Hidrología

The balance between the toxification and detoxification metabolic pathways of potential hepatotoxins will determine the extent of their toxic effects. These pathways may be modified to a remarkable degree by exposure to other agents. It was shown over 50 years ago that administration of agents such as phenobarbital enhances the ability of the liver to metabolise a variety of compounds, accompanied by increases of liver weight, amount of smooth endoplasmic reticulum and the hepatic content of drug-metabolising enzymes63. This phenomenon, referred to as induction, is an adaptive process that provides increased metabolic capacity and occurs in all species from bacteria to mammals15. Some compounds may induce only specific enzymes while others may induce several enzymes simultaneously. Compounds known to induce CYP3A include rifampin64, corticosteroids65, alcohol66, DDT67, midazolam68 and St. John’s Wort69.

Induction of cytochrome P450 enzymes by xenobiotics occurs at the transcriptional level through regulation by a number of ligand-activated transcription factors19. The intracellular receptors most commonly involved include aryl hydrocarbon receptor (AhR) and the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR). AhR is widely distributed in tissues throughout the body70 and typically accepts both endogenous and foreign hydrophobic, planar compounds as ligands71. It primarily regulates expression of genes in the CYP1 family. PXR is thought to be the main environmental ‘xenosensor’ since it is mainly expressed within the liver and small intestine and its main target gene is CYP3A4. It has a large and flexible binding pocket, allowing activation by a range of different sized ligands72 and its own expression is regulated by microRNA73. Upon activation, ligand bound PXR forms a heterodimer with another nuclear receptor retinoid X receptor (RXR), which together can bind to several distinct DNA elements in the target genes74. It is known to induce CYP subfamilies 2A, 2B, 2C and 3A, with its role in the induction of CYP3A4 the most studied75. Several promoter elements have been identified within CYP3A4 such as the ER6 motif in the proximal promoter region 76and the ‘Xenobiotic-response enhancer module’ in the distal region77 that enable PXR binding and up-regulation of transcription. Numerous other cofactors are thought to be involved in this complex process. CAR is closely related to PXR and is only found in mammals, suggesting a recent gene duplication event from the PXR ancestor78. It is expressed mainly in the liver and kidneys, also suggesting a ‘xenosensor’ role. It has been shown that some inducing agents

40 may also activate this receptor through a ligand-independent mechanism79. Upon activation it also dimerises with RXR and binds to many DNA-binding elements, many of which are common to PXR80. Its main targets are members of the CYP2B subfamily, but it is also involved in CYP3A and CYP2C regulation.

Cytochrome P450 activity may also be inhibited by a number of agents. This is a particularly important consideration in clinical practice where harmful drug-drug interactions can be caused by the increased bioavailability of a drug that is normally eliminated by cytochrome P450 enzymes19. Inhibition may be irreversible or reversible depending on whether the inhibitor must first be metabolised by the cytochrome P450 enzyme19. Reversible inhibition is most common and results from competition for the active site of the enzyme. Bonds are quickly formed but easily broken so inhibition is rapid but does not permanently damage the enzyme. Irreversible inhibition can occur when the metabolised intermediary form strong covalent bonds to the protein or the heme of the enzyme and may inactivate it completely. Known CYP3A inhibitors include macrolide antibiotics81, ketoconazole82 and ritonavir83.

Diet can also affect compound metabolism and toxicity84. Fasting can deplete stores of glycogen, reducing availability for the detoxifying glucuronidation pathway, while a low protein diet can decrease the amount and activity of cytochrome P450 expression, reducing the toxicity of compounds activated by these enzymes25. Alcohol can increase toxicity by both inducing cytochrome P450 expression66 and depleting available stores of glutathione62.

Genetic factors play a fundamental part in host susceptibility which is illustrated in the first instance by the great differences observed in the metabolism and toxicity of compounds when given to different strains and species of laboratory animals85, 86. Genetic polymorphisms in Man have been identified within all the major drug-metabolising genes22 and these may translate to differences in phenotype. For some enzymes such as CYP2D6, expression levels varies between individuals by as much as 1000 fold22, due to several missense mutations reducing the function of certain alleles or making them not function completely. In addition some individuals may have more than two functional alleles due to gene duplication events. This is reflected in enzyme activity as illustrated with studies of the drug debrisoquine which showed a multimodal distribution of metabolic activity among participants which corresponded to the number of functional alleles they possessed87. However enzymes that play a more general role in xenobiotic metabolism are far more evolutionarily conserved. CYP3A4 expression levels vary by only 20-fold between individuals with only moderate, but significant, differences in enzyme activity22. Studies of the clearance of CYP3A4

41 substrates reveal a unimodal distribution, indicating the influence of many different genes on CYP3A4 activity88, 89. Polymorphisms among the genes for phase II metabolising enzymes will also play an important role the potential toxicity of a compound. Of these the UDP- glucuronysltransferases (UGT) are the most relevant to drug toxicity after the cytochrome P450 enzymes. Polymorphisms of the isozyme UGT1A1, required for bilirubin glucuronidation, can lead to hyperbilirubinemia, which in most cases is manifested by mild sporadic jaundice called Gilbert’s syndrome90. Mutations in this gene also increases the risk of severe side effects following treatment with the drug irinotecan, since reduced conjugation of its active metabolite in the intestine may lead to life-threatening diarrhoea91.

Age and gender also affects toxin susceptibility. Although clear differences are observed in the expression patterns of drug-metabolising enzymes among rats and mice, no genetic differences in these enzymes between the sexes are observed in humans22. However sex hormones are involved in the regulation of some drug-metabolising enzymes92. Females do appear to be more likely to develop adverse reactions to some drugs, such as tetracycline-induced hepatic injury, particularly when pregnant9. On some of the rare occasions when men experienced an adverse reaction to this drug, they had also been receiving estrogens15. Children and infants appear to be more susceptible to hepatic injury with some drugs such as valproate93 and aspirin15.

The important role of the gut microflora in the metabolism of drugs is increasingly recognised94. The gut microflora consists of more than 400 species of bacteria that increase in density from the duodenum to the colon95. They play a key role in first-pass metabolism and contain many drug- metabolising enzymes. The slower a compound is absorbed by the intestinal mucosa, the more likely it will be biotransformed by the gut bacteria. Since they are mostly anaerobes, hydrolysis and reduction reactions are favoured96. These may provide alternative metabolic pathways to the often toxifying mammalian oxidation reactions97 or may produce toxic metabolites themselves22. Hydrolysis may deconjugate glucuronides, sulfates, and cysteine conjugates which may be excreted in the bile. Through extrahepatic circulation, this may present toxic intermediaries back to the liver. Gut microflora have also been shown to interact with mammalian drug-metabolising systems. Antibiotic treatment suppressed CYP3A11 expression in the mouse98, mediated through lithocholic acid-producing gut bacteria, while re-colonisation of germ-free mice had an inducing effect of both CYP3A11 and CYP2C2999. Among human volunteers, the ratio of sulfate to glucuronide acetaminophen conjugates in the urine was predicted by urinary levels of p-cresol sulphate, a bacteria-originated metabolite, prior to acetaminophen dosing100. Metabonomics, a systems-biology approach that profiles all detectable metabolites in a biofluid in single analytical sweep, has helped

42 to show the size of the contribution of the gut biota to metabolism. Studies have revealed unexpected drug metabolites produced by the gut bacteria97, while a typical analysis of mammalian urine by nuclear magnetic resonance (NMR) spectroscopy reveals a large proportion of detectable metabolites to be microbial in origin101.

1.3 Pyrrolizidine alkaloids