Cytogenetic effects of propyl gallate in CHO-K1 cells.

We investigated whether propyl gallate (PG) can induce sister-chromatid exchanges (SCEs) and chromosomal aberrations (CAs) in CHO-K1 cells. In the absence of an exogeneous metabolizing system, treatments with 0.25-1.5mM PG in plugged flasks for 3h resulted in increases in SCEs, CAs, and endoreduplications (ERDs), which were followed by an increase in the percentage of cells showing cell-cycle delay. At the end of the treatment, a decrease in PG concentration and production of PG dimer and ellagic acid (EA) in the medium were detected, indicating that PG had autoxidized. EA, an oxide of PG, was not genotoxic even at 0.3mM, the maximum concentration soluble in the medium. Several oxygen radical scavengers (superoxide dismutase (SOD), catalase, glutathione and o-phenanthroline (OP)) and an inhibitor of catalase activity (3-amino-1,2,4-triazole (AT)), did not significantly influence PG genotoxicity. When PG autoxidation was suppressed by low pH (6.8) or a 5% CO(2) atmosphere, cell-cycle delay intensified and induction of SCEs and CAs occurred even at the lowest PG dose (0.1mM). When PG (0.5mM) was assayed in the presence of S9 (1.5-9%), gallic acid (GA), a metabolite of PG, was generated in direct proportion to the S9 concentration, while cell-cycle delay and genotoxic effects varied inversely with S9 concentration at the levels over 3%. GA also autoxidized and at >or=0.5mM it induced SCEs. Both catalase and AT suppressed the induction of SCEs by GA or inhibited cell proliferation, indicating that H(2)O(2) participated in the effects. In conclusion, PG in the presence or absence of S9 can induce SCEs, CAs, and ERDs, and the oxides, metabolites and oxygen-free radicals generated during the treatment are partly responsible for these effects.

Metabolism and cytotoxicity of bisphenol A and other bisphenols in isolated rat hepatocytes.

The relation between the metabolism and the cytotoxic effects of bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl)propane) has been studied in freshly isolated rat hepatocytes and isolated hepatic mitochondria. The incubation of hepatocytes with BPA (0.25-1.0 mM) elicited a concentration- and time-dependent cell death, accompanied by losses of intracellular ATP and total adenine nucleotide pools. BPA at a low-toxic level (0.25 mM) in the hepatocyte suspensions was rapidly converted to its major conjugate, BPA-glucuronide, and other minor products without marked loss of cell viability, although at a toxic level (0.5 mM), more than 65% of the compound presented in an unaltered form 2 h after the incubation. Addition of salicylamide (2 mM), non-toxic to hepatocytes during the incubation period, enhanced BPA-induced cytotoxicity and reduced the loss of BPA and the formation of BPA-glucuronide. The addition of BPA to isolated hepatic mitochondria caused a concentration (0-0.5 mM)-dependent increase in the rate of state 4 oxygen consumption in the presence of an FAD-linked substrate (succinate), indicating an uncoupling effect, whereas the rate of state 3 oxygen consumption was inhibited by BPA. Further, the addition of BPA (0.25 mM) reduced state 3 respiration with NAD+-linked substrates (pyruvate plus malate) and/or with the FAD-linked substrate, whereas state 3 respiration with ascorbate plus tetramethyl-p-phenylenediamine (cytochrome oxidase-linked respiration) was not significantly affected by BPA. A comparative study of the toxic effects of BPA and some bisphenols on cell viability (at 1.0 mM) and mitochondrial respiration (at 0.25 mM) revealed that 4,4'-(1,2-diethyl-1,2-ethenediyl)bisphenol (diethylstilbestrol) was more toxic than BPA, followed by 4,4'-methylenediphenol and 4,4'-biphenol. These results indicate that the onset of cytotoxicity caused by BPA may depend on the intracellular energy status and that mitochondria are important targets of the compound. The toxicity caused by the inhibition of ATP synthesis may be related to the concentration of unmetabolised free BPA remaining in the cell suspensions. In addition, the toxic potency of bisphenols to hepatocytes and mitochondria depends on the relative elongation and/or molecular size of the hydrocarbon bridge between the phenolic groups.

Metabolism and toxicity of benzophenone in isolated rat hepatocytes and estrogenic activity of its metabolites in MCF-7 cells.

The metabolism and cytotoxicity of benzophenone and estrogenic activity of its metabolites have been studied in freshly isolated rat hepatocytes and cultured MCF-7 human breast cancer cells, respectively. The incubation of hepatocytes with benzophenone (0.25-1.0 mM) elicited a concentration- and time-dependent cell death, accompanied by loss of intracellular ATP and depletion of adenine nucleotide pools. Benzophenone at a low-toxic level (0.25 mM) in the hepatocyte suspensions was converted to benzhydrol, p-hydroxybenzophenone and its sulfate conjugate, without marked loss of cell viability. The amounts of benzhydrol and sulfate conjugate increased with time. In contrast, addition of 2,6-dichloro-4-nitrophenol (an inhibitor of sulfotransferase; 0.1 mM), nontoxic to hepatocytes during the incubation period, enhanced benzophenone-induced cytotoxicity, and this effect was accompanied by a decrease in the formation of sulfate conjugate and increase in the amount of free p-hydroxybenzophenone. In another experiment, MCF-7 cells, estrogen-responsible breast cancer cells were cultured in estradiol free medium and then exposed to 10 nM-500 microM benzophenone or its metabolites for 6 days. Although at higher concentrations all the compounds were toxic, except for benzophenone and benzhydrol, 10-100 microM p-hydroxybenzophenone significantly increased cell proliferation. These results indicate that benzophenone is enzymaticaly converted to benzhydrol, p-hydroxybenzophenone and its sulphate conjugate in rat hepatocytes. Even if there is less free p-hydroxybenzophenone than benzhydrol and sulfate conjugate in hepatocyte suspensions, p-hydroxybenzophenone itself acts as a weak xeno-estrogen on MCF-7 cells.

Effects of pilsicainide and propafenone on vagally induced atrial fibrillation: role of suppressant effect in conductivity.

The effects of pilsicainide on vagally induced atrial fibrillation and on electrophysiological parameters were compared with those of propafenone in alpha-chloralose-anesthetized dogs. Conduction velocity, effective refractory period, wavelength, averaged atrial fibrillation cycle length and activation sequence in the right atrial free wall were determined before and after drug administration. Pilsicainide (2 mg/kg/5 min and 3 mg/kg/h)(n=10) or propafenone (2 mg/kg/15 min and 4 mg/kg/h)(n=10) was intravenously infused during stable atrial fibrillation sustaining > 30 min. Pilsicainide terminated atrial fibrillation in nine dogs, while propafenone did so in three (p < 0.01). After the drug, conduction velocity was suppressed more in the pilsicainide than in the propafenone group(p < 0.01). There was no difference in effective refractory period after drug between the two groups. Mean wavelength was prolonged from 46.0 to 70.4 mm in the pilsicainide group and from 45.0 to 110.8 mm in the propafenone (p < 0.01 vs. pilsicainide). Activation mapping during atrial fibrillation showed Type II or III atrial fibrillation as previously defined [Konings, K.T.S., Kirchhof, C.J.H.J., Smeets, J.R.L.M., Wellens, H.J.J., Penn, O.C., Allessie, M.A., 1994. High-density mapping of electrically induced atrial fibrillation in humans. Circulation. Vol. 89, pp. 511-521.] before the drug, and changed to Type I before atrial fibrillation termination. Thus, pilsicainide was more effective to terminate vagally induced atrial fibrillation than was propafenone despite a greater effect of propafenone than of pilsicainide on wavelength. In this canine atrial fibrillation model, the suppression of conduction velocity may play an important role in changing the activation pattern of atrial fibrillation and thus, terminating atrial fibrillation.

Acute myocardial infarction and increased soluble intercellular adhesion molecule-1: a marker of vascular inflammation and a risk of early restenosis?

BACKGROUND: Plasma levels of soluble intercellular adhesion molecule-1 (sICAM-1) have been shown to predict activities of inflammatory disorders and malignancies. However, it is unknown whether the plasma level of sICAM-1 is increased in patients with acute myocardial infarction (AMI) with coronary intervention and whether the levels have any diagnostic or predictive values for vascular disease activity in patients with AMI. METHODS: We prospectively observed the time course of the plasma sICAM-1 levels in 20 patients with AMI whose infarct-related coronary artery was successfully recanalized by emergency balloon angioplasty. sICAM-1 was measured by enzyme-linked immunoassay. RESULTS: At admission, 48 hours, 1 week, and 2 weeks after angioplasty, sICAM-1 levels were significantly elevated in patients who had early (3 weeks) restenosis develop compared with those who did not (p < 0.05). At the other time points examined, there was a tendency of higher sICAM-1 levels in patients with than without restenosis (0.06 < p < 0.09). The relation of sICAM-1 levels and total white blood cell counts, neutrophil counts, or numbers of diseased major coronary artery branches was not statistically significant. CONCLUSIONS: A persistent increase in plasma sICAM-1 levels may indirectly implicate vascular inflammation, which could predict the risk of early coronary restenosis after emergency angioplasty in patients with AMI. Hence, measurements of sICAM-1 in patients with AMI would serve as a potentially useful predictor of the risk of early postangioplasty restenosis.

Influence of chronic nitric oxide inhibition of coronary blood flow regulation: a study of the role of endogenous adenosine in anesthetized, open-chested dogs.

The effect of chronic inhibition of endothelium-derived nitric oxide (NO) synthesis on the regulation of coronary blood flow (CBF) is yet to be elucidated. A chronic canine model of inhibited NO synthesis was created and the role of adenosine in the regulation of coronary blood flow in this model was examined. Dogs were fed a diet supplemented with 40 mg/kg per day N(G)-nitro-L-arginine methyl ester (L-NAME group, n=8) or a regular diet without L-NAME supplementation (control group, n=8) for 4 weeks. The experiments were performed in an anesthetized, open-chest state and the results were compared in the L-NAME and control groups. Chronic L-NAME treatment significantly increased arterial pressure. Neither basal CBF in the left anterior descending artery nor heart rate differed between the L-NAME and control groups. In the L-NAME group, the response of CBF to intracoronary acetylcholine and adenosine was blunted, but that to glyceryl trinitrate was not. In addition, myocardial reactive hyperemia following 20 sec coronary occlusion was blunted in the L-NAME group. During atrial pacing at a rate 60 beats/min faster than the sinus rate, CBF increased to a similar degree in the L-NAME and control groups, and systolic wall thickening (SWT) changed similarly in both groups. Intracoronary 8-phenyltheophylline (8-PT), an adenosine receptor blocker, decreased basal CBF in the L-NAME group but not in the control group. In the L-NAME group, pacing-induced increase in CBF was abolished and SWT deteriorated after 8-PT administration. Basal myocardial adenosine release was significantly increased in the L-NAME group compared with the control group. It is concluded that in anesthetized, open-chest dogs with chronic inhibition of NO synthesis, adenosine may play a compensatory role in the regulation of coronary blood flow, as concomitant blockade of adenosine causes deterioration of coronary circulation and cardiac function.

Increased plasma soluble intercellular adhesion molecule-1 levels in patients with acute myocardial infarction.

Intercellular adhesion molecule-1 (ICAM-1) is a major ligand for 2 members of the CD18 family of leukocyte integrin adhesion molecules and mediates adhesion between leukocytes and stimulated endothelial cells. We examined plasma soluble ICAM-1 (sICAM-1) levels in 30 patients with acute myocardial infarction (AMI) within 6 h of symptom onset, 21 patients with unstable angina (UA), 35 patients with stable exertional angina (SEA) and 21 control subjects. Plasma sICAM-1 levels (ng/ml) were significantly higher in both the acute and chronic phases of AMI and in the UA group than in the SEA and the control groups (195 +/- 14, 198 +/- 16 in the acute and chronic phases of AMI, 188 +/- 11 in the UA group vs 142 +/- 7 in the SEA group, 141 +/- 10 in the control group, p < 0.01). Plasma sICAM-1 levels were significantly higher in AMI patients when preceded by unstable angina than when not preceded by unstable angina at any point over the time course except 1 week after admission (p < 0.01 vs admission, 12 h, 2 days, 3 days, 5 days, 2 weeks, 3 weeks. p < 0.05 vs 24 h). These results suggest that the increase in sICAM-1 is associated with repeated episodes of myocardial ischemia and reperfusion not leading to myocardial necrosis. The increase in sICAM-1 may play an important role as an inflammatory component in the pathogenesis of the ischemic myocardium.

Impairment of coronary blood flow regulation by endothelium-derived nitric oxide in dogs with alloxan-induced diabetes.

Diabetes mellitus is a major cause of ischemic coronary artery disease. Endothelial dysfunction is implicated in the pathogenesis of diabetic vascular disease. To examine coronary blood flow (CBF) regulation with endothelium-derived nitric oxide (EDNO) in the diabetic state, we compared the effects of both acetylcholine (ACh) and adenosine (Ado) on left circumflex coronary artery (LCX) blood flow in 12 vehicle-treated and 21 dogs made diabetic with alloxan anesthetized with pentobarbital. All dogs were pretreated with aspirin to inhibit endogenous prostaglandins. None of the hemodynamic parameters were significantly different in the two groups. The percent change in coronary vascular resistance (CVR) after ACh (100 ng/kg) infusion was significantly attenuated in diabetic dogs (-56.5 +/- 1.4%) as compared with vehicle-treated dogs (-64.5 +/- 1.2%) (p < 0.01), whereas the effect of Ado (1 microgram/kg) was not different between the two groups (-71.1 +/- 1.5% in vehicle, -67.0 +/- 1.3% in diabetes). After infusion of incremental doses of NG-nitro-L-arginine methyl ester (L-NAME) 10(-5)-10(-3)M, the effect of ACh was progressively inhibited in both groups and was different no longer between the two groups after the maximal dose. L-Arginine (L-ARG), but not D-ARG, significantly restored the effect of ACh in diabetic dogs but did not affect vehicle-treated dogs. The effect of Ado did not change after L- and D-ARG administration. Cu, Zn-superoxide dismutase (Cu, Zn-SOD) had no effect on any of the effects of ACh and Ado in diabetic dogs. Regulation of CBF with EDNO is impaired in dogs with alloxan-induced diabetes, and this impairment is partially restored by L-ARG.

Cytogenetic effects of piperonyl butoxide and safrole in CHO-K1 cells.

Recently, hepatocarcinogenicity in rats and mice was reported with regard to the methylenedioxyphenyl compound, piperonyl butoxide (PB), which is used as a synergist for pyrethrins and related insecticides. Induction of sister-chromatid exchanges (SCEs) and chromosomal aberrations (CAs) due to PB were investigated using CHO-K1 cells with or without rat liver S9 fraction (S9); at the same time, the effects of safrole (SF), a methylenedioxyphenyl compound and a weak hepatocarcinogen, were also examined. PB (0.25 and 0.3 mM) and SF (0.8 mM) caused a slight but significant increase in SCEs followed by a cell-cycle delay in the 3-h treatment without S9. In the presence of S9 (4.5%), the cytotoxicity of PB or SF was weakened greatly or slightly, the top dose capable of cell division was raised to 0.6 mM (2-fold) or 1 mM, respectively. PB with S9 induced SCE at doses of 0.4 and 0.5 mM, and caused endoreduplications (ERDs, 7%) at a dose of 0.6 mM, while SF caused a dose-related significant increase in SCE at all doses used (0.4-1 mM) with S9. Genotoxicity of the metabolites of PB or SF was cleared by changing the dose of S9 (1.5-9%) while holding the dose of each chemical constant. In the case of SF (0.6 mM), induction of SCE, ERD and cell-cycle delay intensified almost in a dose-effect relationship, and CAs and a high level of ERD (14%) were caused by a 9% dose of S9. The concentration of unchanged SF in the incubated medium was certainly in inverse proportion to the dose of S9. This strongly suggests that the metabolites of SF are genotoxic. In the case of PB (0.3 mM), no positive responses were produced in the cultures, even with a high level of S9, though the amount of unchanged PB left in the incubated medium was very slight. This indicates that the metabolites of PB may not be genotoxic. In conclusion, PB and SF are possible to somewhat induce SCE at high dose(s) in the absence of S9, and the genotoxic effects of SF are more intensified in the presence of S9 than in its absence, while PB is probably no genotoxic in the presence of sufficient metabolic activation.

Effects of angiotensin-converting enzyme inhibitor alacepril in patients with stable effort angina during chronic isosorbide dinitrate treatment.

Nitrate tolerance has been reported to be reversed by certain types of angiotensin-converting enzyme (ACE) inhibitors. We examined whether alacepril, a new long-acting oral ACE inhibitor, has beneficial effects against exercise-induced angina in patients with stable effort angina after substantial isosorbide dinitrate (ISDN) treatment. Thirteen men with stable effort angina were treated with oral ISDN (80 mg/d) for >3 weeks. After this period, efficacy of single oral administration of either alacepril (50 mg) or its placebo on exercise-induced angina and electrocardiographic changes was examined by treadmill exercise test in a double-blind crossover design. Alacepril significantly improved the exercise duration by 9.1% (p=0.03), the time to 1 mm ST-segment depression by 19% (p<0.01), and the maximal ST-segment depression by 33% (p=0.015) compared with placebo. Alacepril did not significantly alter the rate-pressure product, a marker of myocardial oxygen demand, during exercise test compared with placebo. Plasma renin activity was significantly increased (p<0.05) after administration of alacepril, indicating that alacepril significantly blocked ACE activity in our patients. In conclusion, a single oral administration of the ACE inhibitor alacepril (50mg) elicited beneficial effects against exercise-induced myocardial ischemia in patients with stable effort angina during chronic nitrate treatment. These effects may be mediated by increased coronary blood flow.

Enhancement of myocardial reactive hyperemia with manganese-superoxide dismutase: role of endothelium-derived nitric oxide.

OBJECTIVE: To test the hypothesis that superoxide radicals generated during myocardial ischemia and reperfusion influence reactive hyperemia (RH) by reacting with endothelium-derived nitric oxide (EDNO), we examined the effect of manganese (Mn)-superoxide dismutase (SOD) on RH in anesthetized dogs. METHODS: Twelve dogs were pretreated with 8-phenyltheophylline (8PT) to block adenosine's effect. Five dogs were pretreated with 8PT and NG-nitro-L-arginine methyl ester (L-NAME) to block adenosine's and EDNO's effects. Following occlusion of the left circumflex artery (LCX) for 10 and 60 s, RH was observed before and after Mn-SOD. In another group of 6 dogs pretreated with 8PT, RH following 60-s LCX occlusion was observed before and after Mn-SOD and catalase. For comparison with the effect of Mn-SOD, that of copper, zinc (Cu,Zn)-SOD was also examined in another group of 5 dogs. RESULTS: In the dogs pretreated with 8PT, Mn-SOD significantly increased excess flow and repayment of flow debt during RH after 60-s LCX occlusion but did not affect RH after 10-s LCX occlusion. Mn-SOD-induced augmentation of RH following 60-s LCX occlusion was not affected by catalase, while it was completely abolished by L-NAME. In contrast to Mn-SOD, Cu,Zn-SOD showed no effect on RH following 60-s LCX occlusion in the dogs pretreated with 8PT. CONCLUSIONS: Superoxide radicals generated during ischemia for 60 s and reperfusion attenuates myocardial RH through inactivation of EDNO. Mn-SOD shows more beneficial effects on myocardial RH than Cu,Zn-SOD.

Induction of 8-hydroxy-2'-deoxyguanosine in CHO-K1 cells exposed to phenyl-hydroquinone, a metabolite of ortho-phenylphenol.

The induction of 8-hydroxy-2'-deoxyguanosine (8-OHdG), an index of oxidative DNA modification, was investigated in CHO-K1 cells exposed to phenyl-hydroquinone (PHQ), a major metabolite of ortho-phenylphenol (OPP), an antimicrobial. Addition of PHQ at a concentration of 50 microM to CHO cell suspensions (10(6) cells/ml) induced slight elevation of intracellular 8-OHdG levels. Pretreatment of CHO cells with 3-amino-1,2,4-triazole (AT, 20 mM) enhanced PHQ-induced 8-OHdG formation which was accompanied by cell death. Pretreatment of CHO-K1 cells with AT (20 mM) and deferoxamine (DeFe, 20 mM) inhibited the formation of 8-OHdG as well as cell death caused by PHQ. Neither AT nor DeFe affected cell viability or the formation of 8-OHdG in untreated CHO cells during the incubation period. The loss of cellular glutathione induced by the addition of PHQ alone was enhanced by the pretreatment of CHO cells with AT or AT plus DeFe. When PHQ was added to AT-pretreated cell suspensions, the concentration of PHQ decreased with time. This decrease was accompanied by the formation of phenyl-benzoquinone (PBQ). These results suggest that the reactive oxygen species derived from autoxidation of PHQ which converts to PBQ via phenyl-semiquinone elicit DNA damage in CHO cells, especially when the activity of cellular catalase is inhibited.

Role of adenosine in regulation of coronary flow in dogs with inhibited synthesis of endothelium-derived nitric oxide.

Endothelium-derived nitric oxide (NO) regulates coronary blood flow, but it is unclear how NO synthesis inhibition affects myocardial metabolism. In pentobarbital sodium-anesthetized dogs, myocardial oxygen metabolism, adenosine release, lactate extraction rate (LER), and systolic ventricular wall thickening (SWT) at baseline and during atrial pacing were estimated before and after intracoronary NG-nitro-L-arginine methyl ester (L-NAME) infusion. Coronary blood flow and PO2 in the anterior interventricular vein at baseline were both significantly decreased by L-NAME (3 x 10(-4) M in the coronary blood). Coronary flow was increased during pacing, which was not affected by L-NAME. Myocardial adenosine release remained unchanged during pacing before L-NAME, but it was significantly increased after L-NAME infusion. Neither LER nor SWT changed during pacing performed before and after L-NAME. The experiment was also performed in dogs pretreated with 8-phenyltheophyl-line. After L-NAME, pacing-induced increase in coronary flow was suppressed, and both LER and SWT were significantly decreased during pacing. In conclusion, when NO synthesis is inhibited, adenosine release is increased in response to the increase in myocardial oxygen demand. With this compensatory adenosine release, coronary flow is increased and ventricular function is unaffected.

Metabolism and cytotoxicity of propyl gallate in isolated rat hepatocytes: effects of a thiol reductant and an esterase inhibitor.

The relationship between the metabolism and the cytotoxic effects of propyl gallate (PG) has been studied in freshly isolated rat hepatocytes. Addition of PG (0.5-2.0 mM) to the hepatocytes elicited concentration-dependent cell death, accompanied by decreases in intracellular ATP, adenine nucleotide pools, glutathione, and protein thiols. The rapid loss of ATP preceded the onset of cell death. PG in the hepatocyte suspensions was converted to gallic acid, 4-O-methyl-gallic acid, and other minor products over time. In addition, PG was converted to a dimer [dipropyl-4,4',5,5',6,6'-hexahydroxydiphenate (PG-dimer)] and ellagic acid via autooxidation. In comparisons of the toxic effects of PG and its metabolites at concentrations of 2 mM, the parent compound PG was the most toxic. Pretreatment of hepatocytes with diazinon (100 microM), an esterase inhibitor, enhanced PG-induced cytotoxicity. This was accompanied by delay of PG loss and inhibition of gallic acid formation. The cytotoxicity of PG was also enhanced by addition of the thiol reductant dithiothreitol (4 mM), although intracellular levels of glutathione and protein thiols were maintained during the incubation period. Dithiothreitol did not affect the hydrolysis of PG to gallic acid by esterases but did delay the conversion of PG and prevented the formation of PG-dimer. In isolated hepatic mitochondria, PG elicited a concentration-dependent increase in the rate of state 4 oxygen consumption, indicating an uncoupling effect. In contrast, PG-dimer inhibited the rate of state 3 oxygen consumption. Based on the respiratory control index, the order of potency for impairment of mitochondria was PG > PG-dimer > gallic acid = 4-O-methyl-gallic acid = ellagic acid - propyl alcohol. These results indicate (a) that PG-induced hepatotoxicity is mediated by the parent compound and not its metabolites, (b) that toxicity is associated with ATP depletion apparently independently of cellular thiol depletion, and (c) that mitochondria may represent critical targets of PG-induced cytotoxicity.

Cytotoxicity of propyl gallate and related compounds in rat hepatocytes.

The cytotoxic effects of propyl gallate (PG), its related gallates and gallic acid have been studied in freshly isolated rat hepatocytes. Addition of PG (0.5-2.0 mM) to hepatocyte suspension elicited concentration-dependent cell death accompanied by losses of intracellular ATP, adenine nucleotide pools, glutathione (GSH) and protein thiols. The rapid loss of intracellular ATP preceded the onset of cell death caused by PG. In the comparative toxic effects of PG and related gallates at concentration of 1 mM, octyl gallate (OG), dodecyl gallate (DG) and butyl gallate (BG) elicited an abrupt depletion of ATP, followed by an acute cell death. These gallates were more toxic than PG; the toxic effects of PG were similar to those of methyl gallate (MG) and ethyl gallate (EG). In mitochondria isolated from rat liver, PG caused a concentration-dependent increase in the rate of state 4 oxygen consumption, indicating an uncoupling effect. The rate of state 3 oxygen consumption was inhibited by OG and DG. According to the respiratory control index, the order of impairment potency to mitochondria was OG > BG, DG > PG > EG, MG > gallic acid. These results indicate that PG and related gallates are toxic to hepatocytes and that the acute cytotoxicity may be due to mitochondrial dysfunction.

Effect of scavengers of active oxygen species on cell damage caused in CHO-K1 cells by phenylhydroquinone, an o-phenylphenol metabolite.

Phenylhydroquinone (PHQ), a metabolite of o-phenylphenol (OPP), is easily autoxidized to phenylbenzoquinone (PBQ) via the semiquinone (phenylsemiquinone, PSQ) with concomitant production of superoxide anion radicals (O2-.). We have used scavengers of active oxygen species to examine whether or not O2-. produced during oxidation of PHQ is related to cell damage in CHO-K1 cells. PHQ at 10 micrograms/ml (3-h treatment) induced sister-chromatid exchange (SCE), endoreduplication (ERD) and cell-cycle delay in CHO-K1 cells. These effects were inhibited by catalase (280 U/ml), a scavenger of hydrogen peroxide (H2O2), as well as by the reductants, ascorbate (3 mM) and GSH (1 mM). Mannitol (50 mM), a scavenger of hydroxyl radical (OH.), was ineffective and superoxide dismutase (SOD, 150 U/ml), a scavenger of O2-., or SOD plus catalase rather intensified the toxicity as did aminotriazole (20 mM), an inhibitor of catalase. Analyses of incubation solutions by HPLC showed that the extent of cell damage is correlated with PHQ loss; catalase suppressed PHQ loss, whereas SOD promoted it. The correlation was more clearly seen in the time courses of cell death and PHQ loss during incubation of PHQ with each of the scavengers of active oxygen species. These results show that neither O2-. nor OH. participates in the cell damage, but rather H2O2 generated via dismutation of O2-. may participate, probably by accelerating the autoxidation of PHQ and thus causing an increase in the production of toxic intermediates. In fact, conversion of PHQ to PBQ, a reactive product, was demonstrated during incubation with PHQ in phosphate-buffered saline by following the changes in UV-visible spectra of PHQ. Inclusion of H2O2 (0.2 or 1 mM) in the incubation mixture accelerated the PHQ loss. The present results can be explained in terms of the autoxidation mechanism of hydroquinone proposed by O'Brien (1991). Different from the results in the absence of S9 mix, the cell damage induced by 50 micrograms/ml OPP in the presence of S9 mix was not influenced by any of the scavengers of active oxygen species used. We conclude that PHQ causes cytotoxic and genotoxic effects through its autoxidation, both enzymatic and nonenzymatic, and that reactive intermediate(s) such as PSQ and/or PBQ may be ultimately responsible for the effects. H2O2 formed during the oxidation process participates in the damaging effects caused in the absence of S9 mix, probably by accelerating the autoxidation.

Cytotoxic effects of biphenyl and hydroxybiphenyls on isolated rat hepatocytes.

The cytotoxic effects of biphenyl (BP) and its hydroxylated derivatives, o-phenylphenol (OPP), m-phenylphenol (MPP), p-phenylphenol (PPP), 2-biphenylyl glycidyl ether (OPP-epoxide), phenyl-hydroquinone (PHQ), o,o'-biphenol (o,o'-BPol) and p,p'-biphenol (p,p'-BPol), were investigated in freshly isolated rat hepatocytes. OPP, MPP and PPP, at concentration of 0.75 mM, resulted in the loss of intracellular ATP, glutathione (GSH) and protein thiols, causing cell death. OPP-epoxide and BP were less toxic than the OPP isomers. MPP or PPP compared with OPP caused serious impairments in oxidative phosphorylation in mitochondria isolated from rat liver. PHQ (0.75 mM) caused a rapid loss of intracellular ATP which preceded the onset of cell death. PHQ was more toxic than o,o'-BPol or p,p'-BPol. PHQ dissolved in Krebs-Henseleit buffer without hepatocytes was rapidly converted to its corresponding quinone, phenyl-benzoquinone. The cytotoxicity produced by PHQ depends on the rate of formation of reactive intermediates. These results indicate that the addition of a hydroxyl group to the aromatic ring of BP enhances BP-induced cytotoxicity and that the mitochondria are a common target of the OPP isomers and other BP derivatives. In addition, the para- or meta-hydroxyl groups rather than the ortho-hydroxyl group increase the toxicity. The cytotoxicity produced by PHQ depends on the rate of formation of reactive intermediate(s) such as phenyl-benzoquinone.

Effects of diethyl maleate on phenyl-hydroquinone-induced cytotoxicity in isolated rat hepatocytes.

1. The effects of diethyl maleate (DEM) on the cytotoxicity of phenyl-hydroquinone (PHQ) and other hydroquinones were studied in freshly isolated rat hepatocytes. 2. Addition of PHQ (0.5 or 0.75 mM) to hepatocytes resulted in dose-dependent cell death accompanied by the abrupt depletion of both GSH and protein thiols and the accumulation of phenyl-benzoquinone (PBQ). 3. Pretreatment with DEM (1.25 mM), which causes an abrupt depletion of cellular GSH in hepatocytes, delayed the onset of PHQ-induced cytotoxicity. The delay correlated with inhibition of PBQ formation. 4. Although the pH of the cell suspension was increased slightly (mean pH 0.18) by incubation under carbogen flow, the addition of DEM to the cell suspension inhibited both the increase in pH and the formation of PBQ from PHQ. 5. In hepatocyte suspensions without DEM, PHQ cytotoxicity was dependent on pH, and toxicity was associated with oxidation of PHQ and accumulation of PBQ. 6. Among other hydroquinones (0.5 mM), tert-butyl-hydroquinone-induced cytotoxicity was decreased by DEM (1.25 mM), but DEM did not affect the cytotoxicity of 2,5-di(tert-butyl)-1,4-benzohydroquinone. 7. PHQ-induced cytotoxicity correlated with the accumulation of PBQ in the cell, and the inhibition of PHQ-induced cytotoxicity by DEM correlated with pH-dependent changes in PBQ formation.

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.