Use Of Precision-cut Liver Slices Research Articles

Peroxisome Proliferator-Activated Receptor Activation in Precision-Cut Bovine Liver Slices Reveals Novel Putative PPAR Targets in Periparturient Dairy Cows.

Metabolic challenges experienced by dairy cows during the transition between pregnancy and lactation (also known as peripartum), are of considerable interest from a nutrigenomic perspective. The mobilization of large amounts of non-esterified fatty acids (NEFA) leads to an increase in NEFA uptake in the liver, the excess of which can cause hepatic accumulation of lipids and ultimately fatty liver. Interestingly, peripartum NEFA activate the Peroxisome Proliferator-activated Receptor (PPAR), a transcriptional regulator with known nutrigenomic properties. The study of PPAR activation in the liver of periparturient dairy cows is thus crucial; however, current in vitro models of the bovine liver are inadequate, and the isolation of primary hepatocytes is time consuming, resource intensive, and prone to errors, with the resulting cells losing characteristic phenotypical traits within hours. The objective of the current study was to evaluate the use of precision-cut liver slices (PCLS) from liver biopsies as a model for PPAR activation in periparturient dairy cows. Three primiparous Jersey cows were enrolled in the experiment, and PCLS from each were prepared prepartum (−8.0 ± 3.6 DIM) and postpartum (+7.7± 1.2 DIM) and treated independently with a variety of PPAR agonists and antagonists: the PPARα agonist WY-14643 and antagonist GW-6471; the PPARδ agonist GW-50156 and antagonist GSK-3787; and the PPARγ agonist rosiglitazone and antagonist GW-9662. Gene expression was assayed through RT-qPCR and RNAseq, and intracellular triacylglycerol (TAG) concentration was measured. PCLS obtained from postpartum cows and treated with a PPARγ agonist displayed upregulation of ACADVL and LIPC while those treated with PPARδ agonist had increased expression of LIPC, PPARD, and PDK4. In PCLS from prepartum cows, transcription of LIPC was increased by all PPAR agonists and NEFA. TAG concentration tended to be larger in tissue slices treated with PPARδ agonist compared to CTR. Use of PPAR isotype-specific antagonists in PCLS cultivated in autologous blood serum failed to decrease expression of PPAR targets, except for PDK4, which was confirmed to be a PPARδ target. Transcriptome sequencing revealed considerable differences in response to PPAR agonists at a false discovery rate-adjusted p-value of 0.2, with the most notable effects exerted by the PPARδ and PPARγ agonists. Differentially expressed genes were mainly related to pathways involved with lipid metabolism and the immune response. Among differentially expressed genes, a subset of 91 genes were identified as novel putative PPAR targets in the bovine liver, by cross-referencing our results with a publicly available dataset of predicted PPAR target genes, and supplementing our findings with prior literature. Our results provide important insights on the use of PCLS as a model for assaying PPAR activation in the periparturient dairy cow.

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

Real-time ALT and LDH activities determined in viable precision-cut mouse liver slices using hyperpolarized [1-13 C]pyruvate-Implications for studies on biopsied liver tissues.

Precision-cut liver slices (PCLS) are widely used in liver research as they provide a liver model with all liver cell types in their natural architecture. The purpose of this study was to demonstrate the use of PCLS for hyperpolarized metabolic investigation in a mouse model, for potential future application in liver biopsy cores. Fresh normal liver was harvested from six mice. 500μm PCLS were prepared and placed in a 10mm NMR tube in an NMR spectrometer and perfused continuously. 31 P spectra were acquired to evaluate the presence of adenosine triphosphate (ATP) and validate viability in all samples. Hyperpolarized [1-13 C]pyruvate was flushed into the NMR tube in the spectrometer. Consecutive 13 C NMR spectra were acquired immediately after the injection using both non-selective (five injections, two livers) and selective RF excitation (six injections, three livers). The 31 P spectra showed the characteristic signals of ATP, confirming the viability of the PCLS for more than 2.5h in the spectrometer. After each of the [1-13 C]pyruvate injections, both [1-13 C]lactate and [1-13 C]alanine signals were detected. Selective RF excitation aimed at both [1-13 C]lactate and [1-13 C]alanine enabled better visualization and quantification of the metabolic activity. Using this acquisition approach only the newly formed metabolites are observed upon excitation, and their intensities relative to those of hyperpolarized pyruvate enable quantification of metabolite production rates. This rate of lactate and alanine production appeared to be constant throughout the measurement time, with alanine production about 2.3 times higher than lactate. In summary, the viability of PCLS in an NMR spectrometer was demonstrated and hyperpolarized [1-13 C]pyruvate metabolism was recorded. This study opens up the possibility of evaluating alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activities in human liver biopsies, while preserving the tissue architecture and viability. In healthy, well-perfused liver slices the ratio of ALT to LDH activity is about 2.3.

RNA-Seq analysis of transcriptome responses in Atlantic cod (Gadus morhua) precision-cut liver slices exposed to benzo[a]pyrene and 17α-ethynylestradiol

Polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) that activate the aryl hydrocarbon receptor (Ahr) pathway, and endocrine disruptors acting through the estrogen receptor pathway are among environmental pollutants of major concern. In this work, we exposed Atlantic cod (Gadus morhua) precision-cut liver slices (PCLS) to BaP (10 nM and 1000 nM), ethynylestradiol (EE2) (10 nM and 1000 nM), and equimolar mixtures of BaP and EE2 (10 nM and 1000 nM) for 48 h, and performed RNA-Seq based transcriptome mapping followed by systematic bioinformatics analyses. Our gene expression analysis showed that several genes were differentially expressed in response to BaP and EE2 treatments in PCLS. Strong up-regulation of genes coding for the cytochrome P450 1a (Cyp1a) enzyme and the Ahr repressor (Ahrrb) was observed in BaP treated PCLS. EE2 treatment of liver slices strongly up-regulated genes coding for precursors of vitellogenin (Vtg) and eggshell zona pellucida (Zp) proteins. As expected, pathway enrichment and network analysis showed that the Ahr and estrogen receptor pathways are among the top affected by BaP and EE2 treatments, respectively. Interestingly, two genes coding for fibroblast growth factor 3 (Fgf3) and fibroblast growth factor 4 (Fgf4) were up-regulated by EE2 in this study. To our knowledge, the fgf3 and fgf4 genes have not previously been described in relation to estrogen signaling in fish liver, and these results suggest the modulation of the FGF signaling pathway by estrogens in fish. The signature expression profiles of top differentially expressed genes in response to the single compound (BaP or EE2) treatment were generally maintained in the expression responses to the equimolar binary mixtures. However, in the mixture-treated groups, BaP appeared to have anti-estrogenic effects as observed by lower number of differentially expressed putative EE2 responsive genes. Our in-depth quantitative analysis of changes in liver transcriptome in response to BaP and EE2, using PCLS tissue culture provides further mechanistic insights into effects of the compounds. Moreover, the analyses demonstrate the usefulness of PCLS in cod for omics experiments.

Review: Precision Cut Liver Slices for the Evaluation of Fatty Liver and Fibrosis.

Ethanol metabolism in the liver results in oxidative stress, altered cytokine production and fat accumulation in the liver. Thus, it is thought that the accumulation of benign fat into the liver in conjunction with a second hit leads to liver failure. However, we have recently developed the use of precision-cut liver slices (PCLSs) as an in vitro culture model in which to investigate the pathophysiology of alcohol-induced liver injury. In this review, these studies will be discussed and newer data presented. Original investigations into the use of PCLS were obtained from chow fed rats (200-300g). PCLSs were cultured 24-96h in media, 25 mM ethanol, or 25 mM ethanol and 0.5 mM 4- methylpyrazole (4-MP). PCLSs were examined for at different times and evaluated for glutathione (GSH) levels, extent of lipid peroxidation (TBARS assay), cytokine production (ELISA and RT-PCR) and myofibroblast activation. Age-matched rats were fed high fat diets for 13 months, PCLSs were prepared, and evaluated as outlined above. In recently, human and mouse PCLSs were cut, equilibrated, and evaluated using the methods outlined as above. In these studies, it was shown that the PCLSs from rats, mice and human livers retained excellent viability over a 96 hour period of incubation. During this time period, alcohol dehydrogenase, aldehyde dehydrogenase, and cytochrome P4502E1 levels were viable. After 24 hours of ethanol exposure, fatty livers and fibrogenic responses developed and could be prevented/reversed with the 4-MP. In a separate study using overly obese rats, ethanol metabolism was decreased in PCLSs as compared to age-matched controls (AMC). However, higher levels of triglycerides and lipid peroxidation were found in PCLSs from obese rats compared to AMC. Also, increased concentrations of the proinflammatory cytokines (TNF-α and IL-6) were found in the culture supernatants. In contrast, decreased levels of reduced glutathione (GSH) and heme oxygenase I (HO-1) levels were detected. Within 24h of incubation, ethanol metabolism by PCLSs initiates fat accumulation in the liver at which point there is an activation of myofibroblasts. Thus, fatty liver is the first response to ethanol and sensitizes the liver to other products of oxidative stress that result in inflammation and the start of liver failure ending in cirrhosis. Thus, from these studies it appears that PCLSs can be utilized to determine the mechanisms(s) by which ethanol exposure leads to the development and/or progression of alcoholic liver disease (ALD).

A Study of 2,3,7,8-Tetrachlorodibenzo-p-dioxin Induced Liver Injury in Jian Carp (Cyprinus carpio var. Jian) Using Precision-Cut Liver Slices.

The aim of this study was to establish a model for the study of liver injury induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in Jian carp using precision-cut liver slices (PCLS). PCLS were treated with TCDD at concentrations of 0, 0.05, 0.1, 0.3, and 0.6μg/L for 6h, followed by collection of the culture supernatant and PCLS for analysis. Several biochemical indices were analyzed, including glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA). Expression of mRNA was also estimated for cytochrome P4501A (CYP1A), aryl hydrocarbon receptor2 (AhR2), and aryl hydrocarbon receptor nuclear translocator2 (ARNT2). Results showed that some significant effects (p<0.05) in MDA, GSH-Px and PCLS viability were observed at a TCDD concentration as low as 0.05µg/L, and the observed effects increased with exposure concentration. Following exposure to TCDD for 6h at a concentration of 0.3μg/L, significant increases (p<0.01) in the content of GPT, GOT, MDA, and LDH were observed, while SOD activity, GSH-Px activity, and PCLS viability were decreased (p<0.01 or p<0.05). Exposure to 0.3μg/L TCDD also resulted in increased expression of mRNA for CYP1A, AhR2, and ARNT2. Overall, these results provide evidence of TCDD-induced liver injury and oxidative stress in Jian carp. These results also support the use of PCLS as an in vitro model for the evaluation of hepatotoxicity in Jian carp.

Comparison of Biocompatibility and Adsorption Properties of Different Plastics for Advanced Microfluidic Cell and Tissue Culture Models

Microfluidic technology is providing new routes toward advanced cell and tissue culture models to better understand human biology and disease. Many advanced devices have been made from poly(dimethylsiloxane) (PDMS) to enable experiments, for example, to study drug metabolism by use of precision-cut liver slices, that are not possible with conventional systems. However, PDMS, a silicone rubber material, is very hydrophobic and tends to exhibit significant adsorption and absorption of hydrophobic drugs and their metabolites. Although glass could be used as an alternative, thermoplastics are better from a cost and fabrication perspective. Thermoplastic polymers (plastics) allow easy surface treatment and are generally transparent and biocompatible. This study focuses on the fabrication of biocompatible microfluidic devices with low adsorption properties from the thermoplastics poly(methyl methacrylate) (PMMA), polystyrene (PS), polycarbonate (PC), and cyclic olefin copolymer (COC) as alternatives for PDMS devices. Thermoplastic surfaces were oxidized using UV-generated ozone or oxygen plasma to reduce adsorption of hydrophobic compounds. Surface hydrophilicity was assessed over 4 weeks by measuring the contact angle of water on the surface. The adsorption of 7-ethoxycoumarin, testosterone, and their metabolites was also determined after UV-ozone treatment. Biocompatibility was assessed by culturing human hepatoma (HepG2) cells on treated surfaces. Comparison of the adsorption properties and biocompatibility of devices in different plastics revealed that only UV-ozone-treated PC and COC devices satisfied both criteria. This paper lays an important foundation that will help researchers make informed decisions with respect to the materials they select for microfluidic cell-based culture experiments.

Evaluation of the hepatotoxic and hepatoprotective effect of Rwandese herbal drugs on in vivo (guinea pigs barbiturate-induced sleeping time) and in vitro (rat precision-cut liver slices, PCLS) models

Precision-cut liver slices (PCLS) preserve the tissular organization of the organ and represent an in vitro model closer to in vivo conditions than hepatocytes cultures. As this may be an interesting tool not only for the investigation of hepatotoxic and protective effects but also for bioguided fractionations schemes, the usefulness of PCLS was compared with an in vivo test of liver function. Crude extracts derived from five herbs used in Rwanda for hepatoprotective activity were tested on CCl4-treated guinea pigs by the method of barbiturate-induced sleep modification. Aqueous extracts of Ocimum lamiifolium, Crassocephalum vitellinum, Guizotia scabra and Vernonia lasiopus leaves allowed animals to recover barbiturate sleep duration in proportions of 88%, 78%, 61% and 34%, respectively and Microglossa pyrifolia was found inactive. Dried methanolic extracts of the 5 plants were then tested in vitro on rat PCLS for protection against acetaminophen-induced hepatotoxicity. In this model, G. scabra, M. pyrifolia and V. lasiopus were found hepatotoxic by themselves and unable to prevent acetaminophen toxicity. The most active extract, obtained from O. lamiifolium, was subjected to bioassay-guided fractionation by chromatography on Si–C18 to yield two quite active fractions. From a single animal, at least 50 PCLS explants can be prepared, which allows testing large amounts of samples, strengthening ethnopharmacological data on hepatoprotective medicinal plants and investigating hepatotoxic effects.

S1591 Use of Precision-Cut Liver Slices from Asialoglycoprotein Receptor-Deficient and Wild-Type Mice to Examine a Role for This Receptor During Toxin-Induced Liver Injury

S1591 Use of Precision-Cut Liver Slices from Asialoglycoprotein Receptor-Deficient and Wild-Type Mice to Examine a Role for This Receptor During Toxin-Induced Liver Injury

An in vitro method of alcoholic liver injury using precision-cut liver slices from rats

Alcohol abuse results in liver injury, but investigations into the mechanism(s) for this injury have been hampered by the lack of appropriate in vitro culture models in which to conduct in depth and specific studies. In order to overcome these shortcomings, we have developed the use of precision-cut liver slices (PCLS) as an in vitro culture model in which to investigate how ethanol causes alcohol-induced liver injury. In these studies, it was shown that the PCLS retained excellent viability as determined by lactate dehydrogenase and adenosine triphosphate (ATP) levels over a 96-h period of incubation. More importantly, the major enzymes of ethanol detoxification; alcohol dehydrogenase, aldehyde dehydrogenase, and cytochrome P4502E1, remained active and PCLS readily metabolized ethanol and produced acetaldehyde. Within 24 h and continuing up to 96 h the PCLS developed fatty livers and demonstrated an increase in the redox state. These PCLS secreted albumin, and albumin secretion was decreased by ethanol treatment. All of these impairments were reversed following the addition of 4-methylpyrazole, which is an inhibitor of ethanol metabolism. Therefore, this model system appears to mimic the ethanol-induced changes in the liver that have been previously reported in human and animal studies, and may be a useful model for the study of alcoholic liver disease.

Precision-Cut Liver Slices as a New Model to Study Toxicity-Induced Hepatic Stellate Cell Activation in a Physiologic Milieu

Hepatic stellate cell (HSC) activation is a key event in the natural process of wound healing as well as in fibrosis development in liver. Current in vitro models for HSC activation contribute significantly to the understanding of HSC biology and fibrogenesis but still fall far short of recapitulating in vivo intercellular functional and anatomic relationships. In addition, when cultured on uncoated plastic, HSC spontaneously activate, which makes HSC activation difficult to regulate or analyze. We have examined whether the use of precision-cut liver slices might overcome these limitations. Liver slices (8 mm diameter, 250 microm thickness) were generated from normal rat liver and incubated for 3 or 16 h with increasing doses of carbon tetrachloride (CCl4). Rat liver slices remained viable during incubation, as shown by minimal enzyme leakage. Expression of markers for HSC activation and the onset of fibrogenesis in the liver slices was studied using real-time PCR and Western blotting. In unstimulated liver slices, mRNA and protein levels of desmin, heat shock protein 47, and alpha B-crystallin remained constant, indicating quiescence of HSC, whereas Krüppel-like factor 6 expression was increased. In contrast, incubation with CCl4 led to a time- and dose-dependent increase in mRNA expression of all markers and an increased alpha B-crystallin protein expression. In conclusion, we have developed a technique to induce activation of quiescent HSC in rat liver slices. This model permits the study of toxicity-induced HSC activation within a physiological milieu, not only in animal but ultimately also in human tissue, and could contribute to the reduction of animal experiments.

The use of precision-cut liver slices from male Wistar rats as a tool to study age related changes in CYP3A induction and in formation of paracetamol conjugates

Precision-cut liver slices (PCLS) offer a lot of advantages because all heterogeneity and cell-cell interactions within the original tissue matrix are maintained. This in vitro model was used to study the effect of ageing on certain aspects of drug metabolism and liver function in young (3 months), adult (9 months) and old (24 months) Wistar male rats. Protein synthesis, an important liver function, was not modified in young, adult and old rats, suggesting that ageing does not impair liver functionality but it affects some specific targets. Among them, a decrease in total P450 in liver microsomes and the loss of CYP3A23 inducibility in PCLS were clearly observed in old rats as compared to adult rats. Finally, the amount of total paracetamol conjugates was not modified between 9 and 24 months but in old rats, sulfoconjugation of paracetamol, its major route of elimination, was decreased.

Monooxygenation, conjugation and other functions in cryopreserved rat liver slices until 24 h after thawing

For the extensive use of precision-cut liver slices (particularly of human origin) for toxicological investigations successful cryopreservation is necessary. But so far, survival of thawed slices was limited to few hours. This was now overcome by modification of previous procedures. The concentration of DMSO as a cryoprotectant was enhanced to 30%, and washing steps after rapid thawing were omitted. The slices were frozen in liquid nitrogen, thawed at 38°C and incubated immediately in Williams medium E. Protein and potassium contents were stable until 24 h. Glutathione content, amounting to nearly 50% of fresh slices, increased during incubation. High initial lactate dehydrogenase leakage dropped after medium change to less than half during 2–24 h. Testosterone hydroxylation and 7-ethoxycoumarin O-deethylation rates were similar to fresh slices, the latter reaction was inducible by β-naphthoflavone within 24 h. Methylumbelliferone glucuronidation and p-nitrophenol glucuronidation and sulfation were well measurable and either maintained or decreased by about 50% until 24 h. Altogether, the results are encouraging for further experiments to standardise cryopreservation conditions and to investigate the suitability of this cryopreservation protocol with human liver slices.

Intercellular communication and cell proliferation in precision-cut rat liver slices: effect of medium composition and DDT

Gap junctional intercellular communication (GJIC) and cell proliferation were studied in control and 1,1′-bis( p-chlorophenyl)-2,2,2,-trichloroethane (DDT) treated precision-cut liver slices of rat by evaluating connexin 32 (Cx32) expression and 5-bromo-2′-deoxyuridine (BrdU) incorporation. In addition, the effect of different culture media (RPMI and WME) on control and DDT influenced Cx32 expression and cell proliferation was determined. Cx32 expression in control precision-cut liver slices was maintained during 8 h of culturing, but decreased after prolonged culturing. Control cell proliferation was significantly higher when WME was used as culture medium than when RPMI was used. In slices treated with DDT Cx32 expression was decreased. In slices cultured in RPMI medium, this decrease preceded a dose-dependent increase in cell proliferation. These results show the usefulness of precision-cut liver slices in studying cellular proliferation and intercellular communication.

Technical Report: Application of Rat Precision-cut Liver Slices for Toxicity Assessment In Vitro.

The usefulness of precision-cut liver slices for toxicological evaluation in vitro was confirmed with a representative hepatotoxic agent acetaminophen. Liver slices obtained from normal and in vivo phenobarbital-treated rats were incubated with 0, 1.25, 2.5, 5, and 10 mM of acetaminophen for 24 hours. Intracellular potassium concentration dose-dependently decreased and lactate dehydrogenase (LDH) leakage increased in the slices from phenobarbital-treated rats, whereas no changes were detected in the slices from normal rats. The cytotoxicity of acetaminophen was also shown in the slices from normal rats when the slices were preincubated with phenobarbital in vitro for one hour. The acetaminophen cytotoxicity in the liver slices was enhanced by the preincubation with diethyl maleate, a depressant of hepatic glutathione, tributyltin acetate, an inhibitor of glutathione S-transferase, and 2, 6-dichloro-4-nitrophenol, an inhibitor of sulfotransferase. These data suggest that the formation and accumulation of the toxic metabolite are essential to induce the acetaminophen hepatotoxicity. Histopathological observation revealed centrilobular hepatocyte necrosis on the slices from phenobarbital-treated rats 24 hours after the exposure to acetaminophen. These results coincide with those reported in in vivo acetaminophen toxicity. The application of precision-cut liver slices is considered to be useful in toxicological evaluation of chemicals.

Effect of Some Cooked Food Mutagens on Unscheduled DNA Synthesis in Cultured Precision-cut Rat, Mouse and Human Liver Slices

Precision-cut liver slices were prepared from male Fischer 344 rats, female CDF 1 mice and humans (both male and female subjects). Liver slices were cultured for 24 hr in medium containing [ 3H]thymidine and either PhIP, IQ, MeIQ, MeIQ x, Glu-P-1 or Trp-P-1, and then processed for autoradiographic evaluation of unscheduled DNA synthesis (UDS). All six cooked food mutagens examined produced concentration-dependent increases in UDS in human liver slices. PhIP was the most potent compound examined, followed by MeIQ x, IQ and then MeIQ, Glu-P-1 and Trp-P-1. Significant increases in UDS were observed with PhIP, IQ and MeIQ x at concentrations as low as 5 μ m in the culture medium. The same rank order of potency was not apparent in either rat or mouse liver slices. In rat liver slices only MeIQ significantly induced UDS, although positive results were obtained with two other genotoxins, namely 2-acetylaminofluorene and aflatoxin B 1. Apart from MeIQ x, all the cooked food mutagens produced significant increases in UDS in mouse liver slices. This study demonstrates the usefulness of precision-cut liver slices to evaluate species differences in xenobiotic-induced genotoxicity. Both marked compound and species differences in induction of UDS were observed. The data provide further evidence that dietary cooked food mutagens are potential human carcinogens.

Use of Precision-Cut Liver Slices to Evaluate Species Differences in 2-Acetylaminofluorene-Induced Unscheduled DNA Synthesis

Precision-cut liver slices were prepared from male Sprague–Dawley rats (pretreated with or without Aroclor 1254), male Dunkin–Hartley guinea pigs, male cynomolgus monkeys, and humans. Liver slices were cultured for 24 hr using a dynamic organ culture system in medium containing [3H]thymidine and 2-acetylaminofluorene (2-AAF), aflatoxin B1(AFB1), or 6-aminochrysene (6-AC). The liver slices were then harvested and processed for autoradiographic evaluation of unscheduled DNA synthesis (UDS). All three genotoxins induced UDS in liver slices from untreated and Aroclor 1254-treated rats. In human liver slices 2-AAF produced a concentration-dependent induction of UDS and at the highest concentration examined 2-AAF also induced UDS in guinea pig liver slices. However, 2-AAF did not induce UDS in cynomolgus monkey liver slices, although both AFB1and 6-AC induced UDS in liver slices from this species as well as from guinea pigs and humans. The inability of 2-AAF to induce UDS in cynomolgus monkey liver slices appears to be at least partially due to the absence of hepatic CYP1A2 in this species. Precision-cut liver slices appear to be a useful alternative to primary hepatocyte cultures for studies of xenobiotic-induced genotoxicity employing the UDS technique. As shown by this study they may also be employed to evaluate species differences in xenobiotic-induced genotoxicity.

Use of precision-cut liver slices as an in vitro tool for evaluating liver function.

Precision-cut liver slices have been developed as an in vitro tool for assessing liver viability and function and for examining hepatotoxicants. Liver slices from a variety of species (including human) are prepared using mechanical slicers that produce reproducible slices of a uniform thickness, which allows optimum exchange of nutrients, waste, and gases. Slices are incubated in dynamic systems that allow the slices to be maintained viable in culture for 1-10 days. The viability of slices can be assessed by ion content (K+, Na+ ATPase status), intermediary metabolism, energy status (ATP), respiration, biosynthetic ability, and biotransformation activity. In addition, liver tissue slices allow the opportunity for extensive microscopic evaluation (light and electron) as well as newer technologies such as confocal microscopy. Assessment of the toxic potential of a chemical can be performed after a short-term or constant exposure by evaluating the viability parameters. Liver slices have been used extensively for rank-ordering the toxicity of chemicals as well as for examining the mechanisms of liver injury. Liver slices in culture also can be used for an examination of the induction of new enzymes such as cytochrome P-450 and the expression of stress proteins or peroxisomal enzymes. Finally, liver slices offer a system for evaluating whole or cryopreserved liver as well as regeneration of liver tissue after toxic insult. Liver slices have been shown to be a valid in vitro system for examining liver function and offer a bridge between in vivo and cell culture systems.

Use of precision-cut liver slices for studies of unscheduled DNA synthesis

Precision-cut liver slices were prepared from untreated and Aroclor 1254 (ARO)-treated male Sprague-Dawley rats with a Krumdieck tissue slicer. Liver slices were cultured for 24 hr in medium containing [ 3H]thymidine and 0–0.1 m m 2-acetylaminofluorene (2-AAF) using a dynamic organ culture system and processed for autoradiographic evaluation of unscheduled DNA synthesis (UDS). Compared with control (i.e. 0 m m 2-AAF) liver slice cultures, 2-AAF produced a concentration-dependent increase in UDS, the effect being more marked in liver slices from ARO-treated than from untreated rats. With liver slices from untreated rats, 2-AAF produced the greatest increase in UDS in centrilobular hepatocytes. 2-AAF-induced UDS in liver slices from ARO-treated rats was most marked in centrilobular hepatocytes but the effect also extended to other areas of the liver lobule. These results demonstrate that precision-cut liver slices may be a valuable alternative in vitro system to hepatocyte cultures for screening chemicals for potential genotoxicity. Unlike hepatocyte cultures, liver slices permit the study of zonal differences in UDS. Moreover, this technique could be applied to other tissues and the study of species differences in response.