Propyl gallate-induced DNA fragmentation in isolated rat hepatocytes

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. Received: 23 June 1997 / Accepted: 18 August 1997     

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. Received: 25 October 1999 / Accepted: 31 January 2000     

Role of mitochondrial membrane permeability transition in N-nitrosofenfluramine-induced cell injury in rat hepatocytes

The role of mitochondrial membrane permeability transition in N-nitrosofenfluramine-induced cell injury was studied in mitochondria and hepatocytes isolated from rat liver. Mitochondrial permeability transition has been proposed as a common final pathway in acute cell death through mitochondrial dysfunction. In isolated mitochondria, N-nitrosofenfluramine (0.25 to 1.0 mM) in the presence of Ca(2+) (50 microM) elicited a concentration-dependent induction of mitochondrial swelling dependent on mitochondrial permeability transition and the release of cytochrome c, both of which were prevented by pretreatment with a specific inhibitor of mitochondrial permeability transition, cyclosporin A (0.2 microM). The effects of N-nitrosofenfluramine on mitochondria were more potent than those of fenfluramine, which is a sympathomimetic amine with anorectic action. The pretreatment of isolated hepatocytes with cyclosporin A (2 microM) partially but not completely prevented N-nitrosofenfluramine (0.6 mM; a low toxic dose)-induced cell death, loss of cellular ATP, formation of cell blebs and decrease in mitochondrial membrane potential. These results suggest that the onset of N-nitrosofenfluramine-induced cytotoxicity is linked to mitochondrial failure dependent upon induction of mitochondrial permeability transition accompanied by mitochondrial depolarization, the release of cytochrome c and depletion of intracellular ATP through uncoupling of oxidative phosphorylation.     

Role of mitochondrial membrane permeability transition in p-hydroxybenzoate ester-induced cytotoxicity in rat hepatocytes.

The relationship between mitochondrial membrane permeability transition (MPT) and the toxic effects of the alkyl esters of p-hydroxybenzoic acid (parabens) has been studied in mitochondria and hepatocytes isolated from rat liver. MPT has been proposed as a common final pathway in acute cell death through mitochondrial dysfunction. In isolated mitochondria, propyl-paraben (0.1 to 0.5 mM) in the presence of Ca2+ (50 microM) elicited a concentration-dependent induction of mitochondrial swelling dependent on MPT. This was prevented by pretreatment with a specific inhibitor of MPT, cyclosporin A (0.2 microM). For the other parabens tested, the induction of MPT depended on the relative elongation of alkyl side-chains in their molecular structure and was associated with the partition coefficients. In contrast, the induction caused by p-hydroxybenzoic acid was more potent than that of methyl- or ethyl-paraben. The pretreatment of freshly isolated hepatocytes with cyclosporin A (5 microM) and trifluoperazine (10 microM), which inhibit MPT in a synergistic manner, partially but not completely prevented propyl-paraben (1 mM; plus diazinon, 100 microM)-induced cell death, ATP loss, and decreased mitochondrial membrane potential. These results suggest that the onset of paraben-induced cytotoxicity is linked to mitochondrial failure dependent upon induction of MPT accompanied by the mitochondrial depolarization and depletion of cellular ATP through uncoupling of oxidative phosphorylation.     

Evaluation of anti-HSV-2 activity of gallic acid and pentyl gallate

The synthetic n-alkyl esters of gallic acid, also known as gallates, are widely employed as antioxidants by food and pharmaceutical industries. Besides the antioxidant activity, other biological activities have been described for this group of molecules, mainly anticancer, antibacterial and antifungal properties. In the present study, the anti-herpes simplex virus (HSV)-2 activity of gallic acid and pentyl gallate was evaluated followed by the determination of the site of antiviral activity of these compounds. Our results demonstrated that both compounds reduced HSV-2 replication in a concentration-dependent manner when either incubated with the virus prior to the addition of the mixture to cells, or added to and incubated with cells after their infection. In summary, the anti-HSV-2 activity of gallic acid and pentyl gallate was ascribed to their virucidal effect on virus particles, a change that was likely accompanied by partial inhibition of the virus attachment to cells and its subsequent cell-to-cell spread activity. This suggests that these compounds can be regarded as promising candidates for development as topical anti-HSV-2 agents.     

Effects of ochratoxin A on DNA repair in cultures of rat hepatocytes and porcine urinary bladder epithelial cells

In cultured rat hepatocytes the mycotoxin ochratoxin A (OTA) induced unscheduled DNA synthesis (UDS) only in a narrow concentration range. Using a culture medium supplemented with 1% fetal calf serum, at 750 nM OTA a weak induction and at 1 μM OTA a marked induction of DNA repair was observed (15 ± 11 and 38 ± 24% cells in repair, respectively). Concentrations >1 μM OTA were cytotoxic, and <750 nM no induction occurred. In cultures of cells from the urinary bladder (porcine urinary bladder epithelial cells; PUBEC), a target organ of the mycotoxin, OTA induced UDS in a concentration-dependent manner. To inhibit the proliferation of the cultured epithelial cells, which would counteract the detection of DNA repair, epidermal growth factor was omitted and an arginine-deficient medium (ADM) was used. Under these serum-free culture conditions the amount of cells undergoing DNA repair in PUBEC control cultures was ∼7±4%, a value also comparable to those of control cultures of rat hepatocytes. At concentrations between 250 nM and 1 μM OTA a concentration-dependent increase of cells in repair was observed. Above 1 μM OTA was cytotoxic. At this concentration a maximum of ∼61±9% of the cells undergo DNA repair. This amount is comparable to control cultures incubated with 5 or 10 mM ethylmethane-sulphonate (EMS) (49±9 and 69±10% cells in repair, respectively), used as a positive control. These results show that in cultured rat hepatocytes induction of UDS is relatively weak whereas in urothelial cells this effect was significant. Whether this effect is due to OTA metabolites formed locally in the urothelium cannot be excluded since PUBEC have been shown to be able to metabolize xenobiotics independently from the liver. Received: 13 May 1997 / Accepted: 11 June 1997     

Relationship between acute toxicity of (bis)aziridinylbenzoquinones and cellular pyridine nucleotides

The toxicity of aziridinylbenzoquinones may occur by a number of mechanisms, including oxidative stress caused by redox cycling and the activation of the aziridine groups. Isolated hepatocytes were used to assess the relationship between the redox status of NADP(H) associated with oxidative stress, the level of NAD(H) closely linked with DNA repair and the cytotoxicity of three 2,5-bis(aziridinyl)-1,4-benzoquinones (BABQ). Exposure of hepatocytes to the BABQ TW13 (200 μM) and TW25 (100 μM), which are able to arylate and to redox cycle, resulted in increased intracellular NADP⁺ from <0.3 nmol/mg protein to 1.5 nmol/mg protein within 60 min. The increase in intracellular NADP⁺ was followed by the onset of cell death by 180 min. In contrast, exposure to lower concentrations of TW13 (100 μM), TW25 (50 μM) and carboquone (100–200 μM) (which neither arylates nor redox cycles via a one-electron reduction) resulted in a less pronounced (<1.0 nmol/mg) increase in NADP⁺ and there was no evidence of cell death within the 180 min incubation. BABQ had a concentration dependent effect on intracellular NAD⁺. Exposure of hepatocytes to TW13 (200 μM) and TW25 (100 μM) resulted in a decrease in intracellular NAD⁺ from >2.7 to <1.0 nmol/mg protein within 60 min. At concentrations of the BABQ where the level of NAD⁺ remained  >1.0 nmol/mg protein after 30 min, the hepatocytes remained viable at 180 min. These changes in intracellular pyridine nucleotides suggests two mechanisms may be involved in BABQ cytotoxicity. At high concentrations, aziridinylbenzoquinones may cause cytotoxicity via oxidative stress following redox cycling. At lower concentrations, however, the predominant pyridine nucleotide change is a prolonged depletion of NAD⁺, suggesting extensive DNA damage which may lead to delayed cell death. Received: 30 October 1996 / Accepted: 13 January 1997     

Cytotoxicity of ortho-phenylphenol in isolated rat hepatocytes.

The effects of ortho-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 microM) 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.     

Inhibitory effects of catechin gallates on o-methyltranslation of protocatechuic acid in rat liver cytosolic preparations and cultured hepatocytes

Flavonoids including tea catechins and gallic acid esters were characterized for their ability to inhibit o-methyltranslation of protocatechuic acid (PCA) to form vanillic acid (VA) in rat liver cytosolic preparations and cultured hepatocytes. Flavonols and flavones exhibited different behaviors in inhibiting the formation of VA between the cell-free enzymatic preparations and the intact cells. The underlying mechanism of the inhibitory effects of flavonols and flavones on PCA o-methylation in cultured hepatocytes may not be due to the inhibition of the enzyme activity of catechol o-methyl transferase (COMT). Catechin gallates inhibited PCA o-methylation in liver cytosolic preparations with markedly higher potency than other flavonoids. As compared with catechin gallates, ungallated catechins had two to three orders of magnitude lower efficiency in inhibiting cytosolic PCA o-methylation. Gallic acid esters inhibited cytosolic PCA o-methylation with strong potency almost equal to that of catechin gallates. These results suggest that the COMT-inhibitory activity of catechin gallates is derived from the presence of the galloyl moiety at the C3 position in the C-ring. Catechin gallates and gallic acid esters inhibited PCA o-methylation in cultured hepatocytes with two orders of magnitude lower efficacy than that in cytosolic preparations. The inhibitory effects of catechin gallates and gallic acid esters on cellular PCA o-methylation appear to be due to the direct inhibition of COMT activity.     

Effects of alkyl gallates on P-glycoprotein function

In this study, we examined the effects of the food antioxidants, alkyl gallates, on the function of P-glycoprotein (P-gp) and elucidated the importance of alkyl chains and gallic acid moieties on the activity of P-gp. We examined the effects of three alkyl (n-butyl, n-octyl and n-dodecyl) gallates and their related compounds on the cellular accumulation and efflux of rhodamine 123 and daunorubicin in P-gp overexpressing KB-C2 cells. Alkyl gallates increased the cellular accumulation of these P-gp substrates dependent on their alkyl chain lengths by inhibiting the efflux of the substrates. n-Dodecylresorcinol also increased the accumulation, but its effect was less than that of n-dodecyl gallate. However, either lauric acid or n-dodecyl-beta-d-maltoside, which does not have a phenol group, did not increase the accumulation. The results indicated that both the gallic acid moiety and a long alkyl chain play important roles in the modification of P-gp function. The cytotoxicity of daunorubicin was recovered in the presence of alkyl gallates possibly due to their inhibition of P-gp function.     

Toxicity of insecticides on Neotropical stingless bees Plebeia emerina (Friese) and Tetragonisca fiebrigi (Schwarz) (Hymenoptera: Apidae: Meliponini)

Use of pesticides in agroecosystems is considered a major cause of bees diversity losses in the Neotropics, where Plebeia emerina (Friese) and Tetragonisca fiebrigi (Schwarz) (Hymenoptera: Apidae: Meliponini) are wild pollinators of native and crop plants. The aim of this study was to know the acute lethal toxicity of acetamiprid, malathion, phosmet and spinosad insecticides on P. emerina and T. fiebrigi. We obtained the mean concentration and mean lethal dose (LC50 and LD50) and the mean survival of workers after oral and topical exposure to insecticides, respectively. The LC50 values (ng a.i./μl of diet) and the decreasing order of toxicity for P. emerina was spinosad (4.96) > malathion (18.75) > phosmet (97.33) > acetamiprid (4204.06), and for T. fiebrigi also was spinosad (5.65) > malathion (8.39) > phosmet (53.91) > acetamiprid (9841.32), when orally exposed. The LD50 values (ng a.i./bee) and the decreasing order of toxicity for P. emerina was spinosad (1.90) > malathion (10.90) > phosmet (19.54) > acetamiprid (6216.55) and for T. fiebrigi was malathion (29.29) ≥ spinosad (29.79) > phosmet (41.95) > acetamiprid (1421.23), when topically exposed. The mean survival (hours) of contaminated bees by malathion, phosmet, and spinosad, was 11.81, 7.20, and 12.32 for P. emerina and 8.55, 7.20, and 13.34 for T. fiebrigi when orally exposed; and was 4.87, 9.87 and 11.17 for P. emerina, and 4.87, 4.76, and 19.05 for T. fiebrigi when topically exposed. Malathion, phosmet, and spinosad were highly toxic, while acetamiprid was moderately toxic. Our results indicated that the insecticides tested, mainly malathion, phosmet, and spinosad may be harmful to P. emerina and T. fiebrigi, making it essential to propose measures to minimize their impact on wild pollinators.     

Toxicology of gallates: a review and evaluation

The propyl, octyl and dodecyl esters of gallic acid have been studied extensively in a large number of animal experiments involving oral dosing. Experimental data on general toxicity and studies on reproduction, teratogenicity and mutagenicity are also available. Most of the key toxicity studies, however, date back to the 1950s, do not meet current standards of toxicity testing and do not provide evidence for carcinogenic or mutagenic action of the gallates. Mutagenicity studies with octyl gallate and dodecyl gallate are lacking. The biokinetics of propyl gallate apparently differ from those of octyl and dodecyl gallate, the octyl and dodecyl esters being absorbed and hydrolysed to a lesser degree than the propyl ester. In toxicity studies with propyl gallate, growth retardation, anaemia, kidney and liver changes and hyperplasia of the forestomach were the most prominent effects at dose levels above 10,000 mg/kg feed. At 5000 mg/kg feed, liver enzyme induction was seen. In the available studies with octyl gallate or dodecyl gallate as the test compound, effects were found at 3000 mg/kg feed or higher levels. In studies performed with the various gallates, no effects were observed at a dose level of 1000 mg/kg feed, a level that was adopted as the no-effect level by the FAO/WHO Joint Expert Committee on Food Additives (JECFA) in 1976. This committee established an acceptable daily intake (ADI) for man of 0.2 mg/kg body weight (as a sum of propyl, octyl and dodecyl gallates). A re-evaluation of the toxicity of gallates indicates that a 'classic' long-term toxicity study of propyl gallate meeting current standards is required. As yet, the available toxicological evidence indicates that gallates may be used safely as antioxidants.     

Biotransformation and cytotoxicity of a brominated flame retardant, tetrabromobisphenol A, and its analogues in rat hepatocytes

The metabolism and cytotoxic effects of tetrabromobisphenol A (TBBPA), a phenolic flame retardant, and its analogues were studied in freshly isolated rat hepatocytes and isolated hepatic mitochondria, respectively. The exposure of hepatocytes to TBBPA caused not only concentration (0.25-1.0 mM)- and time- (0-3 h) dependent cell death accompanied by the loss of cellular ATP, adenine nucleotide pools, reduced glutathione, and protein thiols, but also the accumulation of oxidized glutathione and malondialdehyde, indicating lipid peroxidation. TBBPA at a weakly toxic level (0.25 mM) was metabolized to monoglucuronide and monosulfate conjugates: the amounts of glucuronide rather than sulfate conjugate predominantly increased, accompanied by a loss of the parent compound, with time. In comparative effects based on cell viability, mitochondrial membrane potential and some toxic parameters, bisphenol A (BPA) was less toxic than TBBPA and tetrachlorobisphenol A (TCBPA), which are not significant differences in these parameters. In mitochondria isolated from rat liver, TBBPA and TCBPA caused an increase in the rate of State 4 oxygen consumption in the presence of succinate, indicating an uncoupling effect and a decrease in the rate of State 3 oxygen consumption in a concentration-dependent manner (5-25 microM). Taken collectively, our results indicate that (i) mitochondria are target organelles for TBBPA, which elicits cytotoxicity through mitochondrial dysfunction related to oxidative phosphorylation at an early stage and subsequently lipid peroxidation at a later stage; and (ii) the toxicity of TBBPA and TCBPA is greater than that of BPA, suggesting the participation of halogen atoms such as bromine and chlorine in the toxicity.     

N-Nitrosofenfluramine induces cytotoxicity via mitochondrial dysfunction and oxidative stress in isolated rat hepatocytes

The cytotoxic effects of fenfluramine, an appetite suppressant, and its N-nitroso derivative, N-nitrosofenfluramine, have been studied in freshly isolated rat hepatocytes and isolated hepatic mitochondria. Exposure of hepatocytes to N-nitrosofenfluramine caused not only concentration (0.25–1.0 mmol L^–1) and time (0–3 h)-dependent cell death accompanied by the loss of cellular ATP, adenine nucleotide pools, reduced glutathione (GSH), and protein thiols, but also the accumulation of oxidized glutathione and malondialdehyde (MDA), indicating lipid peroxidation. There was a time lag for the onset of the accumulation of MDA after the rapid depletion of ATP. Supplementation of the hepatocyte suspensions with N-acetylcysteine (4 mmol L^–1), a precursor of intracellular GSH, partially inhibited N-nitrosofenfluramine (1 mmol L^–1)-induced cytotoxicity. In comparative effects based on cell viability and rhodamine 123 retention, an index of mitochondrial membrane potential, fenfluramine was less toxic than N-nitrosofenfluramine. In mitochondria isolated from rat liver, N-nitrosofenfluramine caused an increase in the rate of state-4 oxygen consumption, indicating an uncoupling effect, and a decrease in the rate of state-3 oxygen consumption in a concentration-dependent manner. These results indicate that (a) mitochondria are target organelles for N-nitrosofenfluramine, which elicits cytotoxicity through mitochondrial dysfunction related to membrane potential and/or oxidative phosphorylation at an early stage and subsequently lipid peroxidation at a later stage; and (b) the toxicity of N-nitrosofenfluramine is greater than that of fenfluramine, suggesting participation of the nitroso group in the toxicity.     

d-Amphetamine-induced hepatotoxicity: possible contribution of catecholamines and hyperthermia to the effect studied in isolated rat hepatocytes

Amphetamines are indirect-acting sympathomimetic drugs widely abused due to their physical and psychostimulating effects. However, the use of these drugs has been associated with numerous reports of hepatotoxicity. While glutathione depletion induced by amphetamines contributes to the exposure of hepatocytes to oxidative damage, other indirect effects attributed to amphetamines may have a role in cell injury. To examine this possibility, Wistar rats were used for plasma measurements of d-amphetamine and catecholamines (noradrenaline, adrenaline and dopamine) (15 min) after i.p. injection of d-amphetamine (5, 20 and 80 mg/kg). Freshly isolated rat hepatocytes were put into contact for 2 h with concentrations of d-amphetamine and catecholamines similar to those found in vivo. Since hyperthermia is a common consequence of acute amphetamine intake, the study using isolated hepatocytes was conducted at 37 °C and also at 41 °C in order to simulate high temperature levels. We found that hyperthermia was an important cause of cell toxicity: in vitro, a rise in incubation temperature from 37 to 41 °C causes oxidative stress in freshly isolated rat hepatocytes, as shown by a depletion of reduced glutathione (GSH; 23%), an increase of oxidized glutathione (GSSG; 157%), the induction of lipid peroxidation with 77% increase of thiobarbituric acid substances TBARS) and the consequent loss of cell viability (≤ 44%). Single treatment of isolated hepatocytes with catecholamines at 37 °C induced lipid peroxidation (29% increase of TBARS) but had no effect on glutathione or cell viability. Conversely, a single treatment with d-amphetamine induced glutathione depletion (≤ 24% depletion of GSH) with no effect on lipid peroxidation or cell viability. Also, d-amphetamine potentiated the induction by catecholamines of lipid peroxidation at 37 °C (≤ 48% increase of TBARS), while concomitant treatment of d-amphetamine and catecholamines potentiated cell death at 41 °C (≤ 56% of cell death) although no effect on viability was seen at 37 °C. It is concluded that the aforementioned modifications induced by d-amphetamine in vivo are cytotoxic to freshly isolated rat hepatocytes. Received: 30 October 1996 / Accepted: 13 January 1997     

Elucidation of mitochondrial effects by tetrahydroaminoacridine (tacrine) in rat, dog, monkey and human hepatic parenchymal cells

Tetrahydroaminoacridine (tacrine) causes morphological and functional changes in the endoplasmic reticulum, ribosomes, and mitochondria in the liver of humans and animals. In order to investigate species differences as well as to understand the morphological changes, we examined the effects of tacrine on respiration and electron transport in mitochondria isolated from rat, dog, monkey, and human liver. Tacrine produced significantly decreased respiratory control ratios (RCR) in all species at concentrations ranging from 5 to 25 μg/ml. Human mitochondria were more sensitive to tacrine effects with RCR decreased 24% at 5 μg/ml while other species were unaffected at this concentration. The tacrine effects were characterized by increased hepatic mitochondrial State 4 respiration in rats and decreased State 3 respiration in humans. Mitochondria from aged rats were more sensitive to the effects of tacrine than mitochondria from young animals, with significantly decreased RCR at 10 μg/ml in aged rats while mitochondria from young rats were unaffected at this concentration. Concomitant with the respiratory changes, mitochondrial DNA synthesis was impaired. Since tacrine undergoes extensive biotransformation, we also explored the possibility that metabolites could exert detrimental effects. The ranking order of potency for decreasing RCR caused by monohydroxylated metabolites was: tacrine >4-OH and 7-OH >2-OH, 1-OH, and velnacrine with the latter group of metabolites having no effect on mitochondrial respiration at concentrations up to 50 μg/ml. In vivo administration of 20 mg/kg tacrine to rats for up to 20 days caused a paradoxical increase in RCR and P/O on Day 1 and decreased RCR on Days 9 and 20, the later findings being consistent with in vitro data. From these data we propose that tacrine does not necessarily have to be metabolized to exert effects on mitochondria at different sites in the electron transport chain that differ among species. These effects are exacerbated in mitochondria from older animals and humans appear to be more sensitive than the laboratory animals studied. Received: 23 September 1997 / Accepted: 17 November 1997     

Ethionine toxicity in vitro: the correlation of data from rat hepatocyte suspensions and monolayers with in vivo observations

The hepato-steatogenic compound ethionine has been used to investigate the correlations between in␣vivo and in vitro toxicity data. The aim was to find a suitable model of toxicity in hepatocyte suspensions or monolayers in vitro, which could predict the known toxicity of ethionine in vivo and which could be implemented in screening compounds of unknown toxicity. Thus a variety of markers of cytotoxicity, metabolic competence and liver-specific functions were investigated in rat hepatocyte suspensions and monolayers and compared with in vivo data in the rat. The following markers were measured in the appropriate system: (1) Neutral red uptake; 3-(4,5 dimethyl)thiazol-2-yl,-2,5-diphenyl tetrazolium bromide (MTT) reduction; lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) leakage (cytotoxicity). (2) ATP levels, protein synthesis and glutathione (GSH) levels (metabolic competence). (3) Urea and triglyceride synthesis and β-oxidation (liver specific functions). Ethionine (0–30 mM) did not affect the markers of direct cytotoxicity, except neutral red uptake, which was reduced by 18 and 30 mM ethionine after 20 h in culture. ATP and GSH depletion occurred in hepatocyte suspensions at the highest concentrations of ethionine (20 and 30 mM) after 1 h. In monolayers, GSH levels were reduced after 4 h, but not 20 h. Urea synthesis was increased in hepatocyte suspensions from 1 to 3 h by 10–30 mM ethionine and reduced after 20 h in cultured hepatocytes (18–30 mM). Protein synthesis was reduced and β-oxidation was increased in ethionine-treated hepatocyte suspensions. Unfortunately, there was no measurable effect on triglyceride accumulation within cells (the major biochemical change in␣vivo) in either system. Ethionine treated hepatocytes in suspension showed the same rate of triglyceride synthesis and transportation out of cells as control cells. Thus, hepatocyte suspensions were able to mimic the early biochemical effects of ethionine in vivo (ATP and GSH depletion, inhibition of protein synthesis) and some effects on urea synthesis, but monolayer cultures appeared to be less sensitive to the toxicity of ethionine. However, neither in vitro system was able to model the effects of ethionine on the accumulation of triglycerides in vivo. Received: 16 June 1998 / Accepted: 29 June 1998     

The toxicokinetics of 1,3-butylene glycol versus ethanol in the treatment of ethylene glycol poisoning.

Ethylene glycol (EG) is a toxic chemical found in antifreeze and heat exchangers. Standard therapy for EG intoxication in administration of ethanol (ETOH) to inhibit its metabolism by alcohol dehydrogenase (ADH). Studies indicate 1,3-butylene glycol (BG) binds to ADH more efficiently than EG and is orally less toxic than EG or ETOH. Male rats were divided into 5 groups of 6 animals. Groups received by oral intubation a single dose of EG (32 mmole/kg), BG (39 mmole/kg) initially and every 6 h up to 72 h, ETOH (39 mmole/kg) initially and every 6 h up to 72 h, or EG initially and then either BG or ETOH every 6 h up to 72 h. Administration of ETOH produced hepatotoxicity and pulmonary pathology as indicated by changes in clinical chemistry, urinalysis, and histopathology, while BG did not. Neither ETOH nor BG produced any apparent nephrotoxicity. ETOH produced ataxia, lethargy and central nervous system depression while BG did not. BG produced a higher concentration of urinary EG indicating a better inhibition of ADH metabolism of EG. Ethanol produced a higher EG blood concentration than BG. Ethanol's higher EG blood concentration may be partially attributed to dehydration and a decreased urine output as well as inhibition of ADH metabolism. Ethanol produced mortality in all animals prior to 72 h. The EG/ETOH combination produced mortality more quickly due to additive toxicity of the combination. Lack of any significant toxicity produced by BG and the production of significant toxicities by ETOH indicates that BG is potentially a better antidote than ETOH.     

The toxic mechanism and metabolic effects of atractyloside in precision-cut pig kidney and liver slices

The toxic and cellular metabolic effects of atractyloside, a diterpenoid glycoside, which causes fatal renal and hepatic necrosis in vivo in animals and humans, have been investigated in tissue slices prepared from male domestic pig kidney and liver. Precision-cut slices (200 μm thick) were incubated with atractyloside at concentrations of 200 μM, 500 μ M, 1.0 mM and 2.0 mM for 3 h at 37 °C and changes in lipid profile and pyruvate-stimulated gluconeogenesis investigated. Lipid peroxidative changes, reduced glutathione (GSH) and ATP content, the release of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), alanine and aspartate aminotransferase (ALT/AST) were also assessed. After 3 h of incubation, atractyloside caused a significant (P < 0.01) and concentration-dependent leakage of LDH and ALP from kidney slices. Only LDH leakage was significantly elevated in liver slices while ALT and AST leakage showed marginal increase. Atractyloside at concentrations of ≥200 μ M caused a significant increase in lipid peroxidation, but only in liver slices. However, atractyloside at concentrations of ≥200 μ M caused a marked depletion of GSH and ATP content in both kidney and liver slices. There was a marked decrease in total and individual phospholipid in kidney but not in liver slices. However, cholesterol and triacylglycerol levels were not affected by atractyloside in both kidney and liver slices. Renal and hepatic pyruvate-stimulated gluconeogenesis were significantly (P < 0.05) inhibited at atractyloside concentrations of ≥500 μM. Accumulation of organic anion p-aminohippuric acid (PAH) was also inhibited in renal cortical slices at atractyloside concentrations of ≥500 μM. These results suggest that the observable in vivo effect of atractyloside can be reproduced in slices and that basic mechanistic differences exist in the mode of toxicity in liver and kidney tissues. The data also raise the possibility that the mechanistic basis of metabolic alterations in these tissues following treatment with atractyloside may be relevant to target selective toxicity. Received: 21 January 1998 / Accepted: 23 March 1998     

Inhibition of murine hepatic cytochrome P450 activities by natural and synthetic phenolic compounds

1. The effect of the phenolic compounds protocatechuic acid, chlorogenic acid, tannic acid,gallates and silybinon ethoxyresorufin O-dealkylase(CYP1A1),methoxyresorufin O-dealkylase (CYP1A2) and pentoxy-O-dealkylase (CYP2B) was examined in mouse liver microsomes from induced animals. 2. All compounds tested could inhibit cytochrome P450-mediated enzyme activities, but to different extents. Tannic acid was the most potent inhibitor, especially toward EROD activity with an IC₅₀ = 2.6 μM. Synthetic dodecyl gallate was also relatively selective toward this enzyme activity with an IC₅₀ = 120 μM. 3. Protocatechuic acid,chlorogenicandsilybin were moreselectivetowards PRODand MROD activities. Their relative inhibitory potency for PROD activity was as follows: chlorogenic acid > protocatechuic acid > silybin > dodecyl gallate > propyl gallate. Protocatechuic acid was a more effective inhibitor of MROD activity than chlorogenic acid, and propyl gallate more effective than dodecyl gallate. Thus, no clear structure-activity or selectivity relationship was observed. 4. Analysis of the kinetics of inhibition revealed that the inhibition in most cases was non-competitive in nature.