Journal of Pharmacology and Experimental Therapeutics TOXICOLOGY

[TOXICOLOGY] Gene Expression Profiles in Livers from Diclofenac-Treated Rats Reveal Intestinal Bacteria-Dependent and -Independent Pathways Associated with Liver Injury

2008-11-12

Diclofenac (DCLF) is a nonsteroidal anti-inflammatory drug that is associated with idiosyncratic adverse drug reactions in humans. Previous studies revealed a crucial role for intestine-derived bacteria and/or lipopolysaccharide (LPS) in DCLF-induced hepatotoxicity. We further explored this mechanism by conducting gene expression analysis of livers from rats treated with a hepatotoxic dose of DCLF (100 mg/kg) with or without oral antibiotic pretreatment. Genes for which expression was altered by DCLF were divided into two groups: genes with expression altered by antibiotic treatment and those unaffected by antibiotics. The former group of genes represented the ones for which DCLF-induced alterations in expression depended on intestinal bacteria. The expression of the latter group of genes was probably changed by direct effect of DCLF rather than by intestinal bacteria. Functional analysis of genes in the former group revealed LPS-related signaling, further suggesting a role for bacterial endotoxin in the liver injury. Functional analysis of genes in the latter group revealed changes in signaling pathways related to inflammation, hypoxia, oxidative stress, the aryl hydrocarbon receptor, and peroxisome proliferator-activated receptor . Neutrophil depletion failed to protect from DCLF-induced hepatotoxicity, suggesting that intestinal bacteria contribute to liver injury in a neutrophil-independent manner. Hypoxia occurred in the livers of rats treated with DCLF, and hypoxia in vitro rendered hepatocytes sensitive to DCLF-induced cytotoxicity. These results support the hypothesis that intestinal bacteria are required for DCLF-induced hepatotoxicity and suggest that hypoxia plays an important role in the pathogenesis.

[TOXICOLOGY] The Molecular Mechanism of "Ryegrass Staggers," a Neurological Disorder of K+ Channels

2008-11-12

"Ryegrass staggers" is a neurological condition of unknown mechanism that impairs motor function in livestock. It is caused by infection of perennial ryegrass pastures by an endophytic fungus that produces neurotoxins, predominantly the indole-diterpenoid compound lolitrem B. Animals grazing on such pastures develop uncontrollable tremors and become uncoordinated in their movement. Lolitrem B and the structurally related tremor inducer paxilline both act as potent large conductance calcium-activated potassium (BK) channel inhibitors. Using patch clamping, we show that their different apparent affinities correlate with their toxicity in vivo. To investigate whether the motor function deficits produced by lolitrem B and paxilline are due to inhibition of BK ion channels, their ability to induce tremor and ataxia in mice deficient in this ion channel (Kcnma1^-/-) was examined. Our results show that mice lacking Kcnma1 are unaffected by these neurotoxins. Furthermore, doses of these substances known to be lethal to wild-type mice had no effect on Kcnma1^-/- mice. These studies reveal the BK channel as the molecular target for the major components of the motor impairments induced by ryegrass neurotoxins. Unexpectedly, when the response to lolitrem B was examined in mice lacking the β4 BK channel accessory subunit (Kcnmb4^-/-), only low-level ataxia was observed. Our study therefore reveals a new role for the accessory BK β4 subunit in motor control. The β4 subunit could be considered as a potential target for treatment of ataxic conditions in animals and in humans.

[TOXICOLOGY] Dehydroalanine Analog of Glutathione: An Electrophilic Busulfan Metabolite That Binds to Human Glutathione S-Transferase A1-1

2008-11-12

Elimination of hydrogen sulfide from glutathione (GSH) converts a well known cellular nucleophile to an electrophilic species, -glutamyldehydroalanylglycine (EdAG). We have found that a sulfonium metabolite formed from GSH and busulfan undergoes a facile β-elimination reaction to give EdAG, which is an ,β-unsaturated dehydroalanyl analog of GSH. EdAG was identified as a metabolite of busulfan in a human liver cytosol fraction. EdAG condenses with GSH in a Michael addition reaction to produce a lanthionine thioether [(2-amino-5-[[3-[2-[[4-amino-5-hydroxy-5-oxopentanoyl]amino]-3-(carboxymethylamino)-3-oxopropyl]sulfanyl-1-(carboxymethylamino)-1-oxopropan-2-yl]amino]-5-oxopentanoic acid); GSG], which is a nonreducible analog of glutathione disulfide. EdAG was less cytotoxic than busulfan to C6 rat glioma cells. GSH and EdAG were equally effective in displacing a glutathione S-transferase (GST) isozyme (human GSTA1-1) from a GSH-agarose column. The finding of an electrophilic metabolite of GSH suggests that alteration of cellular GSH concentrations, irreversible nonreducible glutathionylation of proteins, and interference with GST function may contribute to the toxicity of busulfan.

[TOXICOLOGY] Dual Pathway Activated by tert-Butyl Hydroperoxide in Human Airway Anion Secretion

2008-10-17

We analyzed the mechanisms underlying the ion transport induced by tert-butyl hydroperoxide (t-BOOH), a membrane-permeant oxidant that has been widely used as a model of oxidative stress, in human airway epithelial cells (Calu-3). We found that t-BOOH induced a short-circuit current that was composed of two distinct components, a peaked component (PC) and a sustained component (SC). Both components were reduced by the presence of H-89 (N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline) [10 µM, a protein kinase A (PKA) inhibitor] and clofilium (100 µM, a cAMP-dependent K⁺ channel inhibitor) but not by charybdotoxin (50 nM, a human intermediate conductance Ca²⁺-activated K⁺ channel inhibitor), suggesting that both PC and SC were generated through a common PKA-dependent/Ca²⁺-independent pathway. Notwithstanding, analyses of the physiological properties revealed that PC and SC were attributable to different pathways. PC, but not SC, was correlated with apical membrane Cl^- conductance and was inhibited by the cyclooxygenase (COX)-2 inhibitor NS-398 (N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide; 10 µM). In contrast, SC, but not PC, was composed of a component sensitive to bumetanide (50 µM), an inhibitor of the basolateral Na⁺-K⁺-2Cl^- cotransporter (NKCC1), and was abolished by the cytoskeleton dysfunction induced by cytochalasin D (10 µM) and (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane carboxamide (Y-27632; 20 µM). Collectively, t-BOOH induces PKA-related anion secretion through two independent pathways: rapid activation of apical anion efflux through a COX-2-dependent/cytoskeleton-independent pathway and relatively delayed activation of NKCC1 for basolateral anion uptake through a COX-2-independent/cytoskeleton-dependent pathway.