Efficient analysis of hepatic glucose output and insulin action using a liver slice culture system.

BACKGROUND: Liver slices have been reported to retain histological integrity and metabolic capacity for over 24 hours in flask culture systems, and they have been used for pharmacological and toxicological studies before. However, whether this method is suitable to measure hepatic glucose output is unknown. METHODS: Precision-cut liver slices were prepared from fresh male rat liver. After high-glucose pre-incubation (11.2 mmol/l), medium was changed to low-glucose conditions (0.5 mmol/l). Glucose and lactate levels as well as aspartate aminotransferase activity were monitored for 50 minutes with or without addition of insulin (600 pmol/l) and/or epinephrine (0.5 micromol/l). Slice potassium content and histology were examined to prove liver viability. RESULTS: We observed a stable glucose production from the liver slices of 0.3-0.4 micromol/g liver/min. Epinephrine increased (by 82+/-30%) and insulin decreased (by 80+/-8%) liver slice glucose output. Significant signs of ischemia were not detected. CONCLUSIONS: Hepatic glucose release can be reliably measured in a liver slice culture system, and it is regulated by major hormone systems. This method may be helpful for further characterization of direct insulin action and resistance in a complex tissue as the liver; however, pharmacological applications such as the analysis of drug effects on hepatic glucose metabolism can also be envisioned.     

Cryopreservation of pig and human liver slices

The ability to cryopreserve human liver slices would greatly enhance the opportunities to test potentially hepatotoxic drugs and environmental contaminants as well as the metabolism of these compounds. This study focused on trying to cryopreserve pig and human liver slices. Since the acquisition of human liver tissue is unpredictable and scarce, an animal model was sought to predict problems associated with cryopreservation of human tissue. The pig liver was chosen because of its anatomical and physiological resemblance to human liver. The human liver tissues that did become available were obtained through the Arizona Organ Bank and the National Disease Research Interchange and from surgical liver resections. An in vitro culture system that employed precision-cut liver slices was used in this study. Different types and concentrations of cryoprotectants, cooling rates, and culture media were all tried in an attempt to cryopreserve pig and human liver slices. The viabilities of fresh and cryopreserved liver slices were evaluated using slice K+ retention and protein synthesis. Pig liver slices following cryopreservation retained between 80 and 85% of intracellular K+ content and protein synthesis as compared to controls using 1.4 M Me2SO, a 12 degrees C/min cooling rate, and a rapid rewarming rate of direct submersion of the slice into 37 degrees C fetal calf serum. Human liver slices following cryopreservation retained between 54 and 89% of intracellular K+ content and protein synthesis as compared to controls using the same protocol as for pigs, except that lower cooling rates were giving better results. The large variation seen in cryopreserved human liver slices was due to the length of warm and cold ischemia to which the tissue was exposed before arriving at the laboratory. This study indicated that pig and human liver slices can be cryopreserved and used for future toxicological and metabolic studies.     

Cryopreservation of rat precision-cut liver and kidney slices by rapid freezing and vitrification

Precision-cut tissue slices of both hepatic and extra-hepatic origin are extensively used as an in vitro model to predict in vivo drug metabolism and toxicity. Cryopreservation would greatly facilitate their use. In the present study, we aimed to improve (1) rapid freezing and warming (200 degrees C/min) using 18% Me(2)SO as cryoprotectant and (2) vitrification with high molarity mixtures of cryoprotectants, VM3 and VS4, as methods to cryopreserve precision-cut rat liver and kidney slices. Viability after cryopreservation and subsequent 3-4h of incubation at 37 degrees C was determined by measuring ATP content and by microscopical evaluation of histological integrity. Confirming earlier studies, viability of rat liver slices was maintained at high levels by rapid freezing and thawing with 18% Me(2)SO. However, vitrification of liver slices with VS4 resulted in cryopreservation damage despite the fact that cryoprotectant toxicity was low, no ice was formed during cooling and devitrification was prevented. Viability of liver slices was not improved by using VM3 for vitrification. Kidney slices were found not to survive cryopreservation by rapid freezing. In contrast, viability of renal medullary slices was almost completely maintained after vitrification with VS4, however vitrification of renal cortex slices with VS4 was not successful, partly due to cryoprotectant toxicity. Both kidney cortex and medullary slices were vitrified successfully with VM3 (maintaining viability at 50-80% of fresh slice levels), using an optimised pre-incubation protocol and cooling and warming rates that prevented both visible ice-formation and cracking of the formed glass. In conclusion, vitrification is a promising approach to cryopreserve precision-cut (kidney) slices.     

Use of human organ slices to evaluate the biotransformation and drug-induced side-effects of pharmaceuticals

Human liver and kidney organ slices were used to investigate the biotransformation competence of the slices in combination with several markers of cell viability and function. The immunosuppressant cyclosporin A (CSA) is extensively metabolized in liver slices to the three known primary metabolites and many secondary metabolites. In kidney cortex slices the biotransformation of CSA is far more pronounced in humans than in rats. In human liver slices, levels of CYP3A, the proteins metabolizing CSA, are depressed about 25% by 1 and 10 mol/L CSA within 24 h, indicating that high blood or tissue concentrations will inhibit CSA clearance.A clinical marker for liver damage is the release of cellular -glutathione-S-transferases (GST). In this study the GST levels were used to assess donor organ quality, organ slice incubation conditions, and compound exposure. A marker for cell death in human cells is the solubilization and release of nuclear matrix proteins (Numa). Increases were apparent only after 48 h of culture.A side-effect of CSA is that it induces hypertension and perturbs the lipid profile of transplant recipients. A potential marker for lipid disturbances is levels of serum lipoprotein (a) (Lp(a)), which is synthesized in the liver and found only in humans, apes, and nonhuman primates. CSA increases Lp(a) levels in the human liver slice cultures about 2-fold.This study has demonstrated that the biotransformation capability of the organ slices contributes to the optimization of thein vitro system and to the evaluation of markers for drug induced side-effects or toxicity. Assays were identified that could be used clinically to monitor CSA-induced organ damage or rejection (GST), hypertension (Lp(a)), and toxicity (Numa).Abbreviations GST -glutathione-S-transferases - CSA cyclosporin A - CYP3A cytochrome P4503A - CSX undefined cyclosporin - Lp(a) lipoprotein (a) - IMM SDZ IMM 125 - Numa nuclear matrix proteins     

Cryopreserved rat liver slices: a critical evaluation of cell viability, histological integrity, and drug-metabolizing enzymes

The effects of a cryopreservation procedure on the biochemical, morphological and functional integrity of rat liver slices just after thawing and after 24 h culture were evaluated. Freshly prepared slices were incubated in modified University of Wisconsin solution containing 50% fetal calf serum and 10% dimethyl sulfoxide for 20 min on ice prior to a rapid cooling in liquid nitrogen. After 10-40 days, slices were thawed rapidly at 42 degrees C. Total protein content and (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) (MTT) reduction were well preserved at thawing, whereas ATP content was markedly decreased relative to freshly prepared slices (-83%). The major microscopic findings in sections of just-thawed liver slices consisted of hepatocellular dissociation and minimal apoptosis. The qualitative profile of antipyrine (AP) metabolism was well preserved in cryopreserved slices, but the amounts of phase I and phase II AP metabolites produced over a 3-h incubation period were markedly reduced relative to fresh slices (-58 to -71%). When cryopreserved slices were cultured for 24 h after thawing, the viability was markedly reduced, as reflected by the almost complete absence of MTT reduction and the loss of ATP content. Histological examinations showed extensive cellular necrosis. The amount of AP metabolites produced by cryopreserved slices was similar after a 3- or a 24-h culture period, indicating that AP metabolism capacities were lost at 24 h culture. In conclusion, our results suggest that cryopreserved rat liver slices may be a useful model for short-term in vitro determination of drug metabolism pathways. Further work is required to extend their use for toxicological studies.     

Water crystallization within rat precision-cut liver slices in relation to their viability

This study examined whether tissue vitrification, promoted by partitioning within the tissue, could be the mechanism explaining the high viability of rat liver slices, rapidly frozen after preincubation with 18% Me2SO or VS4 (a 7.5 M mixture of Me2SO, 1,2-propanediol, and formamide with weight ratio 21.5:15:2.4). To achieve this, we first determined the extent to which crystallization or vitrification occurred in cryoprotectant solutions (Me2SO and VS4) and within liver slices impregnated with these solutions. Second, we determined how these events were related to survival of slices after thawing. Water crystallization was evaluated by differential scanning calorimetry and viability was determined by histomorphological examination of the slices after culturing at 37 degrees C for 4 h. VS4-preincubated liver slices indeed behaved differently from bulk VS4 solution, because, when vitrified, they had a lower tendency to devitrify. Vitrified VS4-preincubated slices that were warmed sufficiently rapid to prevent devitrification had a high viability. When VS4 was diluted (to 75%) or if warming was not fast enough to prevent ice formation, slices had a low viability. With 45% Me2SO, low viability of cryopreserved slices was caused by cryoprotectant toxicity. Surprisingly, liver slices preincubated with 18% Me2SO or 50% VS4 had a high viability despite the formation of ice within the slice. In conclusion, tissue vitrification provides a mechanism that explains the high viability of VS4-preincubated slices after ultrarapid freezing and thawing (>800 degrees C/min). Slices that are preincubated with moderately concentrated cryoprotectant solutions (18% Me2SO, 50% VS4) and cooled rapidly (100 degrees C/min) survive cryopreservation despite the formation of ice crystals within the slice.     

Morphological and functional integrity of precision-cut rat liver slices in rotating organ culture and multiwell plate culture: Effects of oxygen tension

We examined the maintenance of functional and morphological integrity of precision-cut rat liver slices cultured in various incubation systems and conditions for 72 h. Slices were incubated (37°C) for 6, 24, 48, and 72 h in supplemented Williams E medium in 6-well plastic culture plates on a gyratory shaking platform (WPCS) or in a rotating organ culture system (ROCS) using 5% CO₂–95% air (WPCS/air or ROCS/air) or 5% CO₂–70% O₂–25% N₂ (WPCS/ O₂ or ROCS/ O₂). Biochemical and functional parameters of slices maintained in WPCS/air or WPCS/ O₂ were almost totally inhibited after 24 h, in keeping with the extensive and diffuse coalescing coagulative necrosis typical of post-ischemic injury affecting almost all the slice surface after 48 h. As compared to freshly isolated slices, slices maintained in ROCS/air for 72 h showed stable ATP and GSH content, increased protein synthesis, and a slight steady decrease in GST activity, while ATP and GST activity remained stable and protein synthesis and GSH content increased in slices incubated in ROCS/ O₂ for 72 h. The extent of coagulative necrosis was markedly lower in longitudinal sections from slices incubated for 72 h in ROCS/ O₂ than in ROCS/air. Transversal sections from slices kept in ROCS/air for 72 h showed a thick central band of necrotic cells edged by two peripheral layers of viable hepatocytes, whereas most of the slice was composed of viable hepatocytes lined by two thin layers of necrotic cells after 72 h in ROCS/ O₂. ROCS/ O₂ emerged as the system best preserving the histological and functional integrity of rat liver slices in long-term culture.     

An organ culture of postnatal rat liver slices

Summary A technique for the organ culture of postnatal and adult rat liver has been developed. Liver slices, 0.3 mm thick, were maintained in Conway units at the interphase between medium and a 95% O₂:5% CO₂ atmosphere. Postnatal liver in culture for up to 72 h had healthy hepatocytes throughout the explants; if adult liver was used the upper 0.2 mm was healthy after 24 h. These slices incorporated tritiated orotate and leucine into trichloroacetic acid-precipitable material. Incorporation of orotate was shown to be spread over the entire slice of neonatal liver. Culturing did not alter the potassium ion content of postnatal liver. Tyrosine aminotransferase activity in liver slices from postnatal, adult, and adrenalectomized adult rats was stimulated by glucocorticoids and dibutyryl cyclic AMP. Cycloheximide and actinomycin D prevented this response. Further, cortisol exerted a permissive effect on the stimulation of tyrosine aminotransferase activity by dibutyryl cyclic AMP in slices from adrenalectomized rats. Induction of urea cycle enzymes by cortisol was demonstrated in cultures of liver from adrenalectomized adult animals. Deceased October 1, 1983. This research was supported in part by a grant from the South African Council for Scientific and Industrial Research.     

Differential effects of estrogen on luteinizing hormone-releasing hormone gene expression in slice explant cultures prepared from specific rat forebrain regions

Five serially sectioned tissue slices (400 microns) from the preoptic area/hypothalamus of postnatal day 4 rats were cultured using a slice explant roller culture technique. After 18 days in culture, these slices thinned sufficiently to allow immunocytochemical and in situ hybridization histochemical assays for LHRH peptide and LHRH mRNA, respectively. Large numbers of neurons containing mRNA encoding LHRH were detected in these slices using in situ hybridization histochemistry (ISHH). These 35S-labeled cells were distributed in the cultured slices in a pattern similar to that found with LHRH immunocytochemistry and ISHH in vivo, indicating that LHRH neurons were maintained in these cultures in an organotypic manner. Densitometric single cell analyses after ISHH of the culture slices were performed using a Loats image analysis system, so as to provide a density value per cell (density/cell). Comparisons of these density values from the slice explants cultured in presence or absence of 10(-7) M estradiol found that: 1) under basal (control) culture conditions there were no consistent differences in the frequency distributions of the density/cell values between all the five slices derived from either male or female rats, 2) mean density/culture values under control conditions did not differ significantly between slices and sexes, 3) the presence of estradiol in the culture media resulted in an overall decrease in density/cell values, with the most significant decrease occurring in slice 3 which is comparable to the level of the organum vasculosum lamina terminalis/rostral preoptic area (OVLT/rPOA) in vivo, and 4) this decrease in density/cell values in slice 3 due to estradiol treatment, was greater in cultures derived from female vs. male tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organotypic slice culture based on in ovo electroporation for chicken embryonic central nervous system

Organotypic slice culture is a living cell research technique which blends features of both in vivo and in vitro techniques. While organotypic brain slice culture techniques have been well established in rodents, there are few reports on the study of organotypic slice culture, especially of the central nervous system (CNS), in chicken embryos. We established a combined in ovo electroporation and organotypic slice culture method to study exogenous genes functions in the CNS during chicken embryo development. We performed in ovo electroporation in the spinal cord or optic tectum prior to slice culture. When embryonic development reached a specific stage, green fluorescent protein (GFP)‐positive embryos were selected and fluorescent expression sites were cut under stereo fluorescence microscopy. Selected tissues were embedded in 4% agar. Tissues were sectioned on a vibratory microtome and 300 μm thick sections were mounted on a membrane of millicell cell culture insert. The insert was placed in a 30‐mm culture dish and 1 ml of slice culture media was added. We show that during serum‐free medium culture, the slice loses its original structure and propensity to be strictly regulated, which are the characteristics of the CNS. However, after adding serum, the histological structure of cultured‐tissue slices was able to be well maintained and neuronal axons were significantly longer than that those of serum‐free medium cultured‐tissue slices. As the structure of a complete single neuron can be observed from a slice culture, this is a suitable way of studying single neuronal dynamics. As such, we present an effective method to study axon formation and migration of single neurons in vitro.     

Release of ethane and pentane from rat tissue slices: effect of vitamin E, halogenated hydrocarbons, and iron overload

The effects of in vitro addition of halogenated hydrocarbons on the susceptibility of various rat tissues to lipid peroxidation, and of iron overload and dietary vitamin E in the intact rat on subsequent lipid peroxidation in rat tissue slices were examined. The ease and speed of tissue slice preparation allowed testing of multiple tissues from the same animals. Total ethane and pentane (TEP) released from the slices was as reliable as and more sensitive than thiobarbituric acid-reactive substances as an index of lipid peroxidation. TEP was released by tissues from vitamin E-deficient rats in the following order of magnitude:intestine = brain = kidney greater than liver = lung greater than heart greater than testes = diaphragm greater than skeletal muscle. The potency of halogenated hydrocarbons for causing increased TEP release from vitamin E-deficient rat liver slices was CBrCl3 greater than CCl4 = 1,1,2,2-tetrabromoethane = 1,1,2,2-tetrachloroethane greater than perchloroethylene. CBrCl3 also stimulated TEP release from kidney, intestine, and heart slices, thus identifying these as potential target organs for CBrCl3 toxicity. Dietary vitamin E decreased TEP release from liver and, to a lesser extent, from kidney. Iron overload in the rat increased TEP release by slices from all tissues tested except the brain.     

Organ slices for the evaluation of human drug toxicity

Human organ slices, an in vitro model representing the multicellular and functional features of in vivo tissue, is a promising model for characterizing mechanisms of drug-induced organ injury and for identifying biomarkers of organ injury. Target organ injury is a significant clinical issue. In vitro models, which compare human and animal tissue to improve the extrapolation of animal in vivo studies for predicting human outcome, will contribute to improving drug candidate selection and to defining species susceptibilities in drug discovery and development programs. A critical aspect to the performance and outcome of human organ slice studies is the use of high quality tissue, and the use of culture conditions that support optimum organ slice survivability, in order to accurately reproduce mechanisms of organ injury in vitro. The attribute of organ slices possessing various cell types and interactions contributes to the overall biotransformation, inflammatory response and assessment of injury. Regional differences and changes in morphology can be readily evaluated by histology and special stains, similar to tissue obtained from in vivo studies. The liver is the major organ of which slice studies have been performed, however the utility of extra-hepatic derived slices, as well as co-cultures is increasing. Recent application of integrating gene expression, with human organ slice function and morphology demonstrate the increased potential of this model for defining the molecular and biochemical pathways leading to drug-induced tissue changes. By gaining a more detailed understanding of the mechanisms of drug-induced organ injury, and by correlating clinical measurements with drug-induced effects in the in vitro models, the vision of human in vitro models to identify more sensitive and discriminating markers of organ damage is attainable.     

Protection and Potentiation of 1-Methyl-4-Phenylpyridinium-Induced Toxicity by Cytochrome P450 Inhibitors and Inducer May Be Due to the Altered Uptake of the Toxin

Abstract: Earlier studies from our laboratory have demonstrated that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity could be modulated by inhibitors and inducer of cytochrome P450 (P450) in an in vitro model consisting of sagittal slices of mouse brain. To understand the molecular mechanisms underlying the role of P450 on MPTP toxicity, it was undertaken to study the effect of the modulators of P450 on the toxicity of the metabolite of MPTP, namely, 1-methyl-4-phenylpyridinium ion (MPP⁺). Incubation of mouse brain slices with various concentrations of MPP⁺ (1–100 µ M ) resulted in dose-dependent inhibition of mitochondrial enzyme NADH-dehydrogenase (NADH-DH) and leakage of the cytosolic enzyme lactate dehydrogenase from the slice into the medium. MPP⁺-induced toxicity was abolished by pretreatment of the slices with inhibitors of monoamine oxidase (MAO; pargyline and deprenyl) or inhibitors of P450 (piperonyl butoxide or SKF-525A) or dopamine uptake blocker (GBR-12909), as measured by the activity of NADH-DH in slices and leakage of lactate dehydrogenase from the slice into the medium. Slices prepared from mice pretreated with phenobarbital (an inducer of P450) potentiated the toxic effects of MPP⁺. Pretreatment of slices with MAO-inhibitor, P450 inhibitors, or dopamine uptake blocker attenuated the uptake of MPP⁺ into the slices. In contrast, MPP⁺ uptake was significantly increased in slices prepared from phenobarbital-pretreated mice. Thus, both MAO and P450 inhibitors abolish the toxicity of MPP⁺ in the sagittal slices of mouse brain by altering the uptake of the toxin into the slices.     

Effects of CYP inhibitors on precocene I metabolism and toxicity in rat liver slices

We present a comprehensive in vitro approach to assessing metabolism-mediated hepatotoxicity using male Sprague–Dawley rat liver slices incubated with the well characterized hepatotoxicant, precocene I, and inhibitors of cytochrome P450 (CYP) enzymes. This approach combines liquid chromatography mass spectrometry (LC MS) detection methods with multiple toxicity endpoints to enable identification of critical metabolic pathways for hepatotoxicity. The incubations were performed in the absence and presence of the non-specific CYP inhibitor, 1-aminobenzotriazole (ABT) and isoform-specific inhibitors. The metabolite profile of precocene I in rat liver slices shares some features of the in vivo profile, but also had a major difference in that epoxide dihydrodiol hydrolysis products were not observed to a measurable extent. As examples of our liver slice metabolite identification procedure, a minor glutathione adduct and previously unreported 7-O-desmethyl and glucuronidated metabolites of precocene I are reported. Precocene I induced hepatocellular necrosis in a dose- and time-dependent manner. ABT decreased the toxicity of precocene I, increased exposure to parent compound, and decreased metabolite levels in a dose-dependent manner. Of the isoform-specific CYP inhibitors tested for an effect on the precocene I metabolite profile, only tranylcypromine was noticeably effective, indicating a role of CYPs 2A6, 2C9, 2Cl9, and 2E1. With respect to toxicity, the order of CYP inhibitor effectiveness was ABT > diethyldithiocarbamate∼tranylcypromine > ketoconazole. Furafylline and sulfaphenazole had no effect, while quinidine appeared to augment precocene I toxicity. These results suggest that rat liver slices do not reproduce the reported in vivo biotransformation of precocene I and therefore may not be an appropriate model for precocene I metabolism. However, these results provide an example of how small molecule manipulation of CYP activity in an in vitro model can be used to confirm metabolism-mediated toxicity.     

Slice culture of the olfactory bulb of Xenopus laevis tadpoles

We report on the development of a slice culture of amphibian brain tissue. In particular, we cultured slices from Xenopus laevis tadpoles that contain the olfactory mucosae, the olfactory nerves, the olfactory bulb and the telencephalon. During 6 days in roller tubes the slices flattened, starting from 250 microm and decreasing to approximately 40 microm, corresponding to about three cell layers. Dendritic processes could be followed over distances as long as 200 microm. Neurons in the cultured slice could be recorded using the patch clamp technique and simultaneously imaged using an inverted laser scanning microscope. We characterized the main neuron types of the olfactory bulb, i.e. mitral cells and granule cells, by correlating their typical morphological features in the acute slice with the electrophysiological properties in both the acute slice and slice culture. This correlation allowed unambiguous identification of mitral cells and granule cells in the slice culture.     

Superoxide dismutase activity in organotypic midbrain-striatum co-cultures is associated with resistance of dopaminergic neurons to excitotoxicity

We have previously demonstrated that dopaminergic neurons in midbrain-striatum slice co-cultures are more resistant to NMDA cytotoxicity than the same neuronal population in single midbrain slice cultures. Here, we show that dopaminergic neurons in midbrain-striatum co-cultures also exhibit resistance to the cytotoxicity of nitric oxide donors, 2,2'-(hydroxynitrosohydrazono)bis-ethanamine (NOC-18) and 3-morpholinosydnonimine (SIN-1). The cytotoxicity of NMDA (30 microM) in single cultures was significantly attenuated by the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (100 microM), whereas the toxicity in co-cultures was not. The levels of tyrosine residue nitration of tyrosine hydroxylase, a hallmark of the occurence of peroxynitrite anion in dopaminergic neurons, were lower in co-cultures than those in single cultures. Single cultures and co-cultures did not show appreciable differences in the number or distribution of NOS-containing neurons as assessed by NADPH diaphorase histochemistry. On the other hand, midbrain slices cultured with striatal slices showed higher levels of superoxide dismutase (SOD) activity as well as increased protein levels of Cu,Zn-SOD, than midbrain slices cultured alone. These results suggested that the generation of NO is involved in NMDA cytotoxicity on dopaminergic neurons, and that increased activity of SOD in co-cultures renders dopaminergic neurons resistant to NMDA cytotoxicity by preventing the formation of peroxynitrite.     

The relative importance of glutathione and metallothionein on protection of hepatotoxicity of menadione in rats.

The effects of induction of metallothionein (MT) on the toxicity of menadione were investigated in rat liver slices. The protective role of hepatic glutathione (GSH) was also studied and compared to that of MT. A 3-h incubation of rat liver slices with menadione (100-300 microM) containing medium (37 degrees C, pH 7.4, 95%O2:5%CO2) resulted in cellular toxicity, as shown by changes in cytosolic K, Ca and GSH concentrations and lactate dehydrogenase (LDH) leakage. A dose-dependent decrease in cytosolic K and GSH was observed concomitant with an increase in cytosolic Ca and LDH leakage after incubation with menadione. Pretreatment of rats with zinc sulphate (ZnSO4) (30 mg/kg body wt.) increased MT levels in liver slices and suppressed the toxicity of menadione. Intracellular GSH concentrations in liver slices were either depleted or increased by injection of rats with buthionine sulfoximine (BSO), (4 mmol/kg body wt.) and N-acetyl-L-cysteine (NAC) (1.6 g/kg body wt.), respectively. Intracellular GSH was found to be crucial in protection against menadione toxicity. Menadione toxicity was increased when the rats were injected with sodium phenobarbital (PB) (4 x 80 mg/kg body wt.). Pretreatment with Zn provided partial protection against menadione toxicity in liver slices from both BSO- and PB-injected rats. These findings suggest that induction of MT synthesis does protect against quinone-induced toxicity, but the role may be secondary to that of GSH. The mechanisms by which MT protect against menadione toxicity are still unclear but may involve protection of both redox cycling and sulphydryl arylation.     

Tissue slices for the evaluation of metabolism-based toxicity with the example of diclofenac

Drug-induced organ injury is a multifaceted process, involving numerous cell types and mediators, and remains a significant safety issue in pharmaceutical development and clinical therapy. Organotypic in vitro models, including precision-cut tissue slices, possess the multi-cellular, structural and functional features of in vivo tissue to facilitate the elucidation of mechanisms of drug-induced organ injury and to characterize species susceptibilities. This study reviews diclofenac-induced hepatotoxicity and presents data comparing the metabolism, specific binding of diclofenac products to cellular proteins and the effects on liver function in rat, monkey and human liver slices. Concentration- and time-dependent increases in specific protein binding demonstrate the progressive nature of the toxicity. Altered liver function correlated with the species differences in the extent of diclofenac metabolism (rat > monkey or human). Liver injury was not detectable within 24 h, unlike specific protein binding, yet it developed by 48 h and lower concentrations of diclofenac exhibited effects by 72 h, demonstrating that continued metabolism and the accumulation of specific protein binding could lead to altered cell function. The decline of liver slice ATP levels at concentrations not affecting GSH levels implicates mitochondrial dysfunction as a primary indicator of hepatotoxicity, of which oxidative stress may be a contributing cause. Diclofenac affected monkey liver slices function at similar concentrations as rat liver slices, while human liver slices exhibited less extensive specific protein binding and required higher diclofenac concentrations to alter cell function.     

Assessment of S-(1,2-dichlorovinyl)-L-cysteine induced toxic events in rabbit renal cortical slices. Biochemical and histological evaluation of uptake, covalent binding, and toxicity

A renal cortical slice system was utilized to investigate the events leading to site-specific nephrotoxicity induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC). DCVC uptake into renal cortical slices was shown to be rapid (5-15 min) as well as time- and concentration-dependent. Of the total amount taken up at 1 h, 40% was subsequently covalently bound. These observations were confirmed by autoradiography, illustrating uptake and binding in the proximal tubule cells. Following these events, toxicity was evidenced by alterations in ATP content and O2 consumption between 4 and 8 h as well as leakage of the brush border enzymes (gamma glutamyl transpeptidase and alkaline phosphatase) as early as 4 h. Light microscopy provided a sequence of histopathological changes from an initial S3 lesion between 4 and 8 h to a lesion encompassing all proximal tubule segments (by 12 h). Electron microscopy demonstrated not only the specificity of DCVC toxicity (at 6 h) but also illustrated mitochondrial damage and loss of brush borders. A comparison of continuous versus short-term exposure to DCVC indicated that an irreversible sequence of events was initiated as early as 30 min. By utilizing an in vitro model which allows correlation of biochemical and histological changes, a sequence of events leading to DCVC induced toxicity was established.