Acrolein toxicity: comparative in vitro study with lung slices and pneumocytes type II cell line from rats.

Toxicological effects of acrolein have been studied in precision-cut rat lung slices and in L2 cells, a rat pneumocyte II cell line. These two models were cultured for 24 h with or without acrolein (0-100 microM in L2 cells; 0-200 microM in lung slices). Treatment with this pneumotoxicant produced a concentration dependent decrease in intracellular ATP levels. Acrolein concentrations higher than 50 microM induced ATP decrease in slices, while this decrease occurred from 10 microM acrolein in L2 cells. Detoxification marker evaluations showed that mostly the glutathione pathway was altered after acrolein treatment in both models. Intracellular glutathione (GSH) levels were drastically increased with an acrolein concentration of 10 microM. This increase was concomitant with glutathione-S-transferase (GST) and glutathione reductase (GRED) activities in L2 cells. After this strong increase, these enzymatic activities as well as GSH levels were quickly decreased. In precision-cut rat lung slices, the induction of the glutathione pathway was less clear-cut. A bell-shaped dose response curve was observed with a maximum for 5 microM acrolein for GST and GRED activities. These differences between acrolein toxic ranges could be explained by the presence of an active detoxification pathway in slices compared to its relative lack in L2 cells.     

Evaluation of the precision-cut liver and lung slice systems for the study of induction of CYP1, epoxide hydrolase and glutathione S-transferase activities

The principal objective was to ascertain whether precision-cut tissue slices can be used to evaluate the potential of chemicals to induce CYP1, epoxide hydrolase and glutathione S-transferase activities, all being important enzymes involved in the metabolism of polycyclic aromatic hydrocarbons. Precision-cut rat liver and lung slices were incubated with a range of benzo[a]pyrene concentrations for various time periods. A rise in the O-deethylation of ethoxyresorufin was seen in both liver and lung slices exposed to benzo[a]pyrene, which was accompanied by increased CYP1A apoprotein levels. Pulmonary CYP1B1 apoprotein levels and hepatic mRNA levels were similarly enhanced. Elevated epoxide hydrolase and glutathione S-transferase activities were also observed in liver slices following incubation for 24h; similarly, a rise in apoprotein levels of both enzymes was evident, peak levels occurring at the same time point. When mRNA levels were monitored, a rise in the levels of both enzymes was seen as early as 4h after incubation, but maximum levels were attained at 24 h. In lung slices, induction of epoxide hydrolase by benzo[a]pyrene was observed after a 24-h incubation, and at a concentration of 1 microM; a rise in apoprotein levels was seen at this time point. Glutathione S-transferase activity was not inducible in lung slices by benzo[a]pyrene but a modest increase was observed in hepatic slices. Collectively, these studies confirmed CYP1A induction in rat liver slices and established that CYP1B1 expression, and epoxide hydrolase and glutathione S-transferase activities are inducible in precision-cut tissue slices.     

Uptake–toxicity relationships of a series of N-substituted N′-(4-imidazole-ethyl)thiourea in precision-cut rat liver slices

A previous study showed that the cytotoxicity of a series of N-p-phenyl-substituted N′-(4-imidazole-ethyl)thiourea in precision-cut rat liver slices increased with increasing electron-withdrawing capacity of the p-substituent and may be related to the V_max/K_m values of bioactivation of the thiourea-moiety by hepatic flavin-containing monooxygenases (FMOs). However, differences in the uptake of xenobiotics into precision-cut liver slices can also have consequences for the rates of metabolism of xenobiotics. In the present study, therefore, we investigated the rate and nature of uptake of 9 N-substituted N′-(4-imidazole-ethyl)thiourea into precision-cut rat liver slices. It was found that a five-fold difference exists among a series of N-substituted N′-(4-imidazole-ethyl)thiourea both in the initial rate of uptake and in the steady-state levels ultimately achieved in the precision-cut rat liver slices. It appeared that the most cytotoxic compounds were also the most readily absorbed compounds. The concentration-dependent initial rate of uptake could be described by a carrier-mediated saturable component and a non-saturable component. At cytotoxic concentrations, the non-saturable component accounted for more than 95% of the total uptake. From this study, it is concluded that differences in rate of uptake of thiourea-containing compounds may be a contributing factor to the differences in bioactivation by FMOs as the basis of the structure–toxicity relationships observed in precision-cut rat liver slices.     

Applicability of rat precision-cut lung slices in evaluating nanomaterial cytotoxicity,apoptosis, oxidative stress,and inflammation

The applicability of rat precision-cut lung slices (PCLuS) in detecting nanomaterial (NM) toxicity to the respiratory tract was investigated evaluating sixteen OECD reference NMs (TiO₂, ZnO, CeO₂, SiO₂, Ag, multi-walled carbon nanotubes (MWCNTs)). Upon 24-hour test substance exposure, the PCLuS system was able to detect early events of NM toxicity: total protein, reduction in mitochondrial activity, caspase-3/-7 activation, glutathione depletion/increase, cytokine induction, and histopathological evaluation. Ion shedding NMS (ZnO and Ag) induced severe tissue destruction detected by the loss of total protein. Two anatase TiO₂ NMs, CeO₂ NMs, and two MWCNT caused significant (determined by trend analysis) cytotoxicity in the WST-1 assay. At non-cytotoxic concentrations, different TiO₂ NMs and one MWCNT increased GSH levels, presumably a defense response to reactive oxygen species, and these substances further induced a variety of cytokines. One of the SiO₂ NMs increased caspase-3/-7 activities at non-cytotoxic levels, and one rutile TiO₂ only induced cytokines. Investigating these effects is, however, not sufficient to predict apical effects found in vivo. Reproducibility of test substance measurements was not fully satisfactory, especially in the GSH and cytokine assays. Effects were frequently observed in negative controls pointing to tissue slice vulnerability even though prepared and handled with utmost care. Comparisons of the effects observed in the PCLuS to in vivo effects reveal some concordances for the metal oxide NMs, but less so for the MWCNT. The highest effective dosages, however, exceeded those reported for rat short-term inhalation studies. To become applicable for NM testing, the PCLuS system requires test protocol optimization.     

Effect of selenium on glutathione metabolism. Induction of gamma-glutamylcysteine synthetase and glutathione reductase in the rat liver

The effect of sodium selenite (Na₂SeO₃, Se) on cellular glutathione metabolism was examined, particularly with respect to its ability to alter the activities of γ-glutamylcysteine synthetase and glutathione disulfide (GSSG) reductase. The treatment of rats with Se (5, 10 and 20 μmoles/kg) caused time- and dose-dependent increases in the activities of the synthetase and the reductase in the liver. The activity of γ-glutamylcysteine synthetase, the rate-limiting enzyme of the glutathione (GSH) biosynthesis, was particularly susceptible to Se treatment. The Se-mediated increases in the activities of the above enzymes were inhibited by puromycin and the increases could not be elicited in vitro. Selenium treatment caused time-dependent perturbations in the levels and ratio of GSSG and GSH in the liver. When compared to the control animals, rats treated for 3 hr with 10 and 20 μmoles Se/kg showed increased cellular levels of GSSG; in contrast, 24 hr after Se treatment the concentration of GSH was increased significantly. The activity of γ-glutamyl transpeptidase, which catalyzes the initial reaction in GSH breakdown, was unaltered by Se treatment. Repeated administration of low doses of Se (7.0 μmoles/kg, three times) also increased the activities of the reductase and the synthetase as well as the cellular levels of hepatic GSH and GSSG. It is suggested that the Se-mediated increases in the activities of γ-glutamylcysteine synthetase and GSSG-reductase represent cellular responses to Se-mediated perturbations in the levels and ratio of GSH and GSSG.     

Tissue-specific effects of fenthion on glutathione metabolism modulated by NAC and BSO in Oreochromis niloticus

Introduction:?The present study was designed to understand the effects of organophosphate (OP) insecticide and avicide fenthion on cellular redox status and the role of reduced glutathione (GSH) on fenthion toxicity in the liver and kidney of Oreochromis niloticus as a model organism. N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) were injected intraperitoneally to fenthion-exposed fish as modulators of GSH metabolism. GSH redox status, GSH-related enzyme activities, and thiobarbituric acid reactive substances (TBARS) contents were then measured spectrophotometrically at 24, 48, and 96 hours. To assess recovery from fenthion exposure, similar analyses were performed on fish transferred to non-treated water for 24, 48, and 96 hours.

Results:?Fenthion increased glutathione S-transferase (GST; EC 2.5.1.18) activity and caused changes in total GSH (tGSH), GSH and oxidized glutathione (GSSG) contents and glutathione peroxidase (GPx; EC 1.11.1.9) specific activity in the liver tissue over time. Increases observed in tGSH and GSSG contents at 24 hours were decreased by fenthion treatment at 96 hours. BSO caused a sharp decline in liver tGSH, GSH, and GSSG contents and an elevation in GST and γ-glutamyl transpeptidase (γ-GT; EC 2.3.2.2) enzyme activities. A significant decrease was observed in tGSH and GSH contents and, also, GST enzyme activities in the kidney at 48-hour fenthion treatment. On the contrary to the liver, a significant increase was observed in tGSH and GSH contents in the kidney by BSO injection. NAC application eliminated the decreasing effects of fenthion on GST activity in this tissue. NAC injection caused decreases in lipid peroxidation (LPO) levels. Decline in tGSH and GSH contents were maintained in the liver during the recovery period, and elevations in LPO levels in the kidney were observed during the same period.

Conclusions:?In conclusion, tissue-specific and time-dependent GSH redox status disturbance of fenthion were observed. BSO revealed the significance of GST-mediated GSH conjugation on the detoxification process of fenthion. NAC seemed useful to avoid the fenthion-related oxidative toxicity.     

Comparison of the toxicity of allyl alcohol,coumarin and menadione in precision-cut rat,guinea-pig,Cynomolgus monkey and human liver slices

 The toxicity of allyl alcohol, coumarin and menadione has been studied in precision-cut liver slice cultures. Liver slices were prepared from male Sprague- Dawley rats, male Dunkin-Hartley guinea-pigs and from samples of Cynomolgus monkey and human liver using a Krumdieck tissue slicer. The liver slices were cultured with the test compounds for 24h in a dynamic organ culture system. Toxicity was assessed by measurement of protein synthesis, potassium content and the MTT assay. At the concentrations examined, menadione produced marked toxicity in liver slices from all four species, whereas rat liver slices were less susceptible to allyl alcohol toxicity. Coumarin produced concentration-dependent toxic effects in rat and guinea-pig liver slices, whereas Cynomolgus monkey and human liver slices were relatively resistant, especially at low coumarin concentrations. At some concentrations of the test compounds examined, the MTT assay appeared to be a less sensitive indicator of toxicity than either protein synthesis or potassium content. These results demonstrate the usefulness of precision-cut liver slices for assessing species differences in xenobiotic-induced toxicity. Received: 21 March 1996/Accepted: 4 June 1996     

Toxicity of 3-methylindole, 1-nitronaphthalene and paraquat in precision-cut rat lung slices

 The toxicity of 3-methylindole, 1-nitronaphthalene and paraquat has been studied in precision-cut rat lung slice cultures. Lung slices were prepared from male Sprague-Dawley rats using an agarose gel instilling technique with a Krumdieck tissue slicer and cultured for 24 h in a dynamic organ culture system. Treatment of rat lung slices with 3-methylindole, 1-nitronaphthalene or paraquat produced concentration dependent decreases in lung slice protein synthesis and potassium content. EC₅₀ values (concentration to produce a 50% inhibition) for protein synthesis were 0.024, 0.27 and 0.57 mM for paraquat, 1-nitronaphthalene and 3-methylindole, respectively. These results demonstrate that precision-cut lung slices are a useful in vitro model system for studying the pulmonary toxicity of xenobiotics. Lung slices offer the potential as a rapid in vitro screen for identifying pulmonary toxicants and to evaluate species differences in response. Received: 10 October 1994 / Accepted: 30 November 1994     

Effect of some indole derivatives on xenobiotic metabolism and xenobiotic-induced toxicity in cultured rat liver slices.

In this study the effect of some indole derivatives on xenobiotic metabolizing enzymes and xenobiotic-induced toxicity has been examined in cultured precision-cut liver slices from male Sprague-Dawley rats. While treatment of rat liver slices for 72 hours with 2-200 microM of either indole-3-carbinol (I3C) or indole-3-acetonitrile (3-ICN) had little effect on cytochrome P-450 (CYP)-dependent enzyme activities, enzyme induction was observed after in vivo administration of I3C. The treatment of rat liver slices with 50 microM 3,3'-diindolylmethane (DIM; a dimer derived from I3C under acidic conditions) for 72 hours resulted in a marked induction of CYP-dependent enzyme activities. DIM appears to be a mixed inducer of CYP in rat liver slices having effects on CYP1A, CYP2B and CYP3A subfamily isoforms. Small increases in liver slice reduced glutathione levels and glutathione S-transferase activity were also observed after DIM treatment. While aflatoxin B1 and monocrotaline produced a concentration-dependent inhibition of protein synthesis in 72-hour-cultured rat liver slices, cytotoxicity was markedly reduced in liver slices cultured with 50 microM DIM. These results demonstrate that cultured rat liver slices may be employed to evaluate the effects of chemicals derived from cruciferous and other vegetables on CYP isoforms. In addition, liver slices can also be utilized to examine the ability of such chemicals to modulate xenobiotic-induced toxicity.     

Identification and quantitation of glutathione in hepatic protein mixed disulfides and its relationship to glutathione disulfide

The amount of glutathione present in hepatic protein mixed disulfides was determined to be 20–30 nmole/g liver. This was established using two specific enzymatic methods: (a) the coupled assay with DTNB and glutathione (GSSG) reductase and (b) a newly developed test using GSH transferase and 1-chloro-2,4-dinitrobenzene for the estimation of GSH released from proteins after borohydride treatment; further, these results were confirmed by HPLC analysis. Thus, authentic glutathione makes up only 2–6% of the value for total protein mixed disulfides. The latter were determined with the generally employed o-phthalaldehyde assay, which is not necessarily specific for GSH. The amount of glutathione mixed disulfides depends linearly on the content of glutathione disulfide in the liver cell in the range studied. By increasing the GSSG levels from 20 to about 60 nmole/g liver with paraquat, nitrofurantoin or t-butyl hydroperoxide, glutathione protein mixed disulfides are increased by a similar amount.     

Effect of reduced glutathione (GSH) on pharmacokinetics and distribution of rifamycin SV in rats

Changes of the pharmacokinetic parameters of rifamycin SV in the blood and changes of the antibiotic concentration in rat organs after its coadministration with glutathione in reduced form (GSH). in oxidized form (GSSG) and with the individual amino acids entering the tripedtide were studied. The use of reduced glutathione together with rifamycin was found to accelerate the distribution phase in the kinetics of the antibiotic in the blood by acceleration of transport of the drug from central compartment (blood) to tissue compartment (tissues), this being reflected by increased K₁₂ and K₁₂/K₂₁ values. Studies of the distribution of rifamycin in individual rat organs have shown GSH to significantly elevate the concentration of the antibiotic in the lungs with concomitant reduction of its level in the liver. The blocking of free sulfhydryl groups of glutathione by its use in oxidized form completely abolished this effect. The administration of rifamycin together with l-cysteine resulted in increased concentration of the antibiotic in both lungs and liver. The two other amino acids entering GSH —glycine and l-glutamic acid — did not cause any changes of rifamycin concentration in the organs. From these results it may be concluded that the mechanism of the observed effect of glutathione on rifamycin concentration differs in the lungs and liver though both effects are mediated by the free sulfhydryl groups of glutathione.     

Protective effect of N-acetyl-L-cysteine against maneb induced oxidative and apoptotic injury in Chinese hamster V79 cells

The role of antioxidant N-acetyl-L-cysteine (NAC) in protection against cellular changes triggered by maneb during in vitro exposure was investigated in cultured Chinese hamster V79 cells. We observed high apoptotic activity and high oxidative stress induced by exposure to maneb evidenced by a statistically significant increase in lipid peroxidation (measured as TBARS - thiobarbituric acid reactive substances) as well as a decrease of glutathione (GSH) and glutathione disulfide (GSSG) ratio (GSH/GSSG). Maneb did not exhibit any effect on protein oxidation (measured by protein carbonyls content). NAC suppressed cellular changes induced by maneb in V79 cells. NAC pre-treatment prevented TBARS production and significantly decreased the number of apoptotic cells. However, protective effect of NAC on GSH and GSSG levels has been shown only in cells exposed to lower concentration of maneb (100 μM).     

Hydrogen peroxide increases the activity of γ-glutamylcysteine synthetase in cultured Chinese hamster V79 cells

Hydrogen peroxide (H₂O₂) caused a rapid and a concentration-dependent increase in the activity of γ-glutamylcysteine synthetase (γ-GCS) in cultured Chinese hamster V79 cells. The increase in the activity was transient and declined rapidly during post-treatment incubation. Inhibition of protein synthesis by cycloheximide, chelation of divalent iron byo-phenanthroline, and scavenging of free radicals by butyl-4-hydroxyanisole failed to suppress the increase in activity of γ-GCS caused by H₂O₂. However, catalase completely inhibited the increase in the activity of the enzyme. H₂O₂ did not change the level of total glutathione (GSH+GSSG) but is oxidized GSH. The increased in levels of GSSG caused by H₂O₂ was enhanced byo-phenanthroline. These results suggest that the increase in activity of γ-GCS caused by H₂O₂ is not an inducible phenomenon, nor it is attributable to the action of free radicals generated by an iron-catalyzed Fenton reaction. Furthermore, the changes in levels of GSH and GSSG caused by H₂O₂ appear not to be responsible for the increase in activity of γ-GCS caused by the hydroperoxide. However, chemical reduction of the enzyme, the activity of which had been increased by H₂O₂, resulted in a decrease, in the activity, suggesting the involvement of oxidation of the enzyme in the increased activity of γ-GCS caused by H₂O₂. The results also suggest that the activity of γ-GCS in cultured V79 cells can be regulated by the cellular oxidation-reduction state.     

Comparative in vitro metabolism of the suspected pro-oestrogenic compound,methoxychlor in precision-cut liver slices from male and female rats

The in vitro metabolism of [¹⁴C]methoxychlor (MXC), a suspected pro-oestrogenic compound, by male and female Fischer rats (F344) was compared in precision-cut liver slices. The results demonstrated time-dependent metabolism of MXC with integrated phase I and II reactions, and the sex differences were detected in the metabolic profiles. In liver slices from male rats, MXC was metabolized to bis-demethylated MXC (bis-OH-MXC) by sequential O-demethylation followed by subsequent O-glucuronidation. The doubly conjugated metabolite, bis-OH-MXC 4-O-sulphate 4′-O-glucuronide was additionally produced. In the case of the female rat, the glucuronides of both mono- and bis-OH-MXC were formed as the main metabolites, and the mono-OH-MXC glucuronide appeared to be specific to the female rat. The ratios of bis-/mono-demethylated metabolite, which include the amounts of corresponding conjugates, were approximately 95/5 for the male rats and 40/60 for the female. These results imply that demethylation to the intermediate metabolite, (S)-mono-OH-MXC, is a key step for the sex-dependent metabolism of MXC in the rats. The phase I metabolites produced were extensively conjugated with D-glucuronic acid in both male and female rats.     

Drug-metabolizing activity of human and rat liver,lung, kidney and intestine slices

1. Organ-specific biotransformation was studied in human and rat liver, lung, kidney and small intestine slices and compared on a protein basis, using four model substances. 2. Deethylation of lidocaine was highest in liver slices from both man and rat, followed by the small intestine. 3. Metabolism of testosterone was highest in liver slices, but a different overall metabolic pattern was found between the different organs. 4. Lung, kidney and intestine slices prepared from human and rat organs showed mainly an unknown metabolite of 7-ethoxycoumarin identified as 4-ethoxy-2-hydroxyphenyl propionic acid (EPPA). 5. The maximal metabolism of 7-ethoxycoumarin in slices was equal with in vivo V_max in the rat. 6. Phase II metabolism of 7-hydroxycoumarin in kidney and intestinal slices was about 60% of the activity in liver slices. 7. In conclusion, organs other than the liver show a surprisingly high drug-metabolizing activity. Thus, the use of precision-cut slices of a combination of drug metabolizing organs in an in vitro test system from both animal and human origin is required for a proper systematic prediction of drug metabolism in man.     

Phenethyl isocyanate is not the metabolite of phenethyl isothiocyanate responsible for mechanism-based inhibition of cytochrome P450

Phenethyl isothiocyanate is a chemopreventive phytochemical present in cruciferous vegetables where it exists as the glucosinolate gluconasturtiin. It is a mechanism-based inhibitor of both rat and human cytochrome P450 enzymes. The principal objective of the present study was to ascertain whether phenethyl isocyanate, formed by the cytochrome P450-mediated oxidative desulphuration of phenethyl isothiocyanate, is the metabolite responsible for the mechanism-based inhibition. Phenethyl isothiocyanate, following incubation with Aroclor 1254-induced rat liver microsomes in the presence of NADPH, markedly suppressed the CYP1A-mediated O-deethylation of ethoxyresorufin; extent of inhibition was directly related to the pre-incubation time and was antagonised by reduced glutathione. When human liver microsomes were used, the inhibitory effect of phenethyl isothiocyanate, which was once again related to the pre-incubation time, was even more pronounced. When the ability of phenethyl isothiocyanate and phenethyl isocyanate to directly inhibit the O-deethylation of ethoxyresorufin in rat microsomes was compared, the latter compound was only moderately more effective. In human microsomes, both compounds were equipotent. In phenobarbital-induced lung microsomes, phenethyl isothiocyanate was a direct and potent inhibitor of the O-depentylation of pentoxyresorufin; pre-incubation of the isothiocyanate had no impact. Human precision-cut liver slices were more effective than rat slices in metabolising phenethyl isothiocyanate. Pre-treatment of rats, however, with phenobarbitone significantly enhanced the metabolism of isothiocyanate. It may be inferred from the present studies that: (a) phenethyl isocyanate is not the metabolite of phenethyl isothiocyanate responsible for its mechanism-based inhibition, and (b) CYP2B is an important catalyst of the metabolism of phenethyl isothiocyanate.     

Comparison of cyclosporine and FK506 effects on glutathione levels in rat cochlea, brain, liver and kidney.

Cyclosporine treatment (50 mg/kg/day, p.o.) caused increases in rat renal reduced glutathione (GSH) levels of 205 and 673%, respectively, after 5 and 10 days. No changes were seen in liver GSH with either dose of cyclosporine. FK506 (2.5 mg/kg/day, p.o., for 7 days) caused an approximately 200% increase in kidney GSH, and an approximately 250% increase in hepatic GSH levels. Oxidized glutathione (GSSG) was never more than 1-2% of the level of the reduced form in any tissue from control animals. Small increases in the ratios of oxidized to reduced glutathione were seen in livers and kidneys from both cyclosporine- and FK506-treated animals. No changes in GSH or GSSG levels were seen in brains or cochleas from any animal.     

LLC-PK CELLS AS A MODEL FOR RENAL TOXICITY CAUSED BY ARSINE EXPOSURE

The mechanisms of arsine (AsH₃) toxicity are not completely understood. Studies were undertaken to determine AsH₃ and arsenite [As(III)] toxicity in a renal tubular epithelial cell line to model kidney dysfunction caused by AsH₃ exposure. The hypothesis was that As(III) is the toxic metabolite responsible for the renal toxicity of AsH₃. There was a concentration- and time-dependent toxic response after As(III) incubation. As(III) produced significant LDH leakage as early as 1 h and intracellular potassium loss at 5 h. AsH₃ produced no changes in these parameters. AsH₃ affected neither potassium nor LDH levels over 24 h and up to 1 mM AsH₃ concentration. In this system, As(III) induced LDH leakage before K+ loss. Oxidative stress-like toxicity effects were also studied by determining levels of glutathione (GSH), glutathione disulfide (GSSG), and heat-shock protein 32 (Hsp32) levels. GSH levels were not markedly affected by any arsenical over a 6-h period or up to 100 µM concentration of the arsenical. However, 100 µM AsH₃ significantly increased GSSG levels as early as 30 min and reached a maximum at 2.5 h. Incubation with 10 µM AsH₃ was sufficient to significantly increase GSSG levels. As(III) had no marked effect on GSSG. Both arsenicals (50 µM) produced a slight increase (about threefold) in Hsp32 levels after 4-h incubation. These results showed that unchanged AsH₃ produced oxidative stress-like toxic effects without producing cell death. However, similar As(III) concentrations induced the stress response and were toxic to the cells. These data indicated that AsH₃ is not directly toxic to LLC-PK₁ cells.     

FACTORS INVOLVED IN HEPATIC GLUTATHIONE DEPLETION INDUCED BY ACUTE ETHANOL ADMINISTRATION

Factors implicated in changes of the hepatic glutathione concentration following acute ethanol administration were examined in rats. Adult female rats were treated with either ethanol (4 g/kg, po) or an isocaloric glucose solution. The hepatic reduced glutathione (GSH) concentration decreased rapidly after ethanol intake with a maximum diminution, approximately 50% of the control value, being observed at t = 6 h. The hepatic GSH concentration gradually increased, and finally rebounded at 24 h after ethanol ingestion. The dose of ethanol induced a transient increase in the oxidized glutathione (GSSG) and GSSG/GSH ratio, which was associated with a significant reduction in GSH rather than elevation in GSSG. The activity of g-glutamylcysteine synthetase (GCS), the rate-limiting enzyme for glutathione synthesis, and the cysteine concentration in liver were also measured. The GCS activity was depressed to approximately 80% of the control value at t = 2.5 h followed by rapid recovery, but no difference in the hepatic cysteine concentration between control and ethanol treated rats was observed for 24 h, suggesting that the reduction in glutathione synthesis may not play a major role in the significant depletion of this tripeptide in liver. The total glutathione concentration was measured both in prehepatic and posthepatic inferior vena cava blood. The glutathione concentration in posthepatic blood was approximately twice as high as that of prehepatic blood in control rats. Acute ethanol administration doubled the elevation of glutathione in posthepatic blood measured at t = 2.5 h. The sinusoidal efflux of glutathione estimated from the increase in blood glutathione concentration was greater than the total amount of its depletion in the liver of rats treated with ethanol. The results suggest that in the liver of rats treated acutely with ethanol, glutathione efflux plays the most important role in the reduction of this tripeptide, which would be aggravated by a transient decrease in glutathione synthesis and by increased consumption in association with its metabolism.     

Comparison of the metabolism of ethylene glycol and glycolic acid in vitro by precision-cut tissue slices from female rat,rabbit and human liver

1.?The metabolism of [1,2-¹⁴C]-ethylene glycol and [1,2-¹⁴C]-glycolic acid was studied in vitro using precision-cut tissue slices prepared from the livers of female Sprague—Dawley rats, New Zealand white rabbits and humans. The time-course for production of metabolites formed from ethylene glycol at concentrations from 3 to 40 mM was determined to compare quantitatively the differences between species in the rates and amounts of formation of glycolic acid, the presumed developmental toxicant of ethylene glycol. The rates of metabolism of glycolic acid to glyoxylic acid at concentrations from 0.05 to 16 mM by liver tissue from the different species were also determined. The apparent V_maJK_m for the metabolic conversions of ethylene glycol to glycolic acid and for glycolic acid to glyoxylic acid in liver tissue from the different species were obtained.

2.?There were qualitative differences in the metabolic profiles and quantitative differences in the formation of glycolic acid between the mammalian liver systems. There was an average of 10-fold less glycolic acid produced by liver slices from rabbits compared with rats. With the human liver, the formation of glycolic acid was not detectable using tissue from three of four human donors. A low level of glycolic acid was detected in one liver slice incubation from one of the four subjects, but only at one extended time point; glyoxylate was detected with liver slices from all four humans.

3.?Liver slices prepared from female Sprague—Dawley rats, female New Zealand White rabbits and three female human subjects all metabolized glycolic acid to glyoxylic acid. Human liver tissue was the most effective at further metabolizing glycolic acid to glyoxylic acid. The ratios of F"_max/-/C_m, representing the relative clearance of glycolic acid from liver tissue, were approximately 14:9:1 for human, rat and rabbit liver, respectively.

4.?Precision-cut liver slices maintained in dynamic organ culture are good predictors of metabolism by liver tissue in vivo. The results of the present study therefore indicate that levels of glycolic acid, if formed in vivo, following exposures to similar concentrationsof ethylene glycol, would be lower in humans than in rabbits and rats.