Kathryn T. Knecht

The role of gut-derived bacterial toxins and free radicals in alcohol-induced liver injury.

Previous research from this laboratory using a continuous enteral ethanol (EtOH) administration model demonstrated that Kupffer cells are pivotal in the development of EtOH‐induced liver injury. When Kupffer cells were destroyed using gadolinium chloride (GdCl3) or the gut was sterilized with polymyxin B and neomycin, early inflammation due to EtOH was blocked. Anti‐tumour necrosis factor (TNF)‐α antibody markedly decreased EtOH‐induced liver injury and increased TNF‐mRNA. These findings led to the hypothesis that EtOH‐induced liver injury involves increases in circulating endotoxin leading to activation of Kupffer cells. Pimonidazole, a nitro‐imidazole marker, was used to detect hypoxia in downstream pericentral regions of the lobule. Following one large dose of EtOH or chronic enteral EtOH for 1 month, pimonidazole binding was increased significantly in pericentral regions of the liver lobule, which was diminished with GdCl3. Enteral EtOH increased free radical generation detected with electron spin resonance (ESR). These radical species had coupling constants matching α‐hydroxyethyl radical and were shown conclusively to arise from EtOH based on a doubling of the ESR lines when 13C‐EtOH was given. α‐Hydroxyethyl radical production was also blocked by the destruction of Kupffer cells with GdCl3. It is known that females develop more severe EtOH‐induced liver injury more rapidly and with less EtOH than males. Female rats on the enteral protocol exhibited more rapid injury and more widespread fatty changes over a larger portion of the liver lobule than males. Plasma endotoxin, ICAM‐1, free radical adducts, infiltrating neutrophils and transcription factor NFκB were approximately two‐fold greater in livers from females than males after 4 weeks of enteral EtOH treatment. Furthermore, oestrogen treatment increased the sensitivity of Kupffer cells to endotoxin. These data are consistent with the hypothesis that Kupffer cells participate in important gender differences in liver injury caused by ethanol.

10) In Vivo Detection of Radical Adducts by Electron Spin Resonance

[10] In Vivo Detection of Radical Adducts by Electron Spin Resonance By RONALD P. MASON and KATHRYN T. KNECHT Introduction Spin trapping is the most direct method for the detection of highly reactive free radicals in vivo. With this electron spin resonance (ESR) technique, a higher steady-state concentration of free radicals (as radical adducts) is achieved, which can overcome the sensitivity problem inherent in the detection of free radicals in biological systems. ~-4 Because the concentration of endogenous radicals in biological tissues is generally near the sensitivity limit of ESR spectroscopy, the spin-trapping technique is not limited by background signals. The technique of spin trapping involves the addition of a primary free radical across the double bond of a diamag- netic compound (the spin trap) to form a radical adduct more stable than the primary free radical. This technique involves the indirect detection of primary free radicals that cannot be directly observed by conventional ESR due to low steady-state concentrations or to very short relaxation times, which lead to very broad lines. All of the reported in vivo spin trapping investigations have used the nitrone spin traps phenyl-tert-butylnitrone (PBN), o~-2,4,6-trimethoxy- PBN [(CH30)3PBN], and 5,5-dimethyl-l-pyrroline N-oxide (DMPO). Al- though the major difficulty of the spin-trapping technique in vivo is the mere detection of a radical adduct, other factors must be considered when spin traps are administered in vivo. For example, spin traps may affect the experimental system by inhibiting (or stimulating) enzymes or by producing toxicity. The latter possibility has not seemed to affect in vivo work to date. There are several recent reviews that address in vivo applica- tions of the spin trapping technique. ~-4 These reviews also discuss the effects of spin traps on enzymes and more general problems of spin trap- ping such as artifacts and ambiguities in the assignment of radical adduct structure. This chapter concentrates on the methodology of sample han- dling for in vivo experiments.

Role of Kupffer Cells, Endotoxin and Free Radicals in Hepatotoxicity Due to Prolonged Alcohol Consumption: Studies in Female and Male Rats

Alcohol ingestion results in increases in the release of endotoxin from gut bacteria or membrane permeability of the gut to endotoxin, or both. Female rats are more sensitive to these changes. Elevated levels of endotoxin activate Kupffer cells to release substances such as eicosanoids, tumor necrosis factor-alpha and free radicals. Prostaglandins increase oxygen uptake and most likely are responsible for the hypermetabolic state in the liver. The increase in oxygen demand leads to hypoxia in the liver, and on reperfusion, alpha-hydroxyethyl free radicals are formed that lead to tissue damage in oxygen-poor pericentral regions of the liver lobule.

Role of free radicals in failure of fatty livers following liver transplantation and alcoholic liver injury.

A critical factor in the extreme shortage of livers for transplantation is frequent failure due to primary non-function of ethanol-induced fatty livers when employed as donor organs (Starzl et all., 1988). Although fatty livers due to ethanol are frequently available in the donor pool since a major source of liver grafts is brain-dead victims of accidents involving alcohol (Butts & Patetta, 1988), surgeons must sometimes discard these organs because of high lipid content. Thus, an examination of the relationship between alcohol, fatty liver, and graft failure following liver transplantation could lead to a larger donor pool of usable organs. With this as a goal, we examined the connection between Kupffer cells and reperfusion injury in ethanol-induced fatty liver since Kupffer cells, which are activated following cold storage and reperfusion (Thurman, Cowper, Marzi, Currin, & Lemasters, 1988), have been implicated in primary non-function. Kupffer cells, when activated, release toxic mediators including cytokines and eicosanoids (Decker, 1990) which may play a role in reperfusion injury following transplantation.

Thiyl Free Radical Metabolites of Thiol Drugs and Glutathione

Lactoperoxidase, a prototypical mammalian peroxidase, is part of an antimicrobial system found in secreted fluids.1 It is believed to act by oxidation of thiocyanate.2 Not surprisingly, it will also metabolize thiol moieties, oxidizing them to free radical intermediates. Thiol compounds are important in pharmacology and toxicology, and we have examined the production of highly reactive thiyl free radicals by this mechanism.