Detoxification of endogenous and exogenous compounds mirka.rovenska@lfmotol.cuni.cz

A) DETOXIFICATION OF AMMONIA Ammonia originates in the catabolism of amino acids that are primarily produced by the degradation of proteins

Removal of nitrogen from amino acids

Ammonia has to be eliminated: Ammonia is toxic, especially for CNS, because it reacts with -ketoglutarate, thus making it limiting for the TCA cycle  decrease in the ATP level Liver damage or metabolic disorders associated with elevated ammonia can lead to tremor, slurred speech, blurred vision, coma, and death Normal conc. of ammonia in blood: 30-60 µM

Transamination Transfer of the amino group of an amino acid to an -keto acid:

L-alanine L-aspartate -ketoglutarate pyruvate oxalocetate glutamate

Transamination is catalyzed by transaminases (aminotransferases) that require participation of pyridoxalphosphate: amino acid pyridoxalphosphate Schiff base

Transaminases differ in their substrate specificity, however, the most of them use -ketoglutarate as an -keto acid, to a lesser extent oxalacetate, thus producing mainly Glu and Asp Transaminations are usually reversible  the actual direction depends on the concentrations of reactants

Principal transaminations: Alanine transaminase (in muscle): AA + pyruvate  -keto acid + Ala Glutamate transaminase: AA + -ketoglutarate  -keto acid + Glu Aspartate transaminase: AA + oxaloacetate  -keto acid + Asp

Net result: Amino acid nitrogen is being accumulated in the form of glutamate or aspartate (by transaminations) Glutamate can be deaminated, releasing ammonia that – in the liver – enters the urea cycle or is used for syntheses Aspartate also enters the urea cycle in the liver

Oxidative deamination of Glu In mitochondria Glu + NAD(P)+ + H2O NAD(P)H + H+ + NH4+ + -ketoglutarate Reaction is reversible – either produces Glu, or releases ammonia, according to the concentrations of reactants glutamate dehydrogenase – uses either NAD+ or NADP+

Transport of ammonia from the other tissues to the liver 1) In tissues, ammonia is built into Gln: Glu + ATP + NH4+  Gln + H2O + ADP + P Reaction is catalyzed by glutamine synthetase; then, Gln is transported to the liver and here deaminated by L-glutaminase: Glu and ammonia are produced: Amide nitrogen, not the -amino nitrogen is removed! an analogous reaction is catalyzed by L-asparaginase

2) The glucose-alanine cycle: Liver Muscle Pyruvate, produced in muscle by glycolysis, is transaminated to Ala, which is transported to the liver and here converted to pyruvate; released NH3 enters the urea cycle, pyruvate is used for gluconeogenesis (muscle lacks enzymes for this pathway). Then, Glc is transported back to the muscle

Thus: Glu accumulated in the liver by the action of transaminases and glutaminase is subjected to oxidative deamination, yielding -ketoglutarate and ammonia which enters the urea cycle Another source of ammonia (beside oxidative deamination of Glu): glutaminase reaction

The overall scheme of transport and removal of ammonia NH4+ NH4+ NH4+ Glutamine Glutamine Glu Glu Alanine Alanine

An alternative fate of Gln: A portion of Gln, not metabolized to Glu and ammonia in the liver, can be released into the bloodstream and taken up by kidney so that the glutaminase reaction takes place here  ammonia is released, but instead of entering the urea cycle, it diffuses into the urine… source of ammonia in the urine Gln produced in the kidney also contributes This process participates in the regulation of the acid-base balance and pH of the urine

Detoxification of ammonia Ammonia that has been accumulated in the liver must be detoxified: a) it is build into Glu (glutamate dehydrogenase reaction) or Gln (glutamine synthetase): -ketoglutarate+NH4++NAD(P)H+H+Glu+H2O+NAD(P)+ Glu + ATP + NH4+  Gln + H2O + ADP + P Glu, Gln are then used for subsequent syntheses: Glu – synthesis of Gln, Pro, Ala, Asp Gln – synthesis of purines and pyrimidines b) the urea cycle converts ammonia to urea which is excreted with the urine…PRINCIPAL

Urea cycle In the liver In mitochondrial matrix, oxidative deamination of Glu takes place, releasing ammonia that is converted to carbamoyl phosphate: NH4+ +HCO3- + 2 ATP  2 ADP +P+ In mitochondria, carbamoyl phosphate reacts with ornithine, yielding citrulline, which is transported to the cytoplasm; ornithin is regenerated (step 4) and transported back to mitochondria carbamoyl phosphate

Carbamoyl phosphate formation is catalyzed by carbamoyl phosphate synthetase I (CPSI), which is activated by N-acetylglutamate: N-Ac-Glu is being synthesized from Glu and AcCoA When AA breakdown (and the need of nitrogen elimination) rises, the conc. of Glu rises, too (due to transaminations)  the concentration of N-Ac-Glu is also increased  activation of CPS I  stimulation of the urea cycle

The urea cycle UREA carbamoyl phosphate transamination oxaloacetate+Glu Asp ornithine transcarbamoylase argininosuccinate- synthetase ATP AMP+PP citrulline ornithine argininosuccinate argininosuccinase arginase UREA fumarate arginine

Chemical balance of the urea cycle: 3 moles of ATP are required for the formation of 1 mole of urea: 2 for the formation of carbamoyl phosphate 1 for the formation of argininosuccinate

Linkages between the urea cycle and the TCA cycle: Via: fumarate transamination of oxaloacetate: Glu + oxaloacetate  Asp + -ketoglutarate Dikarboxylic acids can pass rapidly between the cytosol and mitochondria

NH3 in excess Asp in excess

Relative surplus of ammonia: reaction catalyzed by glutamate dehydrogenase proceeds in the direction of Glu formation  increased flux to aspartate through aspartate transaminase Relative surplus of Asp: these two reactions proceed in the opposite direction to provide ammonia for urea formation

Deficiencies of urea cycle enzymes and their treatment 1) N-acetylglutamate synthetase: administration of carbamoyl glutamate – also activates CPSI 2) CPSI: administration of benzoate and phenylacetate; resulting hippurate and Phe-Ac-Gln are excreted with the urine:

3) ornithine transcarbamoylase: the most common deficiency; treatment is the same as in the case 2) (i.e. removal of nitrogen in the form of Gly and Gln) 1)-3): if not treated, hyperammonemia occurs mental retardation, coma, death 4) Argininosuccinate synthetase: accumulation of citrulline in blood and excretion in the urine (citrullinemia); supplementation with Arg necessary 5) Argininosuccinase: treatment as in case 2) + supplementation with Arg 6) Arginase: rare; Arg accumulates and is excreted; administration of benzoate + low protein diet including essential AA (but excluding Arg) or their keto analogs In all cases, the low nitrogen diet is also applied

NO synthesis NO – biologically active: stimulates vasodilatation NO also serves as a substrate for production of the reactive nitrogen species (RNS) Its formation is catalyzed by nitric oxide synthase: Arg citrulline

B) Metabolism of xenobiotics Drugs, preservatives, pigments, pesticides … Predominantly in the liver Involves two phases

Phase 1 Incorporation of new groups or alteration of groups that are already present in the molecule In the endoplasmic reticulum (ER) Result: increase in the polarity (supports excretion) and: A) decrease in the biological activity (toxicity) B) activation: some compounds only become biologically active once they have been subjected to phase 1 reactions

Toxic effects of some activated compounds – ad B) Cytotoxicity – e.g. by covalent binding to DNA, RNA, proteins Binding to a protein, thus altering its antigenicity  antibodies are produced and can damage the cell Carcinogenesis – phase 1can convert procarcinogens (benzo[]pyren) to carcinogens. Epoxid hydrolase (in ER) can exert a protective effect by converting the reactive, mutagenic and/or carcinogenic epoxides to less reactive diols: diol epoxide

Reactions of phase 1: Hydroxylation Epoxide formation Reduction of carbonyl-, azo- or nitro- compounds Dehalogenation

Hydroxylation Chief reaction of the phase 1 Catalyzed by cytochrome P450s (in human: 35-60 forms): monooxygenases: RH + O2 + NADPH + H+ ROH + H2O + NADP+ Electrons from NADPH+H+ are transferred to NADPH-cytochrome P450 reductase and then to cytochrome P450 that uses them for activation of oxygen. One oxygen atom is then inserted into the substrate.

Isoforms of cytochrome P450 Substrates: drugs, carcinogens, pollutants, but also endogenous compounds (steroids, eicosanoids, fatty acids) Hemoproteins In the endopl. reticulum or inner mitoch. membrane Most abundant in the liver and small intestine; at least 6 isoforms in the liver (in human), each of them exhibiting a broad substrate specificity Certain of them exist in polymorphic forms, some of which exhibit low catalytic activity  poor metabolism and accumulation of corresponding xenobiotic in the body Certain isoforms are involved in the metabolism of polycyclic aromatic hydrocarbons (PAHs), thus playing a role in carcinogenesis (for instance, conversion of inactive PAHs, inhaled by smoking, to active carcinogens in the lung)

Most isoforms are inducible: For instance by phenobarbital and other drugs Mechanisms: in most cases increased transcription Can lead to drug interaction: induction of the distinct isoform by the drug 1 (phenobarbital) can speed up metabolism of drug 2 (warfarin) by this isoform  it is necessary to increase the dose of the drug 2 Ethanol induces the isoform CYP2E1, which metabolizes i.a. some carcinogenic components of tobacco smoke!

Phase 2 – conjugation Products of phase 1 are conjugated with: glucuronate sulphate glutathione Conjugation renders them even more water-soluble and eventually even less active; conjugates are excreted: with the bile (conjugates with Mr  300) or urine (Mr  300)

Glucuronidation UDP-glucuronic acid is the glucuronyl donor: Glucuronate can be attached to oxygen (O-glucuronides) or nitrogen (N-glucuronides) groups Excreted as glucuronides are: benzoic acid, meprobamate, phenol, and also endogenous compounds – bilirubin, steroids glucuronate

Bilirubin excretion Bilirubin is the product of heme catabolism: heme

transported by albumin to the liver M: methyl, V: vinyl, CE: carboxyethyl (propionic) transported by albumin to the liver

conjugation with glucuronate: In the liver – conjugation with glucuronate: ─bilirubin diglucuronide secreted into the bile bacterial enzymes in the gut release bilirubin from diglucuronide and convert it mainly to urobilinogen smaller fraction is reabsorbed, transpor-ted to the kidney and converted to yellow urobilin, excreted in urine most of it is micro- bially converted to red-brown stercobilin, the major pigment of feces

Sulfation Some alcohols, arylamines, phenols, but also steroids, glycolipids, glycoproteins Sulfate donor: PAPS (3´-phosphoadenosine-5´-phosphosulfate):

Conjugation with glutathione Glutathione (GSH) = -glutamylcysteinylglycine: (R = electrophilic xenobiotic) G–S–H + R  G–S–R + H+

Several potentially toxic xenobiotics are conjugated to GSH (certain carcinogens, drugs) Conjugation to GSH prevents binding of these xenobiotics to DNA, RNA or proteins and subsequent cell damage!

Further metabolism of glutathione conjugates: glutamyl and glycinyl of GSH are removed an acetyl group (donated by acetyl-CoA) is added to the amino group of the cysteinyl moiety the product is a mercapturic acid (conjugate of acetylcystein), which is excreted in the urine

mercapturic acid

Other functions of GSH Participates in the decomposition of H2O2: 2 GSH + H2O2  GSSG + 2 H2O It is an important intracellular reductant, helping to maintain essential –SH groups of proteins in the reduced state Participates in the transport of certain amino acids (Cys, Gln) across cell membranes (mainly in the kidney): AA + GSH  -glutamyl-AA + Cys-Gly glutathione peroxidase on the membrane both peptides are transported to the cytosol; here, AA is released

C) Metallothioneines Small proteins (~ 6,5 kDa), cysteine-rich  the –SH groups bind metal ions: Cu2+, Zn2+, Hg2+, Cd2+ In cytosol, mainly of the liver, kidney, and intestine cells Induced by metal ions Binding of metals, regulation of the Zn2+ level, transport (Zn2+)