Detoxification Pathways in the Liver

Detoxification Pathways in the Liver Learning objectives • Functions of the liver • Biotransformation • Phase I reactions the paracetamol toxicity • Phase II reactions Liver overview • Weight of liver = 1.5kg → 2-3% of the body mass • Total O2 consumption = 20-30% • Approx. 300 billion cells in liver (80% are hepatocytes) • Liver is the chemical factory and recycling plant of the body Metabolic functions of the liver • Uptake of nutrients delivered from the digestive tract via the portal vein • Synthesis, storage, interconversion and degradation of metabolites (metabolism) • Regulated supply of energy-rich intermediates and building blocks for biosynthetic reactions • Detoxification of harmful compounds by biotransformation • Excretion of substances with the bile Biotransformation • Metabolism of a non-endogenous chemical (drug or toxin) is an example of biotransformation • Liver is a major organ for biotransformation • Converts chemical to more soluble and less toxic forms (detoxification) to enhances elimination • Sometimes chemicals are converted to more toxic forms (activation) e.g. paracetamol Drug metabolism • Body deals with a foreign compound by making it more soluble, to increase the rate of its excretion through the kidney (via the urine) • Pathways of biotransformation/drug metabolism can be divided into: o Phase I reactions o Phase II reactions Most drugs undergo sequential reactions through phase I and II Phase I reactions: oxidation-reduction • Additions of reactive side groups – a ‘handle’ (-COOH, -OH, -NH2, -SH) • Increases hydrophilicity • One of the commonest phase I enzyme is monooxygenase (MFO – mixed function oxidase) • P450 is the most important monooxygenase P450 MFO – main 2 proteins • NADPH-cytochrome C reductase o One mole of this enzyme contains one mole each of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) o Enzyme is also called NADPH-cytochrome P450 reductase • Cytochrome P450 o Named based on its light absorption at 450nm when complexed with carbon monoxide o Haemoprotein containing an iron atom which can alternate between the ferrous (Fe++) and ferric (Fe+++) states o Electron acceptor – serves as terminal oxidase o Rate-limiting step in the oxidation reactions Monooxygenase transformation • Most common reaction catalysed by cytochrome P450 is a monooxygenase reaction e.g. insertion of one atom of oxygen into an organic substrate (RH) while the other oxygen atom is reduced to water: o RH + O2 + 2H+ + 2e– → ROH + H2O Cytochrome P450 isozymes • At least 57 different isozymes in humans, over 7700 forms in nature • • • • • Catalytically and structurally similar but genetically distinct enzymes (different genes and aa sequences) Different isozymes have different substrate specificities Individuals have several alleles for P450’s and differ in which isozymes they have Because of the number of different isozymes and their different substrates and inhibitors, the metabolism of a drug can be slowed down if an individual takes a second drug metabolized by the same P450 isoenzyme Subset of cytochrome P450’s can be induced, so that more is expressed upon exposure to a compound, and drugs are metabolized faster through this P450 isoenzyme Drug oxidation requires: o Cytochrome P450 o Cytochrome P450 reductase o NADPH o Molecular O2 Phase 1 reactions example • Aromatic hydroxylation • Aliphatic hydroxylation • Furocoumarins in grapefruit juice inhibit certain forms of cytochrome P450 • 2 major clinical problems: o Overdosing in patients taking multiple drugs metabolized by the same P450 isoenzyme (worsened if they have an activity-reducing mutation in the P450 isoenzyme) o Generation of hepatotoxic metabolites (e.g. from paracetamol) Overdosing and CYP2D6 • CYP2D6 is responsible for the metabolism of nearly 25% of all drugs. There are more than 20 drugs known to be 2D6 substrates. They include cardiovascular agents, antidepressants, antipsychotics, and morphine derivatives. • Mutations leading to 2D6 enzyme deficiency are found in 7% to 10% of caucasians. Hepatotoxic metabolites formed in reactions of cytochrome P450 reactions • Paracetamol is the prime example (acetaminophen) o At normal doses, a sulfate or a sugar is attached to OH (Phase II) and the drug is easily removed by the kidney. o At high doses, this pathway cannot keep up, and P450 converts paracetamol to a toxic metabolite which causes liver damage. • Paracetamol is involved in a large proportion of accidental paediatric exposures and deliberate self-poisoning cases • Hepatic failure and death are possible outcomes, although paracetamol remains the most important single cause of acute fulminant hepatic failure in Western countries Phase II reactions – conjugations • These reactions make drugs more polar, more ready for excretion by the kidney (polar molecules cannot diffuse from the urine back into the bloodstream when urine is concentrated) • Main conjugation reactions o Glucuronidation by UDP-Glucuronosyltransferase: (on -OH, -COOH, -NH2, -SH groups) ▪ Requires creation of a high energy intermediate (UDP-glucoronic acid) o Sulfation by Sulfotransferase:(on -NH2, -SO2NH2, -OH groups) o Amino acid conjugation (on -COOH groups) o Glutathione conjugation by Glutathione-S-transferase (to epoxides or organic halides) ▪ Tripeptide Gly-Cys-Glu; conjugated by glutathione-S-transferase (GST) ▪ Glutathione is a protective factor for removal of potentially toxic compounds ▪ Conjugated compounds can subsequently be attacked by ▪ γ-glutamyltranspeptidase and a peptidase to yield the cysteine conjugate → product can be further acetylated to N-acetylcysteine conjugate Detox foods • Broccoli: has been labelled as one of nature’s superfoods o Source of the powerful phytonutrient sulforaphane ▪ Inside liver cells is a protein system that holds the transcription factor NrF2. Sulforaphane, a sulfur- containing molecule found in broccoli can bind to this protein system and release nrf2 ▪ NrF2, being a transcription factor can bind to specific promoter regions of DNA and turn on transcription of genes for detoxification enzymes, one being glutamate cysteine ligase ▪ Glutamate cysteine ligase can then be used to enzymatically synthesize glutathione o Sulforaphanes are Keap crosslinkers and Nrf2 activators ▪ Inducers such as Sulforaphanes crosslink Keap1 by modifying two (C273 and C288) of the 25 cysteine residues of Keap1 ▪ This disrupts the Keap1-Nrf2 complex NrF2 regulated genes • NrF2 regulated genes include the “classical” phase II enzymes: o Glutathione transferases and o UDP glucuronosyltransferases (both conjugate xenobiotics with endogenous ligands) • and also: o Glutathione reductase, o Aldehyde dehydrogenase, o And glutathione S-conjugate efflux pumps.