HLTH 340 (F24) - Section D - Week 8 Xenobiotic Metabolism PDF

HLTH 340 Section D: Toxicokinetics of Metabolism Biotransformation of Xenobiotics  Lipophilic chemicals are particularly well absorbed through the skin, lungs, or GI tract  The lipophilicity of these chemicals can be a barrier to their elimination They can be readily reabsorbed....

HLTH 340 Section D: Toxicokinetics of Metabolism Biotransformation of Xenobiotics  Lipophilic chemicals are particularly well absorbed through the skin, lungs, or GI tract  The lipophilicity of these chemicals can be a barrier to their elimination They can be readily reabsorbed. Therefore, the elimination of xenobiotics often depends on their conversion to water-soluble chemicals through biotransformation. Biotransformation of Xenobiotics Typically we see metabolism (biotransformation) alters the physicochemical properties of xenobiotics: From: Favour absorption across biological membranes (e.g., lipophilicic) To: Favour elimination in urine or bile (i.e., hydrophilic). The terms biotransformation and metabolism are often used interchangeably, particularly when applied to drugs and pharmacokinetics. Biotransformation of Xenobiotics Without metabolism, the many xenobiotics we are exposed to on a daily basis would eventually accumulate to toxic levels. The breakdown products of xenobiotic biotransformation are called metabolites. Metabolism may: Environmental Chemicals: Increase or decrease toxicity Increase toxicity: Bioactivation Decrease toxicity: Detoxification Pharmaceuticals: Increase or decrease therapeutic efficacy Biotransformation of Xenobiotics Some xenobiotics can stimulate the synthesis of enzymes involved in the metabolic process. Referred to as enzyme induction Adaptive and reversible response to xenobiotic exposure. Enzyme induction actually enables some xenobiotics to accelerate their own biotransformation and elimination from the body. Biotransformation of Xenobiotics  Biotransformation processes are controlled by a series of enzyme- catalyzed processes.  Biotransformation is catalyzed by various enzyme systems that can be divided into four categories based on the reaction they catalyze: 1. Hydrolysis (e.g., carboxylesterase) 2. Reduction (e.g., carbonyl reductase) 3. Oxidation (e.g., cytochrome [CYP] P450) 4. Conjugation (e.g., UDP-glucuronosyltransferase [UGT]) Biotransformation of Xenobiotics Biotransforming enzymes are widely distributed throughout the body and are present in several subcellular compartments. In vertebrates, the liver contains the largest and most diverse source of biotransforming enzymes. These enzymes are also located in the skin, lung, nasal mucosa, eye, and GI tract. Biotransformation of Xenobiotics These enzymes can also be found in a variety of other tissue including: the kidney, adrenal, pancreas, spleen, heart, brain, testis, ovary, placenta, plasma, erythrocytes, platelets, lymphocytes, and aorta. Intestinal microflora also play an important role in the biotransformation of certain xenobiotics. Probiotics following antibiotic treatment? Biotransformation of Xenobiotics  Within the liver and most other organs, these metabolic enzymes are located primarily in the: Question: Why would organisms  Endoplasmic reticulum (i.e., microsomes) have evolved these enzyme systems across  Soluble fraction of the cytoplasm (i.e., cytosol) cell compartments?  Other areas of the cell such as the mitochondria, nuclei, and lysosomes.  In general, biotransformation is conducted by a limited number of enzymes with broad substrate specificities.  For example, CYP2D6 and CYP3A4 metabolize over half the orally effective drugs in current use. Biotransformation of Xenobiotics Metabolism Alters structure of parent compound to make one or more metabolites Enzyme Parent Compound Metabolite(s) These metabolic processes are generally classified as: Phase-1 or Phase-2 Phase I versus Phase II Metabolism  Phase I: Chemical modifications that introduce or uncover functional groups on a xenobiotic that provide sites for Phase II metabolism OH Benzene Phenol  Phase II: Synthetic reaction of a xenobiotic (or of a Phase I metabolite of a xenobiotic) with an endogenous substance that introduces polar/ionizable groups to enhance water solubility and hence excretion COOH OH o o HO OH OH Phase I versus Phase II Metabolism Phase I (Functionalization) Phase II (Conjugation) Glucuronidation Oxidative Reactions Sulfation Hydrolytic Reactions Acetylation Reductive Reactions Methylation Amino Acid Conjugation Glutathione Conjugation Metabolism of Benzene Epoxidation Hydroxylation Sulfation General Pathways of Xenobiotic Metabolism Phase-1 metabolism can create reactive metabolites  bioactivation of an inactive parent compound to create a more chemically reactive product  Some reactive metabolites “attack” cellular targets (e.g., DNA) If Phase 2 is overwhelmed: Build up of Phase I product Toxicity if Phase I product is reactive Deficiency in Conjugating Endobiotics: Build up of Phase I product Biotransformation of Lipophiles Phase-1 metabolism (functionalization, degradation)  Comprises mostly oxidation reactions  Primarily catalyzed by cytochrome P450 enzymes  Monooxygenase reaction  a oxygen is added to parent compound  lipophiles are oxidized in this Phase-1 metabolism  hydrophiles are ignored  A modest increase in hydrophilicity with unpredictable toxicity  Functionalization: creates new functional side group on molecule (i.e., -OH, -NH2, -SH, or -COOH)  Degradation: creates pathway for xenobiotic excretion What are Cytochrome P450?  Hemoprotein, like hemoglobin.  Widely distributed across species.  Present in highest amount in liver and small intestines, mainly in the membranes of the smooth endoplasmic reticulum. Cytochrome P450  Substrate specificity.  NADPH is involved in reaction mechanism in cytochrome P450.  Lipids also components of cytochrome P450 system.  Many cytochrome P450 are inducible. Oxidation using P450 Systems Most Phase-1 reactions are performed by CYP450’s P450 enzymes are localized within a mixed-function oxidase (MFO) complex  insoluble enzyme complex embedded within a phospholipid membrane P450 MFO systems can oxidize a wide spectrum of lipophilic substrates Endobiotics  various fatty compounds (e.g., cholesterol)  endogenous steroid hormones (e.g., estrogens, etc.) Xenobiotics  chemicals, drugs, synthetic steroids  Oxidation using P450 Systems CYP450 react with lipophilic xenobiotics to form oxidized metabolites P450 enzymes lipophilic substrates -------------------> oxidized metabolites generally creates oxygen-centered functional groups  R-OH alcohol derivatives  R=O aldehyde derivatives  R-COOH carboxylic acid derivatives  R>O epoxide derivatives also can create non-oxygenated metabolites via ‘unmasking’ reactions (e.g., dealkylation) CYP Enzyme Naming Conventions CYP P450 enzymes are the most important in biotransformation in terms of the catalytic versatility and number of xenobiotics that it metabolizes: 400 isozymes and 36 families The CYP enzymes are arranged in families and subfamilies, and named based on the primary amino acid sequence of the individual enzyme. For Example: CYP2D6 In the CYP family 2 - Subfamily D - Gene number 6 Biotransformation of Lipophiles Phase-2 metabolism (conjugation)  lipophilic xenobiotics (and their metabolites) are conjugated to create more hydrophilic compounds  enzymatic addition of a small endobiotics onto the xenobiotic  e.g., a glucuronide sugar  introduces a hydrophilic conjugating group onto Phase 1 metabolites  commonly provides a detoxication pathway for xenobiotic bioinactivation  usually creates more hydrophilic Phase-2 products  enables excretion via kidney or liver Changes in lipophilicity during biotransformation Log Kow = 2.47 Phase-1 functionalization Log Kow = 1.70 Phase-2 conjugation Log Kow = -0.17 Biotransformation of Lipophiles  Xenobiotic metabolism or biotransformation occurs primarily in the liver.  Metabolism can also occur in non-hepatic tissues, such as: Intestine mucosa Kidney Lungs Bacteria in GI tract  Following oral exposure, xenobiotics are metabolized before reaching the general circulation: First-pass metabolism Xenobiotic Biotransformation in the Liver Phase-1 Metabolism CPT-11  activation of inactive pro-drug to active metabolite (bioactivation)  conversion of active drug to inactive metabolite (detoxication)  conversion of active drug to a toxic metabolite (adverse drug reaction, ADR)  biotransformation of a xenobiotic to a mutagenic or carcinogenic metabolite  biotransformation of a xenobiotic to a less SN-38 harmful metabolite Xenobiotic Biotransformation in the Liver SN-38 Phase-2 Metabolism  usually an inactivation reaction for drugs  usually detoxication reactions for xenobiotics  some important exceptions (where an activated toxic metabolite is produced) Phase-3 Metabolism (active transport)  biotransformation reactions are often coupled with efflux pumps in intestinal mucosa and the liver  e.g., CYP3A4 and P-gp efflux pump SN-38G  Biotransformation of Pesticides by CYP450 Example 1: methoxychlor  formation of hydroxyl groups by the O-dealkylation reaction  bioinactivation reaction (non-toxic)  metabolite can proceed to conjugation in Phase 2  Biotransformation of Pesticides by CYP450 Example 2: malathion  creates oxon group by the oxidative desulfuration reaction  bioactivation reaction (toxic)  malaoxon is the active toxic metabolite Hydrolytic Cleavage By Esterase Enzymes (not P450-dependent)  Biotransformation of Pesticides by CYP450 Example 3: DDT  removal of a chlorine atom by oxidative dehalogenation  no functionalization, so conjugation is not possible (persistent) DDE Reduction reactions (CYP450-dependent)  Reduction reactions are a minor Phase-1 pathway  Metabolites are still lipophilic, no conjugation possible  May allow development of insecticidal resistance to DDT CYP1A1 and CYP1A2 properties  CYP1A1 active in many tissues, especially the lung  CYP1A2 active mainly in liver  Substrates: Polycyclic aromatic hydrocarbon (PAHs), arenes  preferentially oxidizes compounds with 2+ benzene/unsaturated rings  Bioactivation: often creates toxic aryl epoxides as reactive metabolites  PAH /P450 1A1/ reactive metabolites  DNA adducts  genotoxicity  cancer CYP1A1 and CYP1A2 properties Inducible: xenobiotic exposure  2-50x ↑ CYP1A1/2 levels  Polycyclic Aromatic Hydrocarbons (PAHs) Methylcholanthrene (MC) or benzo(a)pyrene (BaP) Persistent organic pollutants (POPs) organochlorine pesticides (e.g., DDT), some PCBs, dioxins and dibenzofurans Hormone metabolism - aromatic steroids (e.g., estrogens)  estrogen hormones (estradiol)  /CYP1A2/  estrogen metabolites (some may be carcinogenic) CYP1A1 and CYP1A2 properties CYP1A1/2 are induced by common environmental factors  Smoking - Tobacco smoke (tar fraction) high in PAHs (e.g., BaP)  Combustion products – e.g., soot, diesel exhaust, BBQ smoke  Some food constituents example: caffeine  Some pharmaceutical drugs Chemical Carcinogenesis: BaP Bioactivation to BPDE benzo(a)pyrene (BaP) (+) trans BPDE ultimate carcinogen  a common type of PAH found in many combustion products BaP epoxide CYP1A1 - cigarette smoke proximate carcinogen - diesel exhaust CYP1A1 BaP is a pro-carcinogen  inactive in parent form Bay-region  lipophilic - good P450 substrate unreactive BPDE metabolites  P450 oxidizes rings in specific regions of BaP molecule BaP pro-carcinogen regioselectivity stereoselectivity Chemical Carcinogenesis: BaP Bioactivation to BPDE (+) trans BPDE ultimate carcinogen Regioselectivity  functional group can BaP epoxide CYP1A1 be inserted on proximate carcinogen different regions of the parent compound  'Bay-region' CYP1A1 metabolites Bay-region unreactive BPDE Stereoselectivity metabolites  position of substituent BaP groups above or pro-carcinogen below the ring Genotoxicity of BPDE reactive metabolite electrophilic reactant (BPDE) (carbonium ion) electrophilic attack adduct:DNA non-reactive metabolite covalent (BaP-tetrol) bond formation bulky adduct Bulky Adduct Impacts DNA Structure and Function CYP2E1 properties CYP2E1 is active in both hepatic and non-hepatic tissues Substrate preference - mainly for small aliphatic compounds  oxidizes small straight-chain or branched-chain compounds  alcohols, acetone and ketone bodies, short-chain fatty acids  chlorinated solvents - chloroform, trichloroethylene (TCE), vinyl chloride  oxidizes benzene (single unsubstituted aromatic ring) Bioactivation  aliphatic epoxides and aldehydes and quinone imines are often reactive metabolites (liver damage)  acetaminophen  NAPQI  liver damage (necrosis)  benzene  benzene epoxide  bone marrow damage (myelotoxicity)  aplastic anemia; leukemia CYP2E1 properties Upregulation of CYP2E1 by small aliphatic compounds Substrate-binding prolongs the enzyme lifetime in the SER  ‘EtOH-inducible P450’  2-10 x increase CYP2E1 levels Levels elevated by many common environmental factors  heavy drinking - ethanol  obesity and obesity-related diabetes (type-2)  some pharmaceutical drugs - example: isoniazid (TB drug) Tylenol Bioactivation and Effects GSH Deficiency: Increases Toxicity Acetaminophen Poisoning Treated with cysteine Direct Phase II possible: Sulfate, Glucuronide Tylenol Bioactivation and Effects Timeline of Toxicity Men  Many common environmental factors  oral contraceptives (birth control pills)  many pharmaceutical drugs - example: erythromycin and similar antibiotics  Common xenobiotics, like Aflatoxin B1 Fungal Toxin Aflatoxin (AFB1) Causes Liver Cancer Mold that grows in hot and damp conditions: Significant public health issue in several countries including: China Mozambique CYP3A4 Bioactivation of AFB1 to AFB1-Epoxide Aflatoxins  Can cause acute hepatotoxicity  Significant differences in species susceptibility. Consider the LD50 for:  Rabbits, Ducks, Turkeys, Trout: 1 mg/kg  Rats: 8 mg/kg  Chickens: 15 mg/kg  Mice: 150 mg/kg  Chronic exposure leads to liver cancer. AFB1 is a pro-carcinogen  AFB1 is bioactivated by CYP3A4 in liver  AFB-epoxide  AFB1-epoxide is an electrophilic reactive metabolite CYP3A4 Bioactivation of AFB1 to AFB1-Epoxide Aflatoxins  Reacts with DNA bases to form covalent adducts  DNA damage  N-7 Position of Guanine  Chronic AFB1 exposure in contaminated foods leads to elevated risk of hepatocellular carcinoma (liver cancer)  Hepatocarcinoma is a significant cause of death in Mozambique in individuals as young as 35 years  Regulated in Canada to < 15 ppb Phase -2 detoxication by conjugation bioactivation by CYP3A4 DNA adduct formation Epoxide Hydrase and the Detoxification of Reactive Metabolites Phase 1 often produces epoxides as toxic reactive metabolites Found in the smooth endoplasmic recticulum Epoxides possibly carcinogenic to many tissues Epoxides cannot be conjugated until epoxide ring is broken open (hydrolysis) Epoxide Hydrase and the Detoxification of Reactive Metabolites Epoxide hydrase (EH) is an essential enzyme transforms epoxides to diols R>0 + H20 --/EH/ R-(OH)2 EH very important to rapid detoxification of epoxide metabolites EH considered potential anti-cancer enzyme Phase I Is More than Just CYP450 Oxidation Reactions Hydrolysis  Water cleaves a bond in an organic molecule  Can be spontaneous or enzymatic. Examples we have already discussed in class:  BaP: Epoxide ring hydrolysis  Carbonium ion  Malathion: esterase-mediated hydrolysis Another Example of Bioactivation due to Hydrolysis  Β-Glucuronidase Activity on Amygdalin  The toxin amygdalin is a cyanogenic glycoside Amygdalin Definitions: Cyanogenic – Hydrolytic cleavage of C--C#N bond releases HCN (a.k.a. Hydrogen Cyanide) Glycoside – Sugar molecule coupled to functional group by a glycosidic bond Consumption of amygdalin containing Hydrolysis of Amygdalin plants: Occurs By: Can release substantial quantities of HCN in the Stomach acids GI tract Bacterial B-glucuronidases Binds irreversibly to Cytochrome A3 of electron transport chain Brush border enzymes Stops ATP production Other Hyperventilation, Biochemical Headache, Nausea, Paralysis, Effects: Selenoenzyme inhibition Coma, Respiratory Arrest Amygdalin Minimum Lethal Dose: 0.5 – 3.5 mg/kg i.e., 30-210 mg for 60 kg adult HCN absorbed through GI tract Plant HCN (mg/100 g) Sublethal Effects: Bitter almond 290 Enlargement of thyroid (i.e., goiter) Goitrogen Peach pit 160 Apricot pit 60 Foods that Can Contain Amygdalin Cassava root 53 Cassava, Almonds, Lima beans Amygdalin in Cassava Cassava is a dietary staple in some parts of Africa – Nigeria is the world’s largest producer  Risk = Hazard * Probability of Exposure Traditional preparation method renders Cassava safe to eat:  Crush cassava in water and ferment (releases Cassava glucuronidase)  Glucuronidase catalyzes amygdalin during fermentation, releasing HCN  Dry and grind into a flour  And water and ferment again  Cook; Discard cooking water (contains HCN not expelled during fermentation  Product: Chickwangue  Safe to eat Toxicology in the News Organic Almonds Recalled from Whole Foods For Containing High Levels of Cyanide http://www.iflscience.com/health-and-medicine/almonds-prove-bitter-organic-purists Bitter almonds imported from Italy or Spain Sold as “Whole Foods Market Organic Raw Almonds” Voluntarily recalled due to “possibility of high levels of cyanide” “Medical Quacks” have recommended amygdalin-containing foods as a potential cancer cure:  Cyanide overdoses and death Toxicology in the News: The Story of Laetrile  Amygdalin was originally isolated in 1830 by two French chemists.  When mixed with certain enzymes, amygdalin breaks down into glucose, benzaldehyde and hydrogen cyanide.  It was tried as an anticancer agent in Germany in 1892, but its use was discontinued due to its ineffectiveness and overall toxicity to the patient.  During the early 1950s, Ernst T. Krebs, Sr., MD, and his son Ernst, Jr., began using a purified form of amygdalin to treat cancer patients.  The chemical they synthesized was laevo-mandelonitrile-beta- glucuronoside. Toxicology in the News: The Story of Laetrile  They called it Laetrile, and marketed it as an anti-cancer treatment. Toxicology in the News: The Story of Laetrile  The Krebs trademarked the modified form as Laetrile for the treatment of “disorders of intestinal fermentation”.  Krebs, Sr., worked as a pharmacist before receiving his medical degree in 1903, marketed one after another drug throughout his lifetime based on claims later considered false and fraudulent.  Krebs, Jr., has often been referred to as “Dr. Krebs”, although he never received an accredited doctoral or medical degree.  Attended a medical college in Philadelphia from 1938 to 1941 but was expelled after repeating his freshman year and failing his sophomore year.  After taking courses in five different colleges and achieving low or failing grades in his science courses, he finally received a Bachelors of Arts degree from the University of Illinois in 1942.  In 1973, after giving a 1-hour lecture on Laetrile, he obtained a “Doctor of Science” degree from American Christian College. Toxicology in the News: The Story of  Laetrile The Krebs theory on Laetrile’s changed multiple times over the years as researchers and the FDA investigated their claims.  The claims of Laetrile efficacy were based on a variety of hypotheses (all proven untrue)  Laetrile has been evaluated by the National Cancer Institute (NCI) in the US numerous times. Each time, their study failed to show anticancer activity.  It continued to be pushed by advocates as an effective anti-cancer treatment, despite many cases of adverse effects.  In 1979, the Food and Drug Administration (FDA) rules that Laetrile products were toxic and ineffective, and banned it for use.  Laetrile was then repositioned as a fight against the establishment, with the government “covering up” Laetrile’s effectiveness in combatting cancer. Toxicology in the News: The Story of Laetrile  In February of 2016, Health Canada issued a warning advising Canadians who purchased Novadalin B17/Amygdalin, what they termed as an unauthorized natural health product claiming to treat cancer, to stop using the product and contact their doctor for appropriate follow-up.  They note in the release that “no drug products containing B17 or amygdalin have been approved by Health Canada”.  The release states that “[t]he concentration of amygdalin is unknown at this time, however, ingesting low to moderate amounts of cyanide may lead to serious adverse health consequences and high doses may be lethal.” Toxicology in the News: The Story of Laetrile  So as long as there are life-threatening and fatal diseases, there will always be individuals eager to offer “alternatives” to scientific treatments, and large numbers of desperate individuals and families willing to try them despite significant health implications.  Leads to the need for relevant regulatory watchdog agencies like Health Canada and the FDA requiring documented trials of a drug or natural food products efficacy to protect the public from snake oil salesmen selling the latest health tonics.