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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