammation. Those injuries are generally the result of phase I bioactivation reactions, with  damage  mediated  by  reactive  drug  metabotiles.  In  contrast,  the  nature  of idiosyncratic  liver  injuries suggests that  most  of these  are  mediated by an  immune mechanism (Pohl, 1990). Idiosyncratic liver injuries are associated with classic signs of hypersensitivity, including fever or rash, and the liver biopsy specimens reveal evidence of monocytic or eosinophilic infiltrates (Kleckner et al., 1975). These reactions tend to occur only after repeated exposure, suggesting the need for initial sensitization and drug rechallenge which elicits reappearance of symptoms. Both humoral and circular immune mechanisms have been implicated in these types of injuries (Bass, 1996). One proposed explanation is the formation of a metabolic macromolecule to generate a neoantigen (Pohl, 1990).

The  phase  II  glucuronidation of these  compounds can  also  produce reactive acylglucuronides, which may bind irreversibly to nucleophilic amino acid side chains in hepatocyte  membrances,  potentially  inducing  a  cell-mediated  or  humoral  immune response (Boelsterl et al., 1995). T lymphocytes or immunoglobin molecules targeted

against a variety of neoantigens have been identified. Some of these  immunoglobin molecules recognize the cytochrome P450 isoenzyme responsible for the metabolism of the offending drug compound (Boelsterl et al., 1995).

Variability in Phase I Enzymatic Activity

Three CYP gene families, designated CYPI, CYP2, and CYP3 encode the cytochrome P450 enzymes that play the major role in human xenobiotic metabolism (Watkins, 1992). Genetic, physiologic, pathophysiologic, and xenobiotic-induced factors that affect cytochrome P450 enzyme activity may help to account for the increased susceptibility of certain individuals to drug-induced liver injury (Dahm and Jones, 1996). Women are at increased risk of drug-induced liver injuries, particularly chronic ones. Oral contraceptives are known inducers of cytochrome P450 enzyme activity, whereas pregnancy has been shown to induce certain isoenzymes, such as P450IIIA4, and inhibit others (Ohkita and Goto, 1990). Cytochrome P450 1A2 activity is gender-related, with males consistently exhibiting higher activity (Horn et al., 1995). However, parity may be an important determinant of P450 1A2 activity. Genetic polymorphisms, characterized by poor and extensive metabolizing phenotypes, have been identified in the P450IIIC18, P450IID6, P450IIEI, and possibly the P450IIIA4 isoforms and can alter susceptibility to xenobiotic-induced liver injury (Bernhardt, 1995). For example, the risk of perhexiline maleate-induced  liver   injury  is   higher   in   individuals  with  the   P450IID6  poor- metabolizing phenotype (Morgan et al., 1984).

Many drug-induced liver injuries are clearly age-related (Neim et al., 1976). The activity of some cytochrome P450  isoenzymes (such as P4501A2 and P450IID6) is reduced by approximately 70% in neonates, followed by a rapid increase in activity during the first few weeks to months after birth to an amount two-to three fold more (for P4501A2) than that of adults. The activity of other isoforms, eg, P450IIIA2 enzymes, can be higher in newborn infants than in adults and certain p450IIA isoforms are primarily expressed only in the developing fetus (Shimada et al., 1996). The classic example in which altered activity of a cytochrome P450 isoenzyme can increase the risk of liver injury is acetaminophen toxicity. Ordinarily >90% of an acetaminophen dose undergoes phase II glucuronidation and sulfation, yielding inactive conjugates that are excreted in

urine and bile (Lee, 1995). About 5% of a dose is oxidized by cytochrome P450IIEI isoenzymes, and to a lesser degree by other P450 isoenzymes, to the hepatotoxic intermediate N-acetyl-p-benzoquinone imine (NAPQI). Hepatocellular damage is ordinarily prevented by phase II glutathione conjugation, which converts NAPQI to the inactive metabolite mercapturic acid. Acute ingestion of approximately 10g of acetaminophen saturates the normal glucuronidation and sulfation pathways, leading to increased production of NAPQ1, which rapidly depletes available glutathione stores. The risk of damage is increased, and the threshold dose lowered, with concomitant use of compounds such as alcohol or Phenobarbital that  are  capable of inducing P450IIEI activity (Nolan et al., 1994).

Variability in Phase II Activity

Another important to host susceptibility is the functional capacity of phase II detoxification pathways. The most common type of phase II reaction is glucuronidation, where glucuronic acid is transferred from uridine diphosphate glucuronic acid (UDPGA) to a drug or phase I metabolite by the enzyme uridine diphosphate glucomyl transferase (UDPGT) (Vessey, 1996). UDPGT enzymes are produced by two gene families. UGT1 and UGT2 (Irshaid and Tephly, 1987). At least six isoenzymes are encoded by UGT1 genes and four isoforms by UGT2 (Ritter et al., 1992; Jansen et al., 1992). The potential for individual variability is given by the fact that inducing agents such as Phenobarbital do not affect the activity of these isoforms equally (Bock et al., 1984). The capacity of the glucuronidation process can be inhibited by the temporary depletion of available UDPGA stores by drugs such as acetaminophen and chloramphenicol (Howell et al.,

1986). Age can also alter UDPGT activity which is low at birth but increases steadily to nearly adult values by age 1-3 months (Onishi et al., 1979). Nutritional deficiencies are another potentially relevant cause of deficient UDPGA stores (Thurman and Kanffman,

1980).  Sulfation  reactions  catalysed  by  three  families  is  cytosolic  sulfatransferase enzymes represent important detoxification pathways for alcohols and phase I intermediates containing phenol groups (Falany  and  Roth,  1993).  The  efficiency of sulfation reactions can be compromised by temporary depletion of inorganic sulfate pools by ingestion of drugs such as salicylamide (Levy, 1986).

Glutathione conjugation is critical in preventing liver injury from several agents, including  acetaminophen  and  bromobenzene  epoxide,  by  acting  as  a  free  radical scavenger (Vessey, 1996). Acetaminophen overdose causes liver injury secondary to the temporary depletion of glutathione stores in the liver. Administration of the antidote N- acetylcysteine prevents further injury by stimulating glutathione synthesis, thereby replenishing liver stores (Smilkstein et al., 1988). Glutathione stores are also senstitive to fasting and alcohol ingestion and, as in most phase II pathways except sulfation, glutathione conjugating activity is depressed in neonates, even though glutathione transferase enzyme activities are apparently within the reference interval (Rollins et al., 1981). Amine or hydrazine-containing drugs or phase I metabolites are detoxified primarily by phase II acetylation reactions, catalysed by cytosolic N-acetyltransferase (NAT) enzymes (Vessey, 1996). NAT-1 and NAT-2 represent the two gene families currently known to exist in the human liver (Grant, 1993). Polymorphism in Nat-2 results in  the  rapid  or  slow  acetylator  phenotype,  which  has  been  implicated  in  host susceptibility  to  liver  damage  by  drugs  such  as  Isoniazid  (Timbrell  et  al.,  1977). Isoniazide undergoes extensive NAT-2 catalysed acetylation to acetylisoniazid, which is then hydroxylated by cytochrome P450 enzymes to the hepatotoxic intermediate acetylhydrazine, a metabolite capable of forming covalent cellular adducts (Woodward et al., 1984). The risk of liver toxicity is higher in slow acetylators, in the elderly, and in association  with  concomitant  use  of  cytochrome  P450  inducers  such  as  alcohol  or rifampin (Dickinson et al., 1981).

1.2.1.4      Classification of Injury and Evaluation of Liver Function

A variety of static and dynamic biochemical markers of liver injury are widely used in the detection of injury, assessment of injury type and severity, determination of functioning liver mass, prognosis, and response to medical management. Each marker has inherent deficiencies in sensitivity or specificity, and no single method appear capable of completely diagnosing the  etiology,  severity,  and  prognosis associated with a  given injury (Kaplan, 1993).

These biochemical markers include:

i.         Serum Aminotransferase Enzymes

Serum activity concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are the most commonly used biochemical markers of hepatocellular necrosis (Friendman, 1996). These enzymes are localized in periportal hepatocytes, reflecting their role in oxidative phosphorylation and gluconeogenosis. ALT is highly specific for the liver, whereas AST is also located in the heart, brain, kidney, and skeletal muscle, making this enzyme less specific for liver injury (Reg, 1978). These enzyme activities presumably increase as a result of cellular membrane damage and leakage. Serum aminotransferase activities are increased in all types of hepatic injury.

The highest increases are observed with acute hepatocellular injuries, such as xenobiotic-induced necrosis or acute viral hepatitis. The degree of increase does not correlate  well with the  extent  of liver  injury.  A decline  in serum activity indicates recovery but in fulminant injury may be a poor prognostic sign, reflecting a major loss of functional hepatocytes (Reg, 1978).

ii.        Serum Alkaline Phosphatase

This is a family of isoenzymes that catalyze the hydrolysis of phosphate esers, generating inorganic phosphate (Millan et al., 1988). Sources of alkaline phosphatase include the liver, bone, leukocytes, kidneys, and placenta. Alkaline phosphatase activities are  markedly  increased  in  children  and  adolescents,  as  well  as  third  trimester  of pregnancy. Serum alkaline phosphate increases to some extent in most types of liver injury. Bile acid account for this increase: They induce alkaline phosphatase synthesis and exert a detergent effect on the canalicular membrane, allowing leakage into serum (Kaplan,  1986).  The  highest  concentrations  are  observed  with  cholestatic  injuries. Alkaline phosphatase activity concentrations cannot be used to differentiate between intrahepatic or extrahepatic etiologies, because serum increases are observed with each type of bile statsis. The specificity of alkaline phosphatase for the liver is poor, for several other conditions (particularly bone diseases, growth spurts or pregnancy) also increase serum values.

iii. Serum Bilirubin

Free bilirubin is not water soluble and must be bound to albumin to facilitate transport to the liver. This indirect or unconjugated bilirubin fraction therefore does not enter the urine. Indirect hyperbillirubinemia is  generally associated with haemolysis. Higher increases or associated abnormalities of other liver enzymes indicate a hepatic aetiology. When hepatic injury is present, the direct bilirubin fraction is at least 50% of the total serum value, but because urine bilirubin is more sensitive indicator of liver injury than is serum; an increase in urinary bilirubin is always indicative of a corresponding increase in serum direct fraction attributable to intrahepatic or extrahepatic cholestasis. The degree of increase in serum bilirubin value has prognostic significance in chronic liver injuries, but not in acute injuries (Dickson et al., 1989).

iv. Serum Bile Acids

Cholic acid and chenodeoxycholic acid are the primary bile acids in humans (Erlinger, 1993). These organic anions are synthesized in hepatocytes from cholesterol, conjugated to glycine or taurine, and excreted into the canaluculus. Measurement of serum bile acid concentrations is a more specific indicator of functional hepatic excretory capacity than serum billirubin (Berk and Javitt, 1978).

An increase in serum bile concentrations in fasting is highly specific for liver injury and serves to exclude congenital or hemolytic causes of hyperbilirubinemia. The greatest increases are observed in acute viral hepatitis or extrahepatic cholestasis. The ratio of cholic to chenodeoxycholic acid decreases with chronic injuries such as cirrhosis and increases with extrahepatic bile obstruction (Linnet and Kelback, 1982).

v. Serum Albumin

Serum albumin, the major plasma protein synthesized in the human liver is a useful maker of hepatic synthetic function (Friedman, 1996). The long elimination half- life  (20  days)  and  ample  storage  pool,  however,  limit  the  utility  of  this  index  in evaluation of chronic liver injuries. Several factors other than liver injury can disrupt

albumin synthesis, including nutritional deficiencies and alterations in plasma oncotic pressure (Keshgegian, 1984).

vi. Prothrombin Time (PT)

PT provides an index of hepatic synthesis capacity that applies to both acute and chronic liver injuries (Keshgegian, 1984). An indicator of the extrinsic clothing cascade, the PT provides an indirect measure of the hepatic synthesis of clothing factors I, II, V, VII, IX and X (Suttie and Jackson, 1977). Other causes of a prolongation of PT include vitamin K deficiency, warfarin therapy, and acquired or congenital clotting factor deficiencies. PT has prognostic value in both acute and chronic liver injury. An extreme or worsening prolongation of the PT in the setting of acute hepatocellular necrosis is associated with an increased risk of fulminant injury (Clark et al., 1973).

1.2.2   Drug-Induced Renal Toxicity

Drugs cause approximately 20% of community and hospital-acquired episodes of acute renal failure (Nash and Hafeez 2002; Bellomo, 2006). Among older adults, the incidence of drug-induced nephrotoxicity may be as high as 66% (Kohl et al., 2000). Older patients have higher incidence of diabetic, cardiovascular disease, take multiple medications, and are exposed to more diagnostic and therapeutic procedures with the potential to harm kidney function (Hoste et al., 2000). Although renal impairment is often reversible when the offending drug is discontinued, the condition can be costly and may require multiple interventions with including hospitalization (Gandhi et al., 2000).

1.2.2.1 Nephrotoxicity

Nephrotoxicity is a poisonous effect of some substances, both toxic chemicals and medication, on the kidney (Galleg, 2000).

Most drug-induced renal impairments are reversible. Renal function returns to baseline provided the impairment is recognized early and the offending medication is discontinued. Failure to  act on available  information relating  to  clinical findings or laboratory results was the main common monitoring error, occurring in 37% of preventable adverse drug events, affecting the kidney.

A  decrease  in  renal  function  by  a  rise  in  serum  creatinine  and  urea  levels following the administration of drugs, signals the possibility of drug-induced renal injury. Although there are no standard guideline used to interpret changes in serum creatinine, a

50% rise from baseline and increase of 0.5 mg/dl (40 Umd/L) or more when baseline serum creatinine is less than 2 mg/dl (180 Umd/L) or an increase of I mg/dl (90 Umdl) or more if baseline creatinine is greater than 2 mg/dl have been used as biochemical criteria for acute renal failure (Werth et al., 2001).

1.2.2.2 Pathogenic Mechanisms

Most drugs found to cause nephrotoxicity exert toxic effects by one or common pathogenic mechanisms. These include altered intraglomerula hemodynamics, tubular cell toxicity, inflammation, crystal nephropathy, rhabdomyolysis, and thrombotic micro- angiopathy (Schnellman and Kelly, 2007).

i. Altered Intraglomerular Hemodynamics

In healthy young adult, approximately 120 ml of plasma is filtered under pressure to the glomerulus per minute, which corresponds to the glomerular filtration rate (GFR). The kidney maintains intraglomerular pressure by modulating the afferent and efferent arteriole, to preserve GFR and urine output. For instance, in patients with volume depletion, renal perfusion depends on circulating prostaglandins to vasodilate the afferent arterioles, allowing more  blood flow through the  glomerulus. Drugs with antiprostaglandin activity (e.g. non-steroidal antiinflammatory drugs [NSAIDs]) or those with antiangiotensin –II activity (e.g angiotensin-converting enzyme [ACE]) inhibitors can interfere with the kidney action to autoregulate glomerular pressure and decrease GFR (Palmer, 2002).

ii. Tubular Cell Toxicity

Renal tubular cells, in particular proximal tubule cells, are vulnerable to the toxic effects of drugs because their role in concentrating and reabsorbing glomerular filtrate exposes them to high level circulating toxins (Perazella, 2005). Drugs that cause tubular cell toxicity do so by impairing mitochondrial function, interfering with tubular transport,

increasing oxidative stress, or forming free radicals (Markowtz and Perazella, 2005). These drugs include aminoglycoside, antiretroviral (Markowitz, 2003).

iii. Inflammation

Drugs can cause inflammatory changes in the glomerulus, renal tubular cells, and the surrounding interstitium, leading to fibrosis and renal scarring. Glomerulonephritis is an inflammatory condition caused by immune mechanisms and is often associated with proteinuria in the nephrotic range (Perazella, 2005) Medications such as gold therapy and lithium have been reported as the causative agents (Markowitz, 2001). Acute intestitial nephritis, which can result from an allergic response to a suspected drug, developing idiosyncratic, non-dose-dependent nephritis (Rossert, 2001). Medications that can cause acute interstitial nephrotis are thought to bind to antigens in the kidney or act as antigens that  are  deposited  in the  interstitium inducing  an  immune reaction (Rossert, 2001). However, classic symptoms of a hypersensitivity reaction (i.e. fever rash, and eosinophilia) are not always observed (Markowitz, 2005). Numerous drugs have been implicated, including antibiotics and proton pump inhibitors especially omeprazole. Chronic interstitial nephritis is less  likely than acute interstitial nephritis to be drug induced and signs of hypersensitivity are often lacking (Appel, 2007). Drugs associated with this mechanism of nephrotoxicity include certain chemotherapy agents, Chinese herbals containing lithum. Early recognition is  important because chronic interstitial nephritis has been known to progress to end-stage renal disease (Appel, 2002).

iv. Crystal Nephropathy

Renal impairment may result from the use of drugs that produce crystals that are insoluble in human urine. The crystals precipitate, usually within the distal tubular lumen, obstructing urine flow and eliciting an interstitial reaction (Markowit, 2005). Common drugs associated with production of crystal precipitation depends on the concentration of the drug in the urine (Perazella, 1999). Patients most at risk of crystal nephropathy are those with volume depletion and renal insufficiency (Perazella, 1999).

v. Rhabdomyolysis

Rhabdomyolysis is a syndrome in which skeletal muscle injury leads to lysis of the myocyte releasing intraculular contents including myoglobin into the plasma leading to myoglobin-induced injury secondary to direct toxicity, tubular obstruction, and alterations  in  GFR  (Coco  et  al.,  2004).  Drugs  may  induce  rhabdomyolysis directly secondary to  a  toxic  effect  on  myocyte  function,  or  indirectly  by  predisposing the myocyte to injury. Drugs and alcohol are causative factors in up to 81% of cases of rhabdomyolysis, and up to 50% of patients subsequently develop acute failure (Prendergast and George, 1993).

vi. Thrombotic Microangiopathy

In thromobotic microangiopathy, organ damage is caused by platelet thrombin in the microcirculation – thrombotic thrombocytopenic purpura (Pisoni et al., 2001). Mechanisms  of renal  injury  secondary to  drug-induced thrombotic  microangiopathy include an immune-mediated reaction or indirect endothelial toxicity (Bellowmo, 2005). Most often associated with this pathogenic mechanism of nephrotoxicity include antiplatelet agent clopidogrel, cyclosporine, mitomycin and quinine (Manor et al., 2004).

1.2.3   Adverse Drug Reaction

An adverse drug reaction is an expression that describes harm associated with the use of medications given at a normal dose (Nebeker et al., 2004). The meaning of this expression differs from the meaning of “side effect” as the latter expression might also imply that the effects can be beneficial (Nebeker et al., 2004). Drug reactions can be classified into immunological and non-immunological aetiologies. Immunological types include Type 1 reaction (1gE medicated), Type II reaction (Cytotoxic), Type III reaction (Immune complex), Type IV T-cell activation and ligand-induced apoptosis. Non- immunological type includes predictable pharmacological side effects, secondary pharmacological side  effects,  drug toxicity,  drug  interaction,  drug  overdose,  pseudo allergic, idiosyncratic.

The majority (75 – 80%) of adverse drugs reaction are caused by predictable, non immunological effect (JCAAI, 1999). The remaining 20 – 25% of adverse drug events are caused by unpredictable effects that may or may not be immune-medicated. Immune- mediated reactions account for 5 –10% percent of all drug reaction and constitute true drug  hypersensitivity, with 1gE  –medicated drug  allergies  falling  into  this  category (Deshazo and Kemp, 1997).

The Gell and Coomb classification system describes the predominant immune medicated immune mechanism that lead to clinical symptoms of drug hypersensitivity, with 1gE medicated drug allergies falling into this category (Deshazo and Kemp, 1997). This classification system includes: Type I reaction (IgE-mediated); Type II reactions (Cutotoxic); Type III reaction (Immune complex); and Type IV reactions (delayed cell- mediated). However, some drug hypersentivity reactions are different to classify because of a lack of evidence supporting a predominant immunologic mechanism. These include certain cutaneous drug reactions ie maculopapular rashes, erythroderma, exfoliative dermalitis, and fixed drug reactions (Yawalkar et al., 2000) and specific drug hypersensitivity syndromes (Pramatarov, 1998).

Unpredictable, non-immune drug reactions can be classified as pseudoallergic, idiosyncratic, or intolerance. Pseudoallergic reactions are the result of direct mast cell activation and degranulation by drugs such as opioids. These reactions may be clinically indistinguishable from type I  hypersensitivity, but  do  not  involve drug-specific IgE. Idiosyncratic reactions are qualitatively aberrant reactions that cannot be explained by the known pharmacologic action of the  drug  and  occur only in  a  small percent  of the population. A classic example of an idiosynacratic reaction is drug-induced haemolysis in persons with glucose-6-phosphate dehydrogenase deficiency. Drug intolerance is defined as a lower threshold to the normal pharmacological action of a drug.

Adverse drug reactions caused by immune and non-immune mechanisms are a major cause of morbidity and mortality worldwide. They are the most common iatrogenic illness, complicating 5-15 percent of therapeutic drug courses (Ditto et al., 2002). In the United State, more than 100,000 deaths are attributed annually to serious adverse drug reactions (Lazarou et al., 1998).

Three to 6 percent of all hospital admissions are because of adverse drug reactions (Einarson, 1993). Epidemiological data support the existence of specific factors that increase the risk of general adverse drug reactions, such as female gender (Barranco,

1998) or infection with human immunodeficiency virus (HIV) or herpes (Bayard et al.,

1992; Descamps et al., 2001). Factors associated with an increased risk for hypersensitivity drug reactions include asthma, systemic erythematosus and use of beta blockers (Lang et al., 1991). The most important drug-related risk factor for drug hypersensitivity concerns the chemical properties and molecular weight of the larger drugs.  Greater  structural complexity (e.g.  non-human proteins)  is  more  likely to  be immunogenic. Most drugs have a small molecular weight (less than 1,000 daltons) but may still become immunogenic by coupling with carrier proteins, such as albumin to form sample chemical-carrier complexes (hapten). Another factor affecting the frequency of hypersensitivity drug reactions is the route of drug administration; tropical, intramuscular, and intravenous routes are most likely to cause hypersensitivity reactions. Oral medications are less likely to result in drug hypersensitivity (Adkinson, 1984).

1.2.3.1            Clinical Manifestations

True hypersensitivity adverse drug reactions are great imitators of disease and may present with involvement of any organ system, including systemic reactions such as anaphylaxis. Drug reactions manifest with dermatological symptoms caused by the immunologic activity of skin (Moscicki et al., 1990).

1.2.3.2            Chemical Evaluation

Drug hypersensitivity reactions not only should be included in the differential diagnosis for patients who have the typical allergic symptoms of anaphylaxis, but also for those with serum sickness-like symptoms, skin rash, fever, pulmonary infiltrates with eosinophili, hepatitis, acute interstitial nephritis, and lupus-like syndromes. A diagnosis of drug hypersensitivity depends on identifying symptoms and physical findings that are compatible with an immune drug reactions signs suggestive of serious adverse drug reactions include the presence of fever, mucous membrane lesions, joint tenderness and swelling, or an abnormal pulmonary examination (Hamilton et al., 1996).

1.2.3.3 Laboratory Evaluation

The goal of diagnosis is to evaluate biochemical or immunological markers that confirm activation of a particular immunopathological pathway to explain the suspected adverse  drug  effect.  Laboratory evaluation  is  guided  by  the  suspected pathogenical mechanism (Holder, 2002). Confirmation of suspected Type I hypersensitivity reactions require the detection of antigen-specific IgE. Skin testing is a useful diagnostic procedure in these patients. It also may be informative when testing high-molecular weight protein substance such as insulin, vaccines, or monoclonal antibodies, and latex (Patterson, 1995; Hamilton et al., 1996). Positive skin testing to such reagents confirms the presence of antigen-specific IgE and  is  supportive of the diagnosis of a Type I  hypersensitivity reaction. Histamine, and betatryptase levels have proved useful in confirming acute IgE medicated reactions but negative results do not rule out acute allergic reaction (Shepherd,

1991). Type II Cytotoxic reactions to a drug result in haemolytic anaemia, thrombocytopenia, or neutropenia with a complete blood count. Haemolytic anemia may be confirmed with a positive direct or indirect coombs’ test, reflecting the presence of complement or drug-hapten on the red cell membrane. In Type III immune complex reactions such as erythrocyte sedimentation rate and c-reactive protein may occur. More specific laboratory testing  for complement or circulating immune complexes can be conducted. Systemic vasculitis induced by medication may be detected by autoantibody test such as antinuclear antibody or antihistone antibody (Adam, 1991). Type IV immune reactions usually present an allergic dermatitis caused by topical medications. In such instances, patch testing for specific drug agents is an appropriate diagnostic step (Adam,

1991).

1.2.4 Drug-induced Neutropenia and Agranulocytosis

Neutropenia  is  a  haematological disorder  characterized  by  an  abnormal  low number of neutrophils, the  most  important type  of white  blood cells,  in the  blood. Neutrophils usually make up 50-70% of circulating white blood cells and serve as the primary defense against infections by destroying bacteria in the blood. Hence patients with neutropenia are more susceptible to bacterial infections which may become life- threatening neutropenic sepsis (Hsieh et al., 2007).

The severity of neutropenia is classified based on the absolute neutrophil count (ANC) measured in cells per microliter of blood (Hsieh et al, 2007). Mild neutropenia (1000 – 1500mm3) minimal risk of infection. Moderate neutropenia (500 – 1000mm3) moderate risk of infection.

Severe neutropenia (<500mm3) severe risk of infection. The causes can be divided

into the following groups.

(a) Decrease production in the bone marrow which could be as a result of cancer, certain medications, hereditary disorder, radiation and vitamin B12 deficiency.

(b) Increase destruction which can occur as a result of autoimmune neutropenia.

(c) Sequestraction eg haemolysis. There is a mild neutropenia in viral infection (Levene et al., 2001). Low neutropenia counts are detected on full blood count. Some investigations are  required  to  arrive  at  a  definite  diagnosis.  Bone  marrow  biosy  is necessary and serial neutrophil count for suspected cyclic neutropenia, tests for antineutrophil antibodies,  autoantibody  screen  and  investigation  for  systemic  Lupas erythematous, vitamin B12 and folate assay and Ham’s test (Levene et al., 2001).

1.2.5   Drug-Induced Leukocytosis

Leukocytosis is defined as a white blood cell count greater than 11,000 per mm3 (11×109/L). An elevated white blood cell count reflects the normal response of bone marrow to infections or inflammatory process. Leukocytosis is a sign of a primary bone marrow abnormality in white  blood cell production, maturation or death (apoptosis)

related to a leukemia or etiology of leukocytosis. The investigation of leukocytosis begins with an understanding of its basic causes which could be as a result of the response of normal bone  marrow to  external stimuli and  the  effect  of a  primary  bone  marrow disorder.

Inflammation associated leukocytosis occurs in tissue necrosis, infarction, burns and arthritis (Jandl, 1996). Leukocytosis may also occur as a result of physical and emotional stress (McCarthy et al., 1987). Other causes of leukocytosis include medications, splenectomy and haemolytic anemia.

Increased numbers of lymphocytes occur with certain acute and chronic infection. Acute infections like cytomegalovirus infection, hepatitis, toxoplasmosis and chronic

infection like tuberculosis where white blood cell count greater than 30,000 per mm3 (30×109/L)   malignancies   of   lymphoid   system   may   also   cause   lymphocytosis. Polycythenia usually presents with excessive numbers of erythroid cells, but increased

white blood cell and platelet counts may be evident. Some patients with polylythenia vera develop myocardial infarction, stroke, venous thrombosis and congestive heart failure. Leukocytosis is also found in patients with essential thrombocythemia, although elevated platelet counts occur in all myeloproliferative disorders prominence of platelets (Curtis et al., 2006).

1.2.6 Thrombocytopenia (DIT)

This can be distinguished from idiopathic thrombocytopenic purpura (ITP), a bleeding disorder caused by thrombocytopenia not associated with a systemic disorder, based on the history of drug ingestion or injection and laboratory findings. DIT disorders can be a consequence of decreased platelet production (bone marrow suppression) or accelerated platelet destruction especially immune mediated destruction. The recurrence of thrombocytopenia following reexposure to drug and laboratory investigation (such as total  blood count  and  platelet  serology test)  is  all  important  factors  for  differential diagnosis (Wazny et al., 2000; Rothe, 2006).

Hundreds of drugs have been implicated in the pathogenesis of DIT. DIT disorder can be a consequence of decreased platelet production or accelerated platelet destruction. A decrease in platelet production is attributable to a generalized myelosuppression, a common and anticipated adverse effect of cytotoxic chemotherapy (Carey, 2003). Chemotherapeutic  agents  can  induce  thrombocytopenia  secondary  to  an  immune mediated mechanism (Curtis, 2006). Accelerated platelet destruction in the presence of the offending drug is most often of immune origin (Curtis, 2006).

i. Drug–Induced Autoantibody

During exposure to a medication in some patients, make drug-dependent antibody and drug-independent antibodies (autoantibodies) are synthesized simultaneously (Lerner et al., 1985; Aster, 2000). These autoantibodies can persist for a long period of time leading to an autoimmune thrombocytopenic purpura (AITP) as it could be case during

the exposure to gold salts (Aster, 2005). The mechanism of this immune-response is unknown but a possibility is that the drug might alter the processing of platelet glycoproteins (GPs) in such a way that one or more peptides not ordinarily seen by the immune system “neoantigens”, are generated could be presented to T cells.

Generation of such peptides through various  mechanisms is an important theme in autoimmunity. In murine models, heavy metal ions such as Hg++ and AU+++ have been shown to alter processing of proteins, leading to presentation of immunogenic peptides (Griem et al., 1995). In several human models, protein specific antibodies and other ligands perturb protein processing, leading to the generation of such peoptides recognized by T cells.

The diagnosis of drug-induced thrombocytopenia is empirical. In patient exposed only to a single drug, recovery after its discontinuation provides evidence that it was caused by drug sensitivity (George et al., 1998).

Many different methods have been used to detect the presence of drug-dependent antibodies  (DDABS).  That  includes  the  use  of  radiolabeled  or  fluorescien-labeled (platelet  immunofluorescence  test;  PIFT)  anti-1gG  to  detect  platelet-bound immunoglobin, enzyme-linked imminospecific assay (ELISA), flow cytometry and immunoprecipitation, western  blotting  (Visentin  et  al.,  1991;  Visentin et  al.,  1990; McFarland, 1993).

1.3 Biochemical Mechanism of Drug Induced Toxicity

Many drugs can be converted in the body to various metabolites that invoke therapeutic and toxicological responses. It appears to involve 2 pathways – direct hepatotoxicity and adverse immune reactions. In most instances, Drug Induced Liver Injury (DILI) is initiated by the bioactivation of drugs to chemically reactive metabolites, which have the ability to interact with cellular macromolecules such as proteins, lipids, and nucleic acids, leading to protein dysfunction, lipid peroxidation, DNA damage, and oxidative stress. Additionally, these reactive metabolites may induce disruption of ionic gradients and intracellular calcium stores, resulting in mitochondrial dysfunction and loss of energy production. This impairment of cellular function can culminate in cell death and possible liver failure.

Hepatic cellular dysfunction and death also have the ability to initiate immunological reactions, including both innate and adaptive immune responses. Hepatocyte  stress  or  damage  could  result  in  the  release  of  signals  that  stimulate activation of other  cells,  particularly those  of the  innate  immune system,  including Kupffer cells (KC), natural killer (NK) cells, and NKT cells. These cells contribute to the progression of liver injury by producing proinflammatory mediators and secreting chemokines to further recruit inflammatory cells to the liver. It has been demonstrated that various inflammatory cytokines, such as tumor necrosis factor (TNF), interferon (IFN) –, and interleukin (IL) –1, produced during DILI are involved in  promoting tissue damage (Blazka et al., 1995; Blazka et al., 1996; Ishida et al., 2002). However, innate immune cells are also the main source of IL-10, IL-6 and certain postglandins, all of which have been shown to play a hepatoprotective role (Bourdi et al., 1994; Naisbitt

2003). Thus, it is the delicate balance of inflammatory and hepatoprotective mediators produced after activation of the innate immune system that determines an individual’s susceptibility and adaptation to DILI.

In addition to the innate immune responses, clinical features of certain DILI cases strongly suggest  that  the  adaptive  immune system is  activated and  involved  in the pathogenesis of liver injury. With regard to the involvement of the adaptive immune system in DILI, this is based on the hapten hypothesis and the p-I (pharmacological interaction of drugs with immune receptors) concept. Evidence to support these hypotheses is gained by the detection of drug-specific antibodies and T cells in some patients with DILI (Bourdi et al., 1994).

1.3.1   Drug-Induced Direct Hepatotoxicity

Direct hepatotoxicity is often caused by the direct action of a drug, or more often a reactive metabolite of a drug, against hepatocytes. One classically studied drug used to examine the mechanisms of hepatotoxicity is acetaminophen (paracetamol) Acetaminophen is a popular over-the-counter analgesic that is safe at therapeutic doses but at overdose can produce centrilobular hepatic necrosis, which may lead to acute liver failure. Acetaminophen is metabolized to a minor electrophilic metabolite, N-acetyl-p- benzoquinoneimine (NAPQI) which during acetaminophen overdose depletes glutathione

and initiates covalent binding to cellular proteins (Liz and Diehl, 2003). These events lead to the disruption of calcium homeostatis, mitochondrial dysfunction, and oxidative stress and may eventually culminate in cellular damage and death (Hshimoto et al., 1995; Tsutsui, 1997).

In  most  instances  of  DILI,  it  appears  that  hepatocyte  damage  triggers  the activation of other cells, which can initiate an inflammatory reaction or an adaptive immune response. These secondary events may overwhelm the capacity of the liver for adaptive repair and regeneration, thereby contributing to the pathogenesis of liver injury.

1.3.2   Drug-induced Immune-Mediated Liver Injury

The innate immune system provides a first  line of defense against  microbial infection, but it is not sufficient in eliminating infectious organisms. The lymphocytes of the adaptive immune system provide a more versatile means of defense and possess “memory,” which is the ability to respond more vigorously to repeated exposure to the same microbe. Moreover, cells of the innate immune system play an integral role in the initiation of adaptive immunity by presenting antigens and are important in determining the subsequent T-cell-or antibody-mediated immune response. Because of the liver’s continuous exposure to pathogens, toxins, tumor cells, and harmless dietary antigens, it possesses a range of local immune mechanisms to cope with these challenges. The liver contains large numbers of both innate and adaptive immune cells, including the largest

populations of tissue macrophages (KC), NK cells, and NKT cells (Liz and Dieh, 2003).

The  liver  also  possesses a  unique+  and  CD8+   T  cells.  Collectively,  the  innate and adaptive immune cells contribute to the unique immune responses of the liver, including removal of pathogenic microorganisms, clearance of particles and soluble molecules from circulation, deletion of activated T cells, and induction of tolerance to food antigens derived from the gastrointestinal tract.

KC play an essential role in the phagocytosis and removal of pathogens entering the liver via portal-venous blood. Upon activation, KC produce various cytokines and other mediators, including prostanoids, nitric oxide, and reactive oxygen intermediates. These KC products play prominent roles in promoting and regulating hepatic inflammation, as well as modulating the phenotype of other cells in the liver, such as NK

and NKT cells (Hashimoto et al., 1995; Tsutsui 1997). Studies of organ transplantation using animal models have further shown that inhibition of KC abrogated the prolonged survival of allografts induced by portal vein infusion of allogeneic donor cells (Callery et al., 1989; Squiers et al., 1990). Collectively, this evidence suggests that KC play an important role in the delicate balance between the induction of immunity and tolerance within the liver.

Unique to the liver are the remarkably high frequencies of NK and NKT cells, which account for 50% of intrahepatic leukocytes (Mehal et al., 2001). These cells act as a first line of defense against certain pathogens and invading tumor cells prior to the adaptive  immune response of B  and  T  lymphocytes. One  characterized function of hepatic NK and NKT cells is their cytotoxic capacity against other cells (Dohert et al.,

1999). This cytotoxicity is further enhanced by IL-12 and IL-18 which are produced by activated KC (Tsutsui 1997). Another function ascribed to NK and NKT cells is their ability to produce high levels of T helper (Th) 1 and Th2 cytokines upon stiumulation (Chen and Paul 1997). NK cells have been shown to represent a major source of IFN- in many types of liver disease (Liz and Diehl, 2003). NKT cells produce either IFN- or IL-

4 cytokines, depending on the differentiation state of the cells and the stimuli (Dohert et al., 1999). It  has also been demonstrated that IL-4 produced by NKT cells may be associated with the initiation and regulation of Th2 responses.

The liver’s adaptive immune responses are unique in that the liver is known to favor induction of immunological tolerance rather than immunity. This is supported by numerous   studies   demonstrating   that   (a)   dietary   antigens   derived   from   the gastrointestinal tract are tolerized in the liver; (b) allogeneic liver organ transplants are accepted across major histocompatibility complex (MHC) barriers (Calne et al., 1969). (c) preexposure to donor cells through the portal vein of recipient animals increased their acceptance of solid tissue allografts (Gorczynski et al., 1994); and (d) preexposure of soluble antigens via the portal vein leads to systemic immune tolerance (Cantor and Dumont, 1967). Several mechanisms have been suggested to account for this tolerance, including apoptosis of activated T cells, immune deviation, and active suppression. The liver has been called the “elephant’s graveyard” for activated T cells (Crispe et al., 2000). These cells accumulate in the liver before undergoing apoptosis. Studies using T- cells undergo apoptosis after a transient accumulation within the liver (Bertolino et al., 1995). Immune deviation may account for liver-induced tolerance, as it has been shown that Th2 cytokine production is preferentially maintained when adoptively transferred Th1 and Th2 cells are recovered from the liver (Klugewitz et al., 2002). It has also been reported that liver sinusoidal endothelial cells (LSEC) are capable of selectively suppressing IFN–producing Th1 cells while concurrently promoting the outgrowth of IL-4-expressing  Th2  cells  (Klugewitz  et  al.,  2002).  Active  suppression  of  T-cell activation resulting in liver-induced tolerance is also likely to occur within the  liver because of its unique anatomy and composition of “tolerogenic” APCs. Within the liver, blood flow slows down through the narrow sinusoids (7-12 m) and is  temporarily obstructed by KC, which resides in the sinusoidal lumen. Because of this reduction in blood flow, circulating T cells can interact with LSEC and KC. Consequently, naïve T cells, within the liver. Current evidence suggests that LSEC and  KC as well as hepatic dendritic cells are important in the induction of tolerance, rather than the activation of T- cell responses. It  has been further demonstrated that  although  LSEC are capable of presenting antigen to T cells, LSEC-activated CD4+ or CD8+ T cells fail to differentiate into Th1 cells or cytotoxic effector cells, respectively (Knolle et al., 1999). In addition, studies have shown that KC and hepatic dendritic cells are not effective APCs when compared with their counterparts in lymphoid tissues (Rubinstein et al., 1986).

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