TOXICOLOGY

• Biotransformation

  • Drug metabolism is the term used to describe the biotransformation of pharmaceutical substances in the body so that they can be eliminated more easily.
  • The majority of metabolic processes that involve drugs occur in the liver, as the enzymes that facilitate the reactions are concentrated there.
• Biotransformation

  • metabolic processes will convert the drug into a more water-soluble compound by increasing its polarity.
  • This is an essential step before the drug can be excreted in the body fluids such as urine or bile.
  • Only a very few drugs can be excreted first without being metabolised.
  • On the whole, as drugs are metabolised their therapeutic effect diminishes, except in the case of pro-drugs.
• Drugs are metabolized through various reactions including:

  • Oxidation - addition and removal of oxygen to a functional group on a drug molecule]
  • Reduction - removal of hydrogen from a functional group on a drug molecule
  • Hydrolysis - addition of a water molecule to drug
  • Conjugation - attachment of an ionised group/lipid to drug e.g. glutathione/methyl/acetyl etc
  • Condensation - two or more drug molecules combine to form a molecular compound
  • Isomerization - reaction changes the drug molecule into its optical isomer, which is either the same or different
• site and enzymes used in biotransformation

  Drug-metabolising enzymes are called ‘microsomal enzymes’ and are found in the smooth endoplasmic reticulum.

  Liver is the most common biotransformation site.

  Non-microsomal enzymes are found in the cytoplasm and mitochondria of hepatic cells.
• stages of metabolism

  • The liver hepatocytes contain all the necessary enzymes for the metabolism of drugs.phase 1 and phase 2.
  • The main enzymes involved in metabolism belong to the cytochrome P450 group. These are a large family of related enzymes housed in the smooth endoplasmic reticulum of the cell.
  • Metabolism is often divided into two phases of biochemical reaction - phase 1 and 2
  • Some drugs may undergo just phase 1 or just phase 2 metabolism, but more often, the drug will undergo phase 1 and then phase 2 sequentially.
• phase 1 metabolism

  • Phase 1 metabolism can involve reduction or hydrolysis of the drug, but the most common biochemical process that occurs is oxidation.
  • Oxidation is catalysed by cytochrome P450 enzymes and results in the loss of electrons from the drug.
  • The drug is now said to be oxidised and after phase 1 reactions, the resulting drug metabolite is still often chemically active.
• CYPs catalyze several types of oxidation reactions including:

  Hydroxylation of an aliphatic or aromatic carbon [Phenytoin -> hydroxyphenytoin]

  Epoxidation of a double bond

  Heteroatom (S-, N-, and I-) oxygenation and N-hydroxylation

  Oxidation/reduction

  Reductive dehalogenation

  Oxidative dehalogenation

  Cleavage of esters

  Dehydrogenation

  Dealkylation [codeine -> morphine]
• Cytochrome P450 Isoforms (CYPs) - An Overview

  NADPH + H+ + O2 + Drug  ®   NADP+ + H2O + Oxidized Drug

  (NADP+ is a coenzyme that functions as a universal electron carrier, accepting electrons and hydrogen atoms to form NADPH, or nicotinamide adenine dinucleotide phosphate. NADP+ is created in anabolic reactions, or reactions that build large molecules from small molecules).
• Cytochrome P450 Isoforms (CYPs) - An Overview

  • Carbon monoxide binds to the reduced Fe(II) heme and absorbs at 450 nm (origin of enzyme family name) 
  • CYP monooxygenase enzyme family is major catalyst of drug and endogenous compound oxidations in liver, kidney, G.I. tract, skin, lungs
  • Oxidative reactions require the CYP heme protein, the reductase, NADPH, phosphatidylcholine and molecular oxygen
  • CYPs are in smooth endoplasmic reticulum in close association with NADPH-CYP reductase in 10/1 ratio
  • The reductase serves as the electron source for the oxidative reaction cycle
• electron flow in cytochrome P450 cycle

  1. ferric ion binds the drug
  2. enzyme/substrate complex is reduced by NADPH. NADPH is oxidised to NADP+ and electron is transferred to the enzyme/substrate complex
  3. Iron give an electron to the oxygen molecule (oxidised – lost an electron). Oxygen has been reduced (gained an electron)
  4. NADPH is oxidised to NADP+ and the electron is accepted by the oxygen atom.
  5. 2 protons are added to the oxygen to break The bond and a water molecule is produced
  6. Here the drug is now modified. Oxygen is attached to the substrate and the product (metabolite) can be released

   
• As mono-oxygenases, the P450s utilize two electrons from NAD(P)H to reductively cleave atmospheric dioxygen, reducing one atom to water and using the second to oxidize the substrate. This is a multi-step mechanism to form reactive iron-oxygen adducts.
• CYP Families

  •Multiple CYP gene families have been identified in humans, and the categories are based upon protein sequence homology

  •Most of the drug metabolizing enzymes are in CYP 1, 2, & 3 families . 

  •CYPs have molecular weights of 45-60 kDa.

  •Frequently, two or more enzymes can catalyze the same type of oxidation, indicating redundant and broad substrate specificity. 

  •CYP3A4 is very common to the metabolism of many drugs; its presence in the GI tract is responsible for poor oral availability of many drugs.
• Factors Influencing Activity and Level of CYP Enzymes

  nutrition - 1A2

  smoking - 1A2

  alcohol - 2E1

  drugs - 2C

  environment - 1A2

  genetic polymorphism- 2C9
• substances that affect drug metabolism

  • Grapefruit juice - CYP 3A4 inhibitor; highly variable effects; furanocoumarins [a specific group of secondary metabolites that commonly present in higher plants, such as citrus plants]; cardiac arrythmias.
  • Brussel sprouts/cigarette smoke induce P450 enzymes.
  • St John’s wort, other herbal products induce P450s [narcotics]
  • Isosafrole, safrole–CYP1A1, CYP1A2 inhibitor;  found in root beer, perfume
• grapefruit juice can markedly augment oral drug bioavailability was based on an unexpected observation from an interaction study between the dihydropyridine calcium channel antagonist, felodipine, and ethanol in which grapefruit juice was used to mask the taste of the ethanol.  post-translational down regulation of cytochrome P450 3A4 (CYP3A4) expression in the intestinal wall. 
• CYP3A RegulationThe molecular basis of a drug-drug interaction

  CYP3A alone is responsible for the metabolism of 50–60% of all prescription drugs.

  • PXR acts as a transcription factor that regulates CYP3A expression in liver and intestine
  • Diverse drugs activate through heterodimer complex with RXR
  • Drug A binds PXR inducing CYP3A expression and accelerates metabolism of Drug B.
  • Protect against xenobiotics
• Human Drug Metabolizing CYPs Located in Extrahepatic Tissues

  1A1- lung and kidney

  1B1- skin and prostate

  2A6- lung and nasal membrane

  2B6- GI tract and lung

  2C- GI tract and lung

  2E1- lung and placenta

  2F1- lung and placenta

  2J2- heart

  3A- GI tract and lung

  4B1- lung and placenta

  4A11- kidney
• Non-CYP drug oxidations

  • Monoamine Oxidase (MAO) - MAO (mitochondrial) oxidatively deaminates endogenous substrates including neurotransmitters (dopamine, serotonin, norepinephrine, epinephrine); drugs designed to inhibit MAO used to affect balance of CNS neurotransmitters (L-DOPA);  MPTP* converted to toxin MPP+ through MAO-B.
  • Diamine Oxidase (DAO) - DAO substrates include histamine and polyamines.
• Non-CYP drug oxidations

  • Alcohol & Aldehyde Dehydrogenase - non-specific enzymes found in soluble fraction of liver; ethanol metabolism
  • Xanthine Oxidase - converts hypoxanthine to xanthine, and then to uric acid.  Drug substrates include theophylline, 6-mercaptopurine.  Allopurinol is substrate and inhibitor of xanthine oxidase; delays metabolism of other substrates; effective for treatment of gout.
• Non-CYP drug oxidations

  Flavin Monooxygenases

  Family of enzymes that catalyze oxygenation of nitrogen, phosphorus, sulfur – particularly facile formation of N-oxides

  Different FMO isoforms have been isolated from liver, lung.

  Require molecular oxygen, NADPH, flavin adenosine dinucleotide (FAD)

  Single point (loose) enzyme-substrate contact with reactive hydroperoxyflavin monoxoygenating agent

  FMOs are heat labile and metal-free, unlike CYPs

  Factors affecting FMOs (diet, drugs, sex) not as highly studied as CYPs
• phase 2 metabolism

  • Phase 2 metabolism involves conjugation - that is, the attachment of an ionised group to the drug. These groups can include glutathione, methyl or acetyl groups. These metabolic processes usually occur in the hepatocyte cytoplasm.
  • The attachment of an ionised group makes the metabolite more water soluble. This facilitates excretion as well as decreasing the pharmacological activity.
• drug metabolites

  • Some drugs are administered in an inactive form called a pro-drug, like enalapril. It is the metabolite that is pharmacologically active, or enalaprilat in this case, which acts as an antihypertensive agent.
  • Some drug metabolites can be toxic such as those produced from paracetamol. These are usually detoxified by phase 2 conjugation joining with glutathione.
• drug metabolites

  However, in an overdose situation where the dose of paracetamol is high, there is not enough glutathione to detoxify the metabolites. The metabolites accumulate causing toxicity and can result in hepatitis. As a solution, compounds are administered to boost the levels of glutathione so that phase 2 metabolism can take place, thus detoxifying the metabolites fully and reducing the risk of liver injury.
• effects on drug metabolism

  Species Differences –Major differences in different species have been recognized for many years.

  Phenylbutazone* half-life is 3 h in rabbit, ~6 h in rat, guinea pig, and dog and 3 days in humans.
• Effects on Drug Metabolism

  Induction - Two major categories of CYP inducers 

  • Phenobarbital is prototype of one group - enhances metabolism of wide variety of substrates by causing proliferation of SER and CYP in liver cells.
  • Polycylic aromatic hydrocarbons are second type of inducer (ex: benzo[a]pyrene).  
  • Induction appears to be environmental adaptive response of organism
  • Orphan Nuclear Receptors (PXR, CAR) are regulators of drug metabolizing gene expression
• additional factlrs effecting drug metabolism

  • Many factors can affect liver metabolism.
  • In aging the numbers of hepatocytes and enzyme activity declines.
  • Diseases that reduce hepatic blood flow like heart failure or shock can also reduce the metabolic potential of the liver.
  • Metabolism could also be altered due to a genetic deficiency of a particular enzyme.
  • Also the use of other drugs as well as dietary and environmental factors can influence liver metabolic function.
• Pharmacology:

  the study of the effect of drugs on the function of living systems

  Toxicology:

  the study of the effect of poisons on the function of living systems
• Chemical agents that cause toxicity include:

  • Drugs 

  • Insecticides/herbicides  

  • Plant toxins  

  • Animal toxins  

  • Chemical weapons 

  • Radioactive elements    
• toxicokinetics

  the effects of the body on the poison

  (relates to Absorption, Distribution, Metabolism, Excretion (ADME)).

  With this information it is possible to predict concentration of toxin that reaches the site of injury and the resulting damage.
• toxicokinetics

  Absorption - ingestion - mercury and dioxin in fish pesticides in produce , salmonella (diary), botulinum (meat) toxins. inhalation  - asbestos, nerve gases

  Distribution  as discussed for therapeutic drugs
• Toxicokinetics

  Metabolism  Phase I by cytochrome P450 (oxidation, reduction,   hydrolysis). Phase II conjugation to allow excretion in urine and bile

  Detoxification: compound rendered less toxic

  Toxification: relatively inert compound converted into toxin

  Excretion  toxins not excreted may be stored in: bone (eg. lead)

  fat (eg. DDE a metabolite of the pesticide DDT dichlorodiphenyl trichloroethane)

   The toxin may be released slowly into the body 
• Adverse drug reactions (ADRs) are noxious or unintended responses occurring at therapeutic doses, according to the World Health Organization (WHO) definition, affecting approximately 5% of all acute hospital admissions.
• Type A ADRs are effects related to known pharmacology but undesirable, common, dose-related, and predictable, with examples including hemorrhage with anticoagulants, respiratory depression with opioids, and sedation with anxiolytic and older antihistamine drugs.
• Type B ADRs are effects unrelated to known pharmacology, rare, unpredictable, often idiosyncratic, and have an individual allergy/genetic basis, with examples including anaphylaxis with penicillin, allergic liver damage by halothane, and bone marrow suppression by chloramphenicol.
• Molecular Mechanisms of Toxicology

  1.  Allergic responses

   - Common form of ADR, usually with a different time course to pharmacological effects

  • 4 basic clinical syndromes – types I, II, III & IV
  • Type I hypersensitivity reaction – IgE-mediated mast cell degranulation
  • Type II antibody-mediated cytotoxic hypersensitivity-

    involve haematological reactions i.e. those pertaining to the blood cells and blood-forming organs

  • Type III immune complex-mediated hypersensitivity
  • Type IV delayed-type hypersensitivity
• Molecular Mechanisms of Toxicology

  2. Receptor, ion channel and enzyme-mediated toxicity

  Molecular drug/toxin targets

  Receptors  (4 major superfamilies) 

  ∙ Ligand-gated ion channels   ionotropic receptors 

    voltage-gated ion channels

  ∙ GPCRs - G protein coupled receptors (metabotropic receptors)

  ∙ Enzyme-linked receptors (tyrosine kinase activity)

  ∙ Nuclear receptors (regulate gene transcription)

  Enzymes   metabolic and catabolic pathways

  Carriers    uptake/transport systems

  Others  proteins involved in vesicle release
• Animal toxins block ion-conduction

  a-bungarotoxin on nicotinic acetylcholine receptor (nAChR)
• Voltage-gated K+ channels are blocked by dendrotoxins

  Because of their high potency and selectivity for potassium channels, dendrotoxins have proven to be extremely useful as pharmacological tools for studying the structure and function of these ion channel proteins.
• Tetrodotoxin acts on Na+ channels to block action potentials

  it has shown efficacy for the treatment of cancer-related pain in phase II and III clinical trials.

  More dangerous than cyanide

  Used in v low doses in Chinese medicine and in Japan – sometimes also for suicide and soldiers not permitted to eat it or eggs

  Captain james cook fed to pigs on boards his ship in 1774 which were fatally poisoned.  Fugu in Japan is a public health concern – a delicacy too.
• Molecular Mechanisms of Toxicology

  3. Biochemical pathways

  i) Cyanide inhibits mitochondrial cytochrome c oxidase to prevent cellular respiration

  (ii) Carbon monoxide: displaces oxygen from haemoglobin causing hypoxia