A study on the mechanisms of acrolein and diaziquone-induced cytotoxicity in isolated hepatocytes

Silva, Jose Manuel (1988) A study on the mechanisms of acrolein and diaziquone-induced cytotoxicity in isolated hepatocytes. Masters thesis, Memorial University of Newfoundland.

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    Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission.
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Abstract

Acrolein may be a causative agent of hepatic necrosis during the biotransformation of allyl alcohol. It has also been implicated in hemorrhagic cystitis of the bladder induced by the antitumor agent cyclophosphamide. The mechanism of cytotoxicity is believed to involve DNA and protein alkylation. AZQ, 2,5-diazirinyl-3,6-bis(carboethoxyamine)-1,4-benzoquinone, is a lipid soluble antitumor agent that has successfully completed Phase II drug trials. The mechanism of cytotoxicity is believed to involve DNA cross-linking. However, the following evidence using isolated hepatocytes suggests that the cytotoxicity may involve oxidative stress. Isolated rat hepatocytes were prepared and incubated with acrolein or AZQ. -- A) Acrolien -- 1. Hepatocyte cytotoxicity induced by acrolein ensued following depletion of cellular GSH. No GSSG was formed. -- 2. The addition of the reducing agent DTT to hepatocytes preincubated with acrolein prevented cytotoxicity. -- 3. Malondialdehyde, a lipid peroxidation metabolite, was formed during either acrolein or allyl alcohol incubation with hepatocytes. -- 4. Although malondialdehyde formation could be prevented by the presence of antioxidants and desferrioxamine in the hepatocyte incubate, cytotoxicity was only delayed. -- 5. The xanthine oxidase inhibitor, allopurinol, did not inhibit acrolein induced lipid peroxidation of protect against cytotoxicity. -- 6. Acrolein readily induced Ca²⁺ release by isolated energized mitochondria. Intramitochondrial NAD(P)H was not affected indicating that oxidative stress was not involved. The reducing agent DTT could prevent the release. A reversible alkylation of proteins involved in Ca²⁺ release could be the mechanism involved. -- These results suggest two mechanisms of cytotoxicity induced by acrolein. One involving lipid peroxidation and another slower mechanism involving alkylation. -- B) AZQ -- 1. Hepatocyte cytotoxicity induced by AZQ ensued following depletion of GSH. -- 2. Incubation of AZQ with isolated rat hepatocytes stimulated cyanide-resistant respiration and stoichiometrically oxidized GSH to GSSG. The GSSG levels remained high, as GSSG was not reduced back to GSH. This was found to be the result of reversible inactivation of GSSG reductase. -- 3. No malondialdehyde was formed. -- 4. If the hepatocytes were compromised with azide to inhibit catalase, cytotoxicity was increased 10-fold. -- 5. AZQ readily induced Ca²⁺ release by isolated energized mitochondria. Ascorbate markedly enhanced the effectiveness of AZQ and catalase delayed the release. H₂O₂ formed by redox cycling may therefore cause mitochondrial Ca²⁺ release. -- These results suggest that under aerobic conditions AZQ participates in futile redox cycling and oxygen activation. The H₂O₂ formed my mediate cell death in compromised cells.

Item Type: Thesis (Masters)
URI: http://research.library.mun.ca/id/eprint/4026
Item ID: 4026
Additional Information: Bibliography: leaves 88-95.
Department(s): Science, Faculty of > Biochemistry
Date: 1988
Date Type: Submission
Library of Congress Subject Heading: Drugs--Toxicology; Liver cells

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