Cultured Liver Slices Research Articles

Morphological and biochemical integrity of human liver slices in long-term culture: effects of oxygen tension.

We tested the effects of low (20% O2) and high (70% O2) oxygen tension on the morphological and biochemical integrity of human liver slices incubated for up to 72 h in supplemented Williams' E medium in a dynamic rotating culture system. High oxygen tension was more effective than low oxygen tension for preserving morphological integrity in long-term culture (48-72 h). After 72 h of culture with 70% O2, the lobular pattern was well preserved, and the survival of hepatocytes (approximately 80%) and other cell types was good. Immunohistochemical studies showed good preservation of the region-specific expression of CYP2EI and CYP3A4 isoenzymes for up to 72 h of incubation in 70% O2. As compared to 20% O2, the oxidized glutathione content and reactive oxygen species production were slightly increased in 70% O2, suggesting that minimal oxidative stress occurred with the high oxygen tension. In conclusion, despite slight oxidative stress associated with high oxygen tension, 70% O2 appeared more appropriate than 20% O2 for preserving the morphological and biochemical integrity of human liver slices cultured in a dynamic organ culture system for up to 72 h.

Viability of Liver Slices Exhibiting Absorption, Metabolism, and Elimination of Substrates in Culture Medium

In an attempt to improve liver slice culture for in vitro tests of newly developed drugs, we found that a relatively thick 2-mm slice was much more viable, long-lived, and metabolically functional than the conventionally used 300-μm-thick slices. Results revealed that some ionic and nonionic compounds (Na125I, [14C]Ala, and [14C]Xyl) dissolved in culture medium were absorbed into the slices, reaching from the cut edges deep into the inner part, in only a short period. Moreover, the slices could be observed for 24 h after incubation so that a normal histological image could be obtained. Each semi-microautoradiographic image was also clearly positive not only at the margin but also in the inner part of each slice. Furthermore, we determined the specific functioning of each donated liver for each substrate added, in uptake, metabolism, and excretion.

Use of precision-cut tissue slices in organ culture to study metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by hamster lung, liver and kidney

The pharmacokinetics of in vitro metabolism of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK; concentration range 0.03–250 μM) and its proximal metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL; 0.04–250 μM), were determined in Syrian golden hamster liver, lung, and kidney tissue slices in organ culture under identical experimental conditions. In the lung, a target organ for NNK animal carcinogenesis, total NNK metabolism was relatively low (maximum 23%) and oxidative metabolism by α-hydroxylation to DNA-reactive intermediates accounted for 13–31% of metabolism. The liver, a non-target organ for NNK carcinogenesis, showed the highest capacity to metabolise NNK (total metabolism 80%), and α-hydroxylation accounted for 12–25% of metabolism. The kidney, another non-target organ, also showed a low capacity for NNK metabolism (maximum 32%) and α-hydroxylation accounted for <3% of metabolism. Detoxification of NNK by pyridyl N-oxidation was similar in lung (5–22%) and liver (5–23%), and negligible in kidney (<2%), while carbonyl reduction of NNK to NNAL was greatest in the kidney (95–100%), followed by liver (59–79%) and lung (47–81%). NNAL is devoid of biological activity in the hamster and total metabolism was about tenfold lower than that of NNK in all tissues (<13% liver; <4% lung and kidney). In the liver, α-hydroxylation was the predominant pathway of NNAL metabolism at almost all concentrations (31–68% of total metabolism), whereas N-oxidation prevailed in the kidney (47–68%). In the lung, a concentration dependent decrease in the relative amount of α-hydroxylation (23–72%) with increasing NNAL concentrations occurred at the expense of N-oxidation (25–72%). Little or no metabolism of NNAL back to NNK was evident in any tissue.

Detection of CYP1A1 protein in human liver and induction by TCDD in precision-cut liver slices incubated in dynamic organ culture.

Cytochrome P4501A1 (CYP1A1) has been implicated in the conversion of numerous polycyclic aromatic hydrocarbons into electrophilic species capable of binding covalently to DNA and has therefore been postulated to be involved in the initiation of carcinogenesis. The expression of CYP1A1 protein appears not to be constitutive, but is readily inducible by aryl hydrocarbon (Ah) receptor ligands in a majority of tissues of experimental animals, especially the liver. To date, there is conflicting evidence for the expression or inducibility of CYP1A1 protein in human liver. In this present study, we report the detection of CYP1A1 in all 20 human liver microsomal samples tested by standard western immunoblotting with chemiluminescent detection using a specific monoclonal antibody (mAb 1-12-3) directed against a marine fish (scup) cytochrome P450E. mAb 1-12-3 has been shown previously to specifically recognize CYP1A1 in mammals. This system consistently demonstrated a detection sensitivity as low as 0.01-0.025 pmol CYP1A1 per lane. In the samples where CYP1A1 protein levels were quantitated, CYP1A1 ranged from approximately 0.4 to 5 pmol CYP1A1/mg microsomal protein. Additionally, the inducibility of CYP1A1 protein was demonstrated by incubating precision-cut human liver slices in dynamic organ culture for up to 96 h in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The specificity of mAb 1-12-3 was tested using several purified human and rat cytochrome P450s to ensure that the protein being detected was CYP1A1. mAb 1-12-3 did not cross-react with human CYP1A2 or CYP3A4 or rat CYP1B1, but did strongly recognize CYP1A1. However, there was a very weak cross-reactivity of mAb 1-12-3 with human CYP2E1, approximately 75-fold less compared with CYP1A1. In order to confirm CYP1A1 as the immunoreactive protein detected in human liver, microsomal samples were subjected to two-dimensional electrophoresis involving isoelectric focusing followed by SDS-PAGE and immunoblotting. Utilizing mAb 1-12-3, the human liver microsomal samples displayed an immunoblotting profile matching that obtained from a microsomal preparation from a AHH-1 TK+/- cell line expressing solely human CYP1A1 and differing from the profile obtained using a polyclonal antibody directed against CYP2E1 and cells expressing CYP2E1. Furthermore, mAb 1-12-3 recognized only one protein of identical mobility on the two-dimensional blots from human liver microsomes and AHH-1 TK+/- cells expressing CYP1A1, while displaying no reaction to cells expressing only CYP2E1. In conclusion, CYP1A1 appears to be expressed in human liver at low levels and is inducible upon exposure to TCDD.

Lack of Effect of Coumarin on Unscheduled DNA Synthesis in Precision-cut Human Liver Slices

In this study the effect of coumarin on unscheduled DNA synthesis (UDS) in precision-cut human liver slices has been examined. Liver slices from tissue samples from four donors were cultured for 24 hr in medium containing [ 3H]thymidine and 0–5.0 m m coumarin using a dynamic organ culture system and processed for autoradiographic evaluation of UDS. As positive controls liver slices were also cultured with three known genotoxic agents, namely 0.02 and 0.05 m m 2-acetylaminofluorene (2-AAF), 0.002 and 0.02 m m aflatoxin B 1 (AFB 1) and 0.005 and 0.05 m m 2-amino-1-methyl-6-phenylimidazo [4,5- b]pyridine (PhIP). UDS was quantified as the net grain count in centrilobular hepatocytes and as the percentage of centrilobular hepatocyte nuclei with more than five net grains. Compared with control liver slice cultures, treatment with 0.05–5.0 m m coumarin had no effect on UDS. In contrast, treatment with 0.02 and 0.05 m m 2-AAF, 0.002 and 0.02 m m AFB 1 and 0.005 and 0.05 m m PhIP produced significant increases in the net grain counts of centrilobular hepatocytes. The greatest induction of UDS was observed in liver slices treated with 0.05 m m PhIP. Treatment with 2-AAF, AFB 1 and PhIP also produced significant increases in the number of centrilobular hepatocyte nuclei with more than five net grains. At the concentrations examined neither coumarin, 2-AAF, AFB 1 nor PhIP had any significant effect on replicative DNA synthesis in 24 hr cultured human liver slices. These results demonstrate that coumarin does not induce UDS in cultured human liver slices. However, all three positive control compounds produced marked significant increases in UDS, thus confirming the functional viability of the human liver slice preparations used in this study. The results of this study suggest that coumarin is not a genotoxic agent in human liver.

P3G195 - Early toxicological screening using rat liver slices in culture

P3G195 - Early toxicological screening using rat liver slices in culture

P3G182 - Effects of oxygen tension on histological integrity and drug-metabolizing enzymes in precision-cut rat liver slices in long-term culture

P3G182 - Effects of oxygen tension on histological integrity and drug-metabolizing enzymes in precision-cut rat liver slices in long-term culture

Use of precision-cut rat liver slices for studies of xenobiotic metabolism and toxicity: comparison of the Krumdieck and Brendel tissue slicers

1. In this study we have compared freshly cut and cultured precision-cut rat liver slices produced by the Krumdieck and Brendel-Vitron tissue slicers. 2. No significant differences were observed in levels of protein, potassium, total glutathione (i.e. GSH and GSSG), reduced glutathione (GSH) and cytochrome P450 and activities of 7-ethoxyresorufin O-deethylase and 7-benzoxyresorufin O-debenzylase in freshly cut rat liver slices produced by the two tissue slicers. However, levels of oxidized glutathione (GSSG) were significantly greater in liver slices produced with the BrendelVitron tissue slicer. 3. Precision-cut rat liver slices produced with both tissue slicers were cultured for 0 (i.e. a 1-h preincubation), 24 and 72 h in a dynamic organ culture system in an atmosphere of either 95% O2/5% CO2 or 95% air/5% CO2. 4. Apart from small differences in glutathione levels in 0 and 24 h cultured liver slices, no significant differences were observed in the parameters measured between liver slices prepared with both tissue slicers and cultured in both gas phases. 5. With liver slices produced by both tissue slicers 50 μM sodium arsenite produced a greater induction of heat shock protein 70 levels in slices cultured for 24 h in a high oxygen than in an air atmosphere. 6. These results suggest that both tissue slicers can readily produce precision-cut liver slices for studies of xenobiotic metabolism and toxicity. However, the data suggest that for any given application of precision-cut tissue slicesit is desirable to establish optimal culture conditions.

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 degrees 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.

Lack of effect of piperonyl butoxide on unscheduled DNA synthesis in precision-cut human liver slices

In this study the effect of piperonyl butoxide (PBO) on unscheduled DNA synthesis in precision-cut human liver slices has been examined. Liver slices prepared from tissue samples from five human donors were cultured in medium containing [ 3H]thymidine and 0–2.5 mM PBO using a dynamic organ culture system. After 24 h the liver slices were processed for autoradiographic examination of UDS. As positive controls, liver slices were also cultured with three known genotoxic agents, namely 2-acetylaminofluorene (2-AAF), aflatoxin B 1 (AFB 1) and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP). UDS was quantified as the net grain count in centrilobular hepatocytes and as the percentage of centrilobular hepatocyte nuclei with >5 and >10 net grains. Compared to control liver slice cultures PBO had no effect on UDS. In contrast, treatment with 0.02 and 0.05 mM 2-AAF, 0.002 and 0.02 and 0.02 mM AFB 1 and 0.005 and 0.05 mM PhIP produced significant increases in net grain counts of centrilobular hepatocytes. The greatest induction of UDS was observed in liver slices treated with 0.05 mM PhIP. Treatment with 2-AAF, AFB 1 and PhIP also produced increases in the number of centrilobular hepatocyte nuclei with >5 and >10 net grains. At the concentrations examined neither PBO, 2-AAF nor PhIP had any significant effect on replicative DNA synthesis in 24 h cultured human liver slices. In cultured liver slices treated with 0.02, but not 0.002, mM AFB 1 a significant reduction in the rate of replicative DNA synthesis was observed. These results demonstrate that PBO does not induce UDS in cultured human liver slices. However, all three positive control compounds produced marked significant increases in UDS, thus confirming the functional viability of the human liver slice

Validation of Precision-Cut Liver Slices in Dynamic Organ Culture as anin VitroModel for Studying CYP1A1 and CYP1A2 Induction

The utilization of precision-cut liver slices in dynamic organ culture as anin vitromodel was validated by comparing the induction of the biomarker responses followingin vitro(rat liver slice) andin vivoexposure of rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The biomarker responses investigated were cytochrome P450s 1A1 and 1A2 (CYP1A1 and CYP1A2) mRNA, protein, and activities. Precision-cut rat liver slices were incubated in dynamic organ culture for 24 hr with medium containing 0.001–10 nMTCDD or medium without TCDD (control). The resultant mean TCDD concentration in the slices ranged from 19 to 80,925 ppt (wet wt), respectively. A concentration-dependent induction of CYP1A1 mRNA, protein, and activities and a more modest induction of CYP1A2 mRNA was observed in liver slices at all medium concentrations of TCDD. The O-demethylation of 7-methoxyresorufin, a marker for CYP1A2 activity, was induced at TCDD medium levels of 0.01 nMand greater, whereas a detectable increase in CYP1A2 protein occurred only at the higher concentrations. Comparable liver concentrations of TCDD (8–64,698 ppt wet wt) were achieved at 24 hr following a singlein vivoexposure of rats to TCDD at doses ranging from 0.002 to 5 μg/kg po. Concentration–effect and dose–response relationships for induction of CYP1A1 and CYP1A2 were similar followingin vitroandin vivoexposure to TCDD, although the magnitude of induction was greater forin vivoexposure. The data support the use of liver slices in dynamic organ culture for assessing the relativein vivopotency of a compound to induce CYP1A1 and CYP1A2. Human tissue can also be readily utilized in thisin vitromodel to predict the biological and toxicological effects of a givenin vivoexposure to TCDD.

96/04655 Chemical desulfurization methods of organic sulfur of Eocene coal, Jaintla Hills, Meghalaya

Hepatic Uptake and Metabolism of 2,3,7,8-Tetrachlorodibenzo-p-dioxin and 2,3,7,8-Tetrachlorodibenzofuran. Olson, J. R., McGarrigle, B. P., Gigliotti, P. J., Kumar, S., and McReynolds, J. H. (1994). Fundam. Appl. Toxicol. 22, 631-640.The pharmacokinetics of TCDD and related compounds is congener, dose, and species specific, with urinary and biliary excretion being dependent on the metabolism of these compounds. Isolated hepatocytes and liver slices in suspension culture and hepatic microsomes were used as in vitro models to assess the hepatic uptake and metabolism of [3H]- and [14C]TCDD and [3H]TCDF (0.01-1.0 μM) in control and induced (5 μg TCDD/kg, 3 days earlier) male Sprague-Dawley rats. TCDD pretreatment, with an increase in cytochromes P450 1A1 and 1A2 (CYP1A1, CYP1A2), produced an increase in the hepatic uptake of TCDD, while no increase in the hepatic uptake of TCDF was observed. The results are consistent with CYP1A2 serving as a hepatic binding protein for TCDD but not for TCDF. The rates of metabolism of TCDD and TCDF were directly proportional to their concentrations, indicating that the reaction follows first order kinetics at concentrations from 0.01 to 1.0 μM. Very limited metabolism of TCDD and TCDF was observed in control rat liver (0.45 and 3.2 pmol/hr/g hepatocyte wet wt at 0.1 μM, respectively). TCDD induced its own rate of metabolism about two- to fivefold at 1.0 μM but no induction was observed at 0.01 and 0.1 μM. In contrast, TCDD markedly induced the rate of TCDF metabolism at all substrate concentrations. While the results support the role of rat CYP1A1 in TCDF metabolism, the data suggest that CYP1A1 or CYP1A2 may not metabolize TCDD. These results also support the hypothesis that the more rapid metabolism and excretion of TCDF accounts for the relative resistance of the rat to the acute toxicity of TCDF. Comparative studies in rat and human liver microsomes found that TCDF metabolism exhibited first order kinetics in both species. Furthermore, the rate of TCDF metabolism in human liver microsomes was similar to that of control rat liver microsomes. Together the results suggest that TCDF will be far more persistent in rats, and possibly humans, following exposure at low doses which do not significantly induce cytochrome P450 1A1 and/or 1A2.

Liver slice culture for assessing hepatotoxicity of freshwater cyanobacteria

1. A modified mouse liver slice culture technique was established and the viability of the system was assessed on the basis of leakage of cytosolic enzymes viz. lactate dehydrogenase (LDH), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartic aminotransferase (AST) and slice histology. 2. This system was employed for toxicity screening of five algal species of Indian origin on the basis of the EC50 for LDH leakage (dose of cyanobacteria resulting in leakage of 50% of enzyme) of a known toxic cyanobacterial strain Microcystis aeruginosa (PCC 7820). On the basis of both in vitro and in vivo toxicity none of the five species screened exhibited toxicity. 3. The toxicity of PCC 7820 was compared with a purified cyanobacterial hepatotoxin, Microcystin-LR. Various biochemical indices and histological changes confirm the hepatotoxic nature of the toxins. 4. The toxins did not induce glutathione-mediated lipid peroxidation but they did cause significant mitochondrial damage based on an MTT assay. 5. The study illustrates the utility of this in vitro system in identifying naturally occurring toxic cyanobacteria, particularly hepatotoxic species.

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.

Role of Tumor Necrosis Factor-α in Cadmium-Induced Hepatotoxicity

Liver and kidney injury following acute or chronic exposure to cadmium is well characterized. While hepatocytes and endothelial cells of the sinusoids are thought to be the primary cellular targets in the liver, ultrastructural changes may vary depending upon the exposure regimen and the time following administration. Since acute and chronic liver disease is often associated with the presence of cytokines, we investigated the role of proinflammatory cytokines in cadmium-induced hepatotoxicity. Supernatants from cultured liver slices obtained from acute or subchronic cadmium-exposed rats and mice were collected and cytokine secretion was examined. In addition, mRNA transcripts for IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, MIP-2, IFN-gamma, and ICAM-1 from livers of treated mice were quantitated by reverse transcription-polymerase chain reaction. Modest increases in secretion of TNF-alpha, IL-1 alpha, and IL-6 were observed in response to cadmium which were enhanced in LPS-primed mice. Additionally, cadmium exposure increased IL-1 alpha, IL-1 beta, TNF-alpha, MIP-2, IL-6, and ICAM-1 mRNA transcripts in the liver. Immunohistochemical analysis revealed that TNF-alpha was associated with nonparenchymal cells in livers of cadmium-treated mice. Cadmium exposure produced a marked increase in plasma hepatocellular enzyme levels (i.e., AST, LDH, SDH), acute phase proteins (i.e., serum amyloid A), and foci formation in the liver, while focal inflammation and serum amyloid A (SAA) secretion, but not plasma enzymes, were further increased in cadmium-exposed mice primed with LPS. SAA secretion and focal inflammation were prevented by pretreatment with antibodies to TNF-alpha, indicating that these pathological manifestations are cytokine dependent. These data indicate that TNF-alpha, released from nonparenchymal cells as well as associated cytokines, are responsible for certain manifestations observed with cadmium-induced hepatotoxicity.

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 mumol/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 alpha-glutathione-S-transferases (alpha GST). In this study the alpha 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 the in vitro system and to the evaluation of markers for drug induced side-effects or toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of peroxisome proliferators and inducers of xenobiotic metabolism on marker enzyme activities in cultured rat liver slices

Previous work has demonstrated that precision-cut liver slices represent a valuable in vitro system for studying the metabolism and toxicity of xenobiotics. We have investigated their use for enzyme induction studies. Liver slices were prepared from male Sprague-Dawley rats and by using a dynamic culture system could be maintained in RPMI 1640 medium for up to 72 hr. The addition of 0.02–0.5 m m concentrations of ciprofibrate, nafenopin and Wy-14,643 for 48 and 72 hr produced a concentration and time dependent induction of palmitoyl-CoA oxidation and carnitine acetyltransferase activities. Ultrastructural examination of liver slices revealed an increase in the number of peroxisomes. Liver slices were also cultured with 0.2–5 m m phenobarbitone, 5–50 μ m β-naphthoflavone and 0.5–20 μg Aroclor 1254/ml and effects on the metabolism of 7-ethoxycoumarin, 7-benzoxyresorufin and 7-ethoxyresorufin determined. All three compounds produced a concentration and time dependent induction of mixed-function oxidase enzyme activities. The results of this study demonstrate that rat liver slices can be cultured for up to 72 hr and appear to retain functional viability in that they respond to some known peroxisome proliferators and inducers of xenobiotic metabolism. Cultured liver slices may thus be a useful alternative in vitro system to hepatocytes for screening compounds for effects on various enzyme activities.

Adenine and pyridine nucleotide concentrations and relationships to 2,6-dinitrotoluene metabolism in cultured rat liver slices

The relationships between nucleotide (ATP, ADP, AMP, NADPH, NADP +, NADH and NAD +) concentrations and the metabolism of the hepatocarcinogen 2,6-dinitrotoluene (2,6-DNT) in cultured slices of rat liver were investigated. ATP, NADPH and NADH concentrations in freshly prepared rat liver slices at the beginning of culture were 48–67% lower than those measured in freeze-clamped rat liver ( in vivo values). ATP concentrations in cultured liver slices increased with time of incubation, and after 4 hr ATP concentrations in liver slices were 25% greater than in vivo values. In contrast, NADPH and NADH concentrations did not recover with time of culture, and after 4 hr NADPH and NADH concentrations in liver slices were 50 and 24% of in vivo values, respectively. The addition of ethanol (50 m m) to cultured liver slices increased NADH concentrations by 132%, relative to untreated liver slices. Treatment of liver slices with ammonium chloride (10 m m), 6-aminonicotinamide (1 m m), or the substitution of fructose for glucose in the incubation medium significantly decreased NADPH concentrations by 64, 44 and 63%, respectively. All treatments significantly decreased ATP concentrations; fructose was the most effective agent tested and decreased liver slice ATP concentrations by 69%. These results indicate that the changes in liver slice nucleotide concentrations that occur during culture are not irreversible, and we suggest that these changes are a homoeostatic response to culture conditions and not a reflection of tissue damage or cytotoxicity. Rates of 2,6-dinitrotoluene metabolism by rat liver slices were unaffected by fructose or ammonium chloride, despite the decrease in NADPH concentrations produced by these agents. These results suggest that NADPH concentrations are not rate-limiting to 2,6-DNT metabolism by rat liver slices.

Hepatic Uptake and Metabolism of 2,3,7,8-Tetrachlorodibenzo-p-dioxin and 2,3,7,8-Tetrachlorodibenzofuran

Hepatic Uptake and Metabolism of 2,3,7,8-Tetrachlorodibenzo-p-dioxin and 2,3,7,8-Tetrachlorodibenzofuran. Olson, J. R., McGarrigle, B. P., Gigliotti, P. J., Kumar, S., and McReynolds, J. H. (1994). Fundam. Appl. Toxicol. 22, 631-640.The pharmacokinetics of TCDD and related compounds is congener, dose, and species specific, with urinary and biliary excretion being dependent on the metabolism of these compounds. Isolated hepatocytes and liver slices in suspension culture and hepatic microsomes were used as in vitro models to assess the hepatic uptake and metabolism of [3H]- and [14C]TCDD and [3H]TCDF (0.01-1.0 μM) in control and induced (5 μg TCDD/kg, 3 days earlier) male Sprague-Dawley rats. TCDD pretreatment, with an increase in cytochromes P450 1A1 and 1A2 (CYP1A1, CYP1A2), produced an increase in the hepatic uptake of TCDD, while no increase in the hepatic uptake of TCDF was observed. The results are consistent with CYP1A2 serving as a hepatic binding protein for TCDD but not for TCDF. The rates of metabolism of TCDD and TCDF were directly proportional to their concentrations, indicating that the reaction follows first order kinetics at concentrations from 0.01 to 1.0 μM. Very limited metabolism of TCDD and TCDF was observed in control rat liver (0.45 and 3.2 pmol/hr/g hepatocyte wet wt at 0.1 μM, respectively). TCDD induced its own rate of metabolism about two- to fivefold at 1.0 μM but no induction was observed at 0.01 and 0.1 μM. In contrast, TCDD markedly induced the rate of TCDF metabolism at all substrate concentrations. While the results support the role of rat CYP1A1 in TCDF metabolism, the data suggest that CYP1A1 or CYP1A2 may not metabolize TCDD. These results also support the hypothesis that the more rapid metabolism and excretion of TCDF accounts for the relative resistance of the rat to the acute toxicity of TCDF. Comparative studies in rat and human liver microsomes found that TCDF metabolism exhibited first order kinetics in both species. Furthermore, the rate of TCDF metabolism in human liver microsomes was similar to that of control rat liver microsomes. Together the results suggest that TCDF will be far more persistent in rats, and possibly humans, following exposure at low doses which do not significantly induce cytochrome P450 1A1 and/or 1A2.

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.