Gregory A. Moore

2,5-Di(tert-butyl)-1,4-benzohydroquinone - a novel inhibitor of liver microsomal Ca2+ sequestration

Treatment of rat liver microsomes with 2,5‐di(tert‐butyl)‐1,4‐benzohydroquinone caused a dose‐related inhibition (K i⋍1 μM) of ATP‐dependent Ca2+ sequestration. This was paralleled by a similar impairment of the microsomal Ca2+ ‐stimulated ATPase activity. In contrast, the hydroquinone failed to induce Ca2+ release from Ca2+ ‐loaded liver mitochondria (supplied with ATP), and inhibited neither the mitochondrial F1F0‐ATPase nor the Ca2+ ‐stimulated ATPase activity of the hepatic plasma membrane fraction. The inhibition of microsomal Ca2+ sequestration was not associated with any apparent alteration of membrane permeability or loss of other microsomal enzyme activities or modification of microsomal protein thiols. These findings suggest that 2,5‐di(tert‐butyl)‐1,4‐benzohydroquinone is a potent and selective inhibitor of liver microsomal Ca2+ sequestration which may be a useful tool in studies of Ca2+ fluxes in intact cells and tissues.

Quinone toxicity in hepatocytes without oxidative stress

The toxicity of quinones is believed to be mediated via redox cycling involving formation of semiquinone radicals which autoxidize to form active oxygen species. However, when the cytotoxicity of benzoquinones was compared using freshly isolated rat hepatocytes, benzoquinones which did not mediate oxidative stress were highly toxic. Thus, the benzoquinone analogs in decreasing order of cytotoxicity were 2-CH3-, 2-Br-, unsubstituted, 2,6-(CH3)2-, 2,5-(CH3)2-, and 2,3,5-(CH3)3-benzoquinone. Cellular thiols were rapidly depleted and glutathione (GSH) was converted to a quinone conjugate without oxidation to glutathione disulfide. No increase in cyanide-resistant respiration was observed and benzoquinone-induced cytotoxicity was not enhanced by inactivation of catalase or glutathione reductase. In contrast, duroquinone [2,3,5,6-(CH3)4-benzoquinone], which stimulated cyanide-resistant respiration and GSH oxidation, was only cytotoxic when catalase or glutathione reductase was inactivated. These results suggest that alkylation and/or oxidative stress may be important mechanisms in the cytotoxicity of benzoquinone derivatives.

On the relationship between Ca2+ efflux and membrane damage during t-butylhydroperoxide metabolism by liver mitochondria

The release of Ca2+ from rat liver mitochondria induced by t‐butylhydroperoxide metabolism is caused by pyridine nucleotide oxidation and precedes alterations in membrane permeability which appear to result from, rather than cause, Ca2+ release.

Quinone toxicity in hepatocytes: Studies on mitochondrial Ca2+ release induced by benzoquinone derivatives

Hepatocyte cytotoxicity caused by substituted benzoquinones was associated with increased cytosolic Ca2+ concentration. p-Benzoquinone-induced hepatotoxicity was enhanced when the hepatocytes were loaded with Ca2+ by preincubation with ATP. A similar order of potency of the substituted benzoquinones in releasing Ca2+ from isolated mitochondria and inducing hepatocyte cytotoxicity was found; in decreasing order, this was 2-Br-, unsubstituted-, 2-CH3-, 2,6-(CH3O)2-, 2,6-(CH3)2-, 2,5-(CH3)2-, 2,3,5-(CH3)3-, and 2,3,5,6-(CH3)4-benzoquinones (duroquinone). The cellular products of quinone metabolism, hydroquinones and glutathione conjugates, did not cause mitochondrial Ca2+ release. Benzoquinone-induced mitochondrial Ca2+ release was preceded by GSH conjugate formation and NAD(P)H oxidation but followed by mitochondrial swelling. With duroquinone, a slow GSH and NADPH oxidation preceded Ca2+ release, but GSH oxidation did not occur with Se-deficient mitochondria lacking glutathione peroxidase activity. Cyanide-insensitive respiration was also observed with duroquinone but not with benzoquinone, suggesting that duroquinone undergoes redox cycling. GSH was depleted by both arylation and oxidation with 2,6-(CH3O)2-, 2,6-(CH3)2-, 2,5(CH3)2-, and 2,3,5-(CH3)3-benzoquinones. Benzoquinone concentrations that totally depleted GSH did not cause Ca2+ release until intramitochondrial NAD(P)H was oxidized. Ca2+ release was also prevented when NAD(P)H generation was stimulated by the presence of isocitrate or 3-hydroxybutyrate. This suggests that mitochondrial Ca2+ release is associated with NAD(P)H oxidation catalyzed by NADH dehydrogenase with benzoquinone or by the glutathione peroxidase-glutathione reductase system with duroquinone.

Role of sulfhydryl groups in benzoquinone-induced Ca2+ release by rat liver mitochondria

Incubation of rat liver mitochondria with benzoquinone derivatives in the presence of succinate plus rotenone has been shown to cause NAD(P)H oxidation followed by Ca2+ release. Further investigation revealed: (1)p-Benzoquinone-induced Ca2+ release was not initiated by a collapse of the mitochondrial membrane potential. However, Ca2+ release and subsequent Ca2+ cycling caused limited increased membrane permeability. (2) p-Benzoquinone-induced NAD(P)H oxidation and Ca2+ release were prevented by isocitrate, 3-hydroxybutyrate, and glutamate but not by pyruvate or 2-oxoglutarate. (3) Inhibition of pyruvate and 2-oxoglutarate dehydrogenases by p-benzoquinone was attributed to arylation of the SH groups of the cofactors, CoA and lipoic acid. Isocitrate dehydrogenase was also inhibited by p-benzoquinone, but the cofactors NAD(P)H and Mn2+ protected the enzyme. Glutamate dehydrogenase was not inhibited by p-benzoquinone. (4) Arylation of mitochondrial protein thiols by p-benzoquinone was associated with an inhibition of state 3 respiration, which was attributed to the inactivation of the phosphate translocase. In contrast, state 4 respiration, and the F1.F0-ATPase and ATP/ADP translocase activities were not inhibited. It was concluded that inhibition of mitochondrial NAD(P)H dehydrogenases by arylation of critical thiol groups will decrease the NAD(P)+-reducing capacity, and possibly lower the NAD(P)H/NAD(P)+ redox status in favor of Ca2+ release.

Cytotoxic effects of postharvest fungicides, ortho-phenylphenol, thiabendazole and imazalil, on isolated rat hepatocytes.

The cytotoxic effects of ortho-phenylphenol (OPP), imazalil (IMZ) and thiabendazole (TBZ) on isolated rat hepatocytes were investigated. Addition of IMZ and OPP to hepatocyte suspensions at a concentration of 0.75 mM resulted in acute cell death, accompanied by depletion of intracellular levels of glutathione and protein thiols. Both compounds rapidly depleted cellular ATP which consistently preceded the cell death. In addition, the cell death caused by IMZ was accompanied by the accumulation of intracellular malondialdehyde, indicating initiation of lipid peroxidation. During a 3-hr incubation period, TBZ did not affect these parameters. In mitochondria isolated from rat liver, IMZ and OPP impaired respiration related to oxidative phosphorylation. Based on these results, the order of toxic potency is IMZ > OPP > TBZ.

Relationship between metabolism and cytotoxicity of ortho-phenylphenol in isolated rat hepatocytes

The relationship between the metabolism and the cytotoxicity of ortho-phenylphenol (OPP) was investigated using isolated rat hepatocytes. Addition of OPP (0.5-1.0 mM) to the hepatocytes caused a dose-dependent toxicity; 1.0 mM OPP caused acute cell death. Pretreatment of hepatocytes with SKF-525A (50 microM, a non-toxic level) enhanced the cytotoxicity of OPP (0.5-1.0 mM). This was accompanied by inhibition of OPP metabolism. Conversely, OPP at low concentrations (0.5 or 0.75 mM) was converted sequentially to phenyl-hydroquinol (PHQ) and then to glutathione (GSH) conjugate in the cells. The concentrations of both metabolites, especially PHQ-GSH conjugate, were very low in hepatocytes exposed to 1.0 mM OPP alone as well as with SKF-525A. The cytotoxicity induced by 0.5 mM OPP was enhanced by the addition of diethylmaleate (1.25 mM) which continuously depletes cellular GSH. In contrast, additions to hepatocytes of 5 mM of dithiothreitol, cysteine, N-acetyl-L-cysteine or ascorbic acid significantly inhibited the cytotoxicity induced by 0.5 mM PHQ; GSH, protein thiols and ATP losses were also prevented. Further, these compounds depressed the rate of PHQ loss in hepatocyte suspensions. These results indicate that the acute cytotoxicity caused by the high dose (1.0 mM) of OPP is associated with direct action by the parent compound; at low doses (0.5-0.75 mM) of OPP, the prolonged depletion of GSH in hepatocytes enhances the cytotoxicity induced by PHQ.

Relationship between mitochondrial dysfunction and toxicity of propyl gallate in isolated rat hepatocytes

The relationship between cytotoxicity and mitochondrial dysfunction caused by propyl gallate (PG) has been studied in hepatocytes freshly prepared from fasted rats. Hepatocytes isolated from fasted (18 h) rats were significantly more susceptible to the toxicity of PG than hepatocytes from fed rats. The addition of fructose (15 mM), an alternative carbohydrate source, to hepatocyte suspensions resulted in the prevention of PG (1 mM)-induced cell killing accompanied by decrease in intracellular ATP loss during a 3 h-incubation period. Despite this, fructose did not completely prevent an abrupt loss of intracellular glutathione caused by PG, but effectively inhibited the loss of protein thiol levels. Fructose elicited a concentration (0.5-20mM)-dependent protection against the cytotoxicity of 1.5 mM PG. The incubation of hepatocytes with sodium azide (4 mM), an inhibitor of oxidative phosphorylation, enhanced the toxicity induced by PG (1 mM), but coincubation with fructose delayed the onset of toxicity. Neither azide alone nor fructose plus azide did affect the cell viability during the incubation period. Furthermore, the addition of 2 mM salicylamide, nontoxic to hepatocytes during the incubation period, enhanced PG (1 mM)-induced cytotoxicity and decreased the loss of free PG. These results indicate that the onset of cytotoxicity caused by PG may depend on the intracellular energy status and that mitochondria are critical target for the compound. In addition, the toxicity caused by the inhibition of mitochondrial ATP synthesis is related to the concentration of PG remaining in cell suspensions.

Cytotoxicity of orthro-phenylphenol in isolated rat hepatocytes

Abstract The effects of otrho -phenylphenol (OPP) and its metabolites, phenyl-hydroquinol (PHQ) and phenyl-benzoquinone (PBQ), on isolated rat hepatocytes were investigated. Addition of OPP (0.5–1.0 mM) to cells caused a dose-dependent cell death accompanied by the depletion of intracellular levels of ATP, glutathione (GSH) and protein thiols. GSH loss correlated with the formation of oxidized GSH. In addition, PHQ and especially PBQ (both at 0.5 mM) resulted in acute cell death with rapid depletion of ATP, GSH and protein thiols, and further low doses of PBQ (10–50 μM) elicited serious impairment of mitochondrial functions related to oxidative phosphorylation and Ca fluxes in isolated liver mitochondria. These results indicate that mitochondria are a target for these compounds and that OPP is itself toxic to hepatocytes even when metabolism is inhibited. The loss of cellular GSH and protein thiols accompanied by the impairment of mitochondrial function may be the main mechanisms of cytotoxicity induced by OPP and its metabolites.

Propyl gallate-induced DNA fragmentation in isolated rat hepatocytes

Abstract Incubation of isolated rat hepatocytes with propyl gallate (PG) at concentrations of ≥1 mM induced cell killing, whereas PG at ≤0.5 mM did not cause cell death during a 3-h incubation. PG at ≥0.5 mM elicited the ladder formation of soluble low-molecular weight DNA fragments with integer multiples of approximately 180 bp and specific nuclear DNA cleavages detected cytopathologically by labeling of a digoxigenin-nucleotide complex to new 3′-OH ends. Both of these PG-induced changes observed in hepatocytes are characteristic features of apoptosis. In contrast, the pretreatment of N-acetylcysteine (4 mM), a precursor of intracellular glutathione (GSH) and antioxidant, prevented PG (0.5 mM)-induced formation of soluble DNA fragments and loss of cellular GSH, ATP, and formation of blebbing. These results suggest that when the concentration of PG is decreased, the effects of PG on hepatocytes change from acute necrotic to apoptotic mode, and that the onset of DNA fragmentation is associated with GSH depletion.