Introduction to the Toxicology of the Liver (9-Aug-1999)

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1 In: Veterinary Toxicology, V. Beasley (Ed.) Publisher: International Veterinary Information Service ( Ithaca, New York, USA. Introduction to the Toxicology of the Liver (9-Aug-1999) V. Beasley Department of Veterinary Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA. The liver is uniquely positioned as the body's primary detoxification center for ingested agents. The blood travels from the digestive tract via the portal vein directly to the liver where it percolates through the sinusoids with their porous endothelial linings. Thus, the hepatocytes are able to take up and process not only a range of nutrient substances but also many natural and manmade toxicants. In most cases, the entry of toxicants into hepatocytes is a passive process, but in some cases, such as with the water soluble algal toxins termed microcystins, active transport processes are involved in hepatocyte uptake. Hepatic metabolism = biotransformation = usually makes xenobiotics more water soluble. As a result of biotransformation, more of the xenobiotic tends to be filtered by the glomerulus and less is reabsorbed by the kidney; thus, more leaves in the urine. Also, the metabolites tend to undergo increased active secretion by the kidney and the liver. Processes of Metabolism are Comprised of Phase I and Phase II Reactions Phase I Reactions Oxidation, reduction, hydrolysis. Some Specific Phase I Processes A. Oxidation by P450 Enzymes P450 + NADPH(H) + O2 + RH Many oxidation and hydrolytic reactions + some reduction reactions; many but not all of the Phase I reactions are catalyzed by microsomal cytochrome P450 enzymes. Microsomes = (laboratory altered) pieces of smooth endoplasmic reticulum. P450 are membrane-bound enzymes. Therefore, substances metabolized must be somewhat lipophilic. ROH2 + H2O (e - source) (substrate)* Uses one O atom from O2 to oxidize RH. The other O atom is reduced to H2O. Because of these two functions of the enzyme system, it is called "mixed function oxidase". *Substrate is often a xenobiotic, or an endogenous compound such as a hormone or fat soluble vitamin. B. Reduction by P450 Enzymes 1. Same initial reaction Adds e - (electron) first as in above reaction, except here (especially if the animal is hypoxic), there is no subsequent addition of oxygen. Instead a reactive (often toxic) free radical is formed:

2 = - R. tissue binding may cause liver necrosis e.g., halothane if hypoxic-increased chance of hepatotoxicity. Normally a lot is breathed off and some is oxidatively metabolized by liver. With hypoxia increased amount of reduction to free radical metabolites. 2. Cyclic compounds may get addition of e- by P450 but then e- falls off e- may hook onto oxygen to form an oxygen radical. Oxygen radicals react with lipids lipid peroxidation (see subsequent part of this section); or e- reacts with other molecules forms - R. (free radicals). C. Hydrolysis as a Result of P450 Enzymatic Reactions (Includes epoxide hydrolase) Cyclic compound with double bond P450 metabolism often fast Reactive strained epoxide which oftenreacts with macro-molecules causing cytotoxic or DNA-damaging effects Epoxide hydrolase often slow Diole (not highly toxic) D. Hydrolysis by Soluble Esterases (e.g., nonspecific carboxylesterases of liver, pseudocholinesterase of blood.) (Not a P450 reaction.) E. Oxidation by Cytosolic Enzymes (e.g., alcohol dehydrogenase.) (Not a P450 reaction.) Others. Summary: Agents Affecting Mixed Function Oxidases Induction of Cytochrome P450 Enzymes 1. Regarded by some toxicologists as a toxic effect in itself. 2. Can cause notable liver enlargement. 3. Agents that induce (to induce means to increase the amounts of) certain isoenzymes of P450 can increase the toxicity of xenobiotics bioactivated by those enzymes. For example, P450IA enzymes are inducible by methylcholanthrene, and phenobarbital induces P450 IIB isoenzymes. Such compounds increase the toxicity of xenobiotics which are bioactivated by the specific isoenzymes that they induce. Specific examples include: aflatoxin B1, nitrosamines, bromobenzene, and carbon tetrachloride. Conversely, if the xenobiotic is detoxified by P450 without notable activation, induction of P450 may protect the animal. A specific examle would be the tolerance of an animal to a drug after being on phenobarbital as an anticonvulsant. Note - Different P450 isoenzymes occur in different locations in the liver which can result in local activation and local damage. Generally, centrilobular areas tend to have some of the highest P450 activities overall, which can predispose to centrilobular toxic effects. Inducers of microsomal enzymes, e.g., PCBs, phenobarbital, halogenated dioxin, dieldrin or other chlorinated hydrocarbon insecticides. Causes an increase in liver size and weight. May shorten the duration of effect of other drugs, e.g., endogenous steroids. May increase susceptibility to hepatotoxic agents which act primarily after conversion to reactive metabolites by liver enzymes or May lessen toxicity of agents whose metabolites are less toxic than parent compounds. Inhibition of Cytochrome P450 Enzymes Inhibitors of P450 generally do not reduce the amounts of enzyme present. They simply reduce the activity of the enzyme. If a xenobiotic is bioactivated by P450 isoenzymes, then agents that inhibit the action of these isoenzymes protect against toxicity. Conversely, if a xenobiotic is simply detoxified by a reaction with P450 (and not activated to significant degree in the process), inhibitors of P450 increase its toxicity. Chloramphenicol is bioactivated by P450s into reactive intermediates, especially dichloroacetamido groups that acetylate and irreversibly inhibit cytochrome P450IIB enzymes.

3 Fluoroquinolone antibacterials including enrofloxacin, ciprofloxacin, and norfloxacin, inhibit cytochrome P450IA. The inhibition is considered to be reversible. Macrolide antibiotics, such as erythromycin also inhibit P450s. Inhibitors of microsomal enzymes, e.g., chloramphenicol, piperonyl butoxide, N-octylbicycloheptene dicarboxiimide, sesame oil, and the initial (temporary) effect of inducers such as phenobarbital on the first day of dosing: May prolong the effect of drugs metabolized by P450s to inactive forms. May prevent liver lesions associated with agents otherwise converted by P450s in the liver to highly (locally) reactive metabolites. May increase the systemic toxicity of agents which are metabolized by P450s in the liver to products less toxic than the present compound. Phase II (Addition Reactions) = Conjugation Reactions (for information on species differences see Appendix C). Glucuronidation - Often involved and is a system with a relatively high capacity - except in cats and neonates of many species. Cats have a glucuronyl transferase, but for many xenobiotics, it is ineffective. Sulfation - Low capacity (especially in pigs) - other species tend to have a low amount of sulfate in pool. Conjugation Glutathione (GSH) - Low capacity in all species but still very important in interrupting (hooking onto) highly reactive intermediates (metabolites) formed by P450 (Phase I) reactions. Amino acids - Conjugation with various other amino acids (species dependent). Acetyl group - Acetylation may make more or less water soluble depending upon the polarity of the original compound. Methyl group - Methylation tends to make xenobiotics more lipid soluble; and sometimes more volatile. Some Specific Phase II Reactions A. Glutathione, Glutathione Conjugation, and Glutathione Depletion Glutathione = GSH = a tripeptide composed of glycine, glutamic acid, and cysteine. Glutathione contains a nucleophilic thiol that usually detoxifies: 1. Electrophilic carbon-containing metabolites. 2. Metabolically produced oxidizing agents. GSH tends to detoxify many reactive intermediates produced by cytochrome P450 enzymes. 3. GSH also intercepts reactive intermediates formed by spontaneous epoxide breakdown. Glutathione-S-transferases Conjugate GSH to numerous substrates. To be a substrate for GSH-S-transferase, the xenobiotic must: 1. Be somewhat hydrophobic. 2. Contain an electrophilic carbon. 3. React nonenzymatically with GSH to some degree. Glutathione Conjugation via Specific Glutathione-S-Transferase Reactions Substitution Reactions (Methyl iodide) Glutathione-S-alkyltransferase: CH3I + GSH CH3 -SG + HI Glutathione-S-aryltransferase: 3,4-Dichloronitrobenzene + GSH GSH loses H and 3,4-Dichloronitrobenzene loses a Cl, forming the-sg conjugate + HCl

4 Glutathione-S-aralkyltransferase: Benzyl chloride + GSH GSH loses H and Benzyl chloride loses a Cl, forming the -SG conjugate + HCl Addition Reactions Glutathione-S-alkenetransferase: Diethyl maleate + GSH Double bond in Diethyl maleate is broken forming the -SG conjugate on the carbon atom previously involved in the C=C bond. Glutathione-S-epoxidetransferase: 1,2 Epoxyethylbenzene + GSH epoxide is broken and -SG conjugate is formed on carbon atom previously involved in the epoxide bond. Further processing of GSH conjugates: GSH conjugates are subsequently cleaved to cysteine derivatives primarily by renal enzymes and then acetylated, thus forming N- acetylcysteine derivatives (also called mercapturic acid conjugates). GSH depletion and depletion or lack of other Phase II processes: Examples of compounds transformed to reactive intermediates and then bound to GSH include: bromobenzene, chloroform, acetaminophen. Such toxicants may deplete GSH. Without adequate GSH (readily depleted, low-capacity system), the reactive metabolites produced by cytochrome P450 enzymes often bind to other "vital cellular constituents" may cause local hepatotoxic or local pulmonary toxic effects. Alternatively, one compound may deplete GSH and another may be converted to a reactive, toxic intermediate. Depletion or lack of function of other conjugation reactions (such as glucuronidation) also can predispose animals to liver (Or Other Organ) damage. An example includes hepatotoxicy in cats caused by phenolics.

5 Glutathione Peroxidase: The enzyme reduces peroxides and lipid peroxides, for example: GSH peroxidase* H2O2 + GSH H2O GSH peroxidase* R-OOH + GSH -ROH (lipid peroxide) normally reactive and self-perpetuating Selenium is a limiting component in glutathione peroxidase structure and thus, in its function. (This is a major reason why Se has antioxidative and anticancer effects). GSH is a cofactor for glutathione peroxidase; and depletion of GSH can therefore diminish the body's ability to defend against lipid peroxidation (see below). B. Lipid Peroxidation Lipid peroxidation of subcellular membranes is an important event in the pathogenesis of many toxicants. This process is believed to be important in the effects of white or yellow phosphorus and those of carbon tetrachloride on the liver and other organs. The double bonds of polyenoic unsaturated fatty acids in the subcellular membranes are susceptible to attack by reactive chemicals (whether the compound is inherently reactive or if it is made reactive in the organism [i.e., by P450]). This attack can result in the loss of a hydrogen atom, which yields a free radical on one of the carbons of the lipid chain. This step is termed the initiation phase of lipid peroxidation. The free radical creates instability of the lipid molecule and these undergo a series of transformations, including shifting of double bonds to form the diene configuration. Then the free radicals react with molecular oxygen to form oxygen peroxy radicals. The peroxy radicals remove a hydrogen from a methylene group of a nearby unsaturated fatty acid, which thereby produces one hydroperoxy-lipid and a new radical.

6 This chain reaction is called propagation phase of lipid peroxidation; and in this process, a linear spread of lipid peroxidation occurs. The hydroperoxides are also unstable and therefore they decompose to form additional free radicals. These reactions tend to continue until they are controlled by cellular defense mechanisms or until all the available unsaturated lipids are depleted. Due to lipid peroxidation in subcellular membranes, there is a loss in structure and function in the damaged organelles. Also, the lipid peroxides themselves and their breakdown products are toxic to the cells. C. Disruption of Hepatic Blood Flow Severe hepatic necrosis from any agent can cause secondary "venoocclusive" lesions (i.e., collapse of the sinusoids). Examples include acute toxicoses from: pyrrolizidine alkaloids, nitrosamines, and especially microcystins and nodularin (from blue-green algae). Ultimately, both the sinusoidal endothelium and the hepatocytes are damaged. By contrast, some toxicants cause coagulative necrosis and the sinusoidal endothelium is spared. An example is acute carbon tetrachloride toxicosis. Note - Some cardiotoxins and other agents that cause right sided heart failure may indirectly cause centrilobular necrosis because of passive congestion of the liver. This should not be confused with primary toxic hepatic injury. D. Carcinogenesis Liver carcinogens include (among others): aflatoxin, certain other hepatotoxic mycotoxins, some pyrrolizidine alkaloids, dialkylnitrosamines ("nitrosamines"), some organochlorine pesticides, certain PCBs, carbon tetrachloride, chloroform, and vinyl chloride. Fumonisin-containing corn (includes mutagens as well as the non-mutagenic but hepatotoxic fumonisin) causes hepatocarcinogenesis. If there is an alteration in DNA (mutagenesis), and if the cell is altered so that it has cancer potential, the process is termed initiation. Subsequently, a change in turnover rate of cells is a common cause of promotion. Initiation followed by promotion can result in development of malignant tumor development (i.e., cancer). Biotransformation of xenobiotics to reactive intermediates increases the likelihood of the formation of adducts to DNA (alkylation of DNA = one form of mutagenesis) or other types of DNA damage (some of which can sometimes comprise an initiation reaction). Hepatotoxicity and necrosis tends to be followed by regeneration, which increases the turnover rate (thereby causing tumor promotion). General Appearance of Animals with Liver Failure Acute Liver Failure. Animals may experience abdominal pain, vomiting, and hypovolemic shock if hemorrhage into liver is extensive and if it occurs over a short enough period of time. Hepatic necrosis may also contribute to shock and occasionally DIC. Animals in acute liver failure may also die from hypoglycemia because of inability of the damaged liver to provide glucose. Animals may also develop secondary hepatoencephalopathy from NH3. Some develop jaundice. Upon palpation, liver pain and enlargement may be evident. Subacute Liver Failure. Intermittent GI upset. Appetite may be reduced. Fecal color is usually reasonably normal at least initially. Poor growth, production, hair coat, etc. Jaundice often. Possible liver pain, enlargement on palpation. Chronic Liver Failure. Recurrent GI upset is common. Chronic weight loss often occurs. Hypoproteinemia may cause fluid accumulation, polyuria if severe.

7 Palpable or (more often) shrunken liver, possible cirrhosis. Jaundice is variable. Secondary photosensitization in some grazing animals. Aspects of Therapy for Liver Toxicosis (General) A. Measures to Limit Toxicant Absorption B. Measures to Support the Animal: 1. Fluids and antiemetics (e.g., Tigan suppositories) to avoid dehydration from vomiting, diarrhea, unwillingness to drink. 2. Measures to alleviate shock if present. 3. Nutritional support-frequent small feedings with low-fat, high-carbohydrate, low-volume but high-quality protein diet (to decrease workload on liver and to decrease deamination that results in formation of ammonia). 4. Oral antibiotics to limit deamination of protein by gut bacteria. 5. Other therapies as per liver failure. 6. Other therapies as per liver failure. Introduction to the Toxicology of the Liver References 1. Arias IM. (1988) The Liver: Biology and Pathobiology. Raven Press. 2. Casarette and Doull. Toxicology. The Basic Science of Poisons. 3. Vancutsem PM, Babish JG. (1996) In vitro and in vivo study of the effects of enrofloxacin on hepatic cytochrome P-450. Potential for drug interactions. Vet Human Toxicol 38: All rights reserved. This document is available on-line at Document No. A