Characterization of rat small intestinal and colon precision-cut slices as an in vitro system for drug metabolism and induction studies.

The aim of this study was to characterize rat small intestinal and colon tissue slices as a tool to study intestinal metabolism and to investigate gradients of drug metabolism along the intestinal tract as well as drug-induced inhibition and induction of biotransformation. Tissue morphology and the intestinal mucus layer remained intact in small intestinal and colon slices during 3 h of incubation, while alkaline phosphatase was retained and the rate of metabolism of three model compounds (7-hydroxycoumarin, 7-ethoxycoumarin, and testosterone) appeared constant. Phase I and phase II metabolic gradients, decreasing from stomach toward colon were shown to be clearly different for the model compounds used. Furthermore, the observed slice activities were similar or even higher compared with the literature data concerning metabolism of in vitro intestinal systems. Preincubation with beta-naphthoflavone for 24 h induced the O-deethylation of 7-ethoxycoumarin from nearly undetectable to 140 pmol/min/mg protein in small intestine (fresh slices, 43 pmol/min/mg protein) and to 100 pmol/min/mg protein in colon slices (fresh slices, undetectable). Ketoconazole inhibited metabolism of testosterone by 40% and that of 7-ethoxycoumarin by 100%. In conclusion, we showed that the intestinal slice model is an excellent model to study drug metabolism in the intestine in vitro, since we found that the viability parameters remain constant and the measured enzyme activities are relevant, sensitive to inhibitors, and inducible. Therefore, it is a promising tool to study intestinal drug metabolism in human intestine in vitro in the future.     

Metabolic and structural viability of precision-cut rat lung slices in culture

1. The principal objective was to evaluate the functional and structural integrity of precision-cut rat lung slices in culture over 72 h. 2. Lung slices metabolized 7-ethoxycoumarin in a time-dependent fashion, the major metabolites being the sulphate and glucuronide of 7-hydroxycoumarin with very low levels of the free compound. Prior treatment of rats with beta-naphthoflavone elevated markedly the rate of metabolism. The optimum slice thickness, as exemplified by the metabolism of 7-ethoxycoumarin, was about 600 microm. 3. Lung slices retained metabolic viability towards 7-ethoxycoumarin for 8 h, but after this point a marked decline in metabolic activity was noted. However, very low levels of activity were still evident following a 72 h incubation. 4. Morphological examination of lung slices revealed nuclear degeneration and loss of tissue architecture following 24h incubation. When cellular integrity was assessed using lactate dehydrogenase, a time-dependent leakage was evident with maximum loss occurring within 24h; longer incubations did not result in further leakage. 5. It is concluded that precision-cut rat lung slices, of 600 microm thickness, can be maintained metabolically viable in culture for some 8 h.     

A new technique for preparing precision-cut slices from small intestine and colon for drug biotransformation studies

INTRODUCTION: A new technique was developed to prepare precision-cut slices from small intestine and colon with the object of studying the biotransformation of drugs in these organs. METHODS: Rat intestinal slices were prepared in two different ways. In the first method, slices were punched out of the small intestine. In the second method, precision-cut slices were made from agarose-filled and -embedded intestines, using the Krumdieck tissue slicer. This method was also applied to colon tissue. Viability of the slices was determined by analysis of intracellular ATP and RNA levels and morphology. Drug metabolizing activity was studied using lidocaine, testosterone, and 7-ethoxycoumarin (7-EC) as phase I substrates, and 7-hydroxycoumarin (7-HC) as a phase II substrate. RESULTS: Precision-cut slices made from agarose-filled and -embedded intestine better preserved ATP levels than tissue that was punched out of the intestinal wall. After 24 h of incubation, morphology in precision cut-slices showed was quite well preserved while punched out tissue was almost completely autolytic after incubation. In addition, total RNA amount and quality was much better maintained in precision-cut slices, when compared to punched out tissue. Both intestinal slices and punched-out tissue showed high, and comparable, phase I and phase II biotransformation activities. DISCUSSION: It is concluded that preparing precision-cut 0.25 mm slices out of agarose-filled and -embedded intestine provides an improvement, compared with punched-out tissue, and that both intestinal and colon slices are useful preparations for in vitro biotransformation studies.     

Innovative methods to study human intestinal drug metabolism in vitro: precision-cut slices compared with ussing chamber preparations.

Predictive in vitro methods to investigate drug metabolism in the human intestine using intact tissue are of high importance. Therefore, we studied the metabolic activity of human small intestinal and colon slices and compared it with the metabolic activity of the same human intestinal segments using the Ussing chamber technique. The metabolic activity was evaluated using substrates to both phase I and phase II reactions: testosterone, 7-hydroxycoumarin (7HC), and a mixture of cytochrome P450 (P450) substrates (midazolam, diclofenac, coumarin, and bufuralol). In slices of human proximal jejunum, the metabolic activity of several P450-mediated and conjugation reactions remained constant up to4hof incubation. In the colon slices, conjugation rates were virtually equal to those in small intestine, whereas P450-mediated conversions occurred much slower. In both organs, morphological evaluation and ATP content implied tissue integrity within this period. P450 conversions using the Ussing chamber technique showed that the metabolic rate (sum of metabolites measured in apical, basolateral, and tissue compartments) was constant up to 3 h. For 7HC conjugations, the metabolic rate remained constant up to 4 h. The distribution of the metabolites in the compartments differed between the substrates. Overall, metabolic rates were surprisingly similar in both techniques and appear similar to or even higher than in liver. In conclusion, this study shows that both human intestinal precision-cut slices and Ussing chamber preparations provide useful tools for in vitro biotransformation studies.     

Metabolic and structural viability of precision-cut rat lung slices in culture

1. The principal objective was to evaluate the functional and structural integrity of precision-cut rat lung slices in culture over 72?h.

2. Lung slices metabolized 7-ethoxycoumarin in a time-dependent fashion, the major metabolites being the sulphate and glucuronide of 7-hydroxycoumarin with very low levels of the free compound. Prior treatment of rats with β-naphthoflavone elevated markedly the rate of metabolism. The optimum slice thickness, as exemplified by the metabolism of 7-ethoxycoumarin, was about 600?µm.

3. Lung slices retained metabolic viability towards 7-ethoxycoumarin for 8?h, but after this point a marked decline in metabolic activity was noted. However, very low levels of activity were still evident following a 72?h incubation.

4. Morphological examination of lung slices revealed nuclear degeneration and loss of tissue architecture following 24?h incubation. When cellular integrity was assessed using lactate dehydrogenase, a time-dependent leakage was evident with maximum loss occurring within 24?h; longer incubations did not result in further leakage.

5. It is concluded that precision-cut rat lung slices, of 600?µm thickness, can be maintained metabolically viable in culture for some 8?h.

     

Liver slices in dynamic organ culture. II. An in vitro cellular technique for the study of integrated drug metabolism using human tissue

1. Precision cut human liver slices in dynamic organ culture have been used to study the integrated metabolism of 7-ethoxycoumarin and the conjugation of 7-hydroxycoumarin. 2. The metabolism of 7-ethoxycoumarin and 7-hydroxycoumarin was monitored for 6 h. For both substrates there was a time-dependent increase in metabolites present in the incubation medium. The low levels of free 7-hydroxycoumarin found in the medium when 7-ethoxycoumarin was the substrate suggests good coupling of phase I and phase II metabolism. 3. With suitable incubation conditions, i.e. change of medium containing new substrate every 2 h, the metabolism of both 7-ethoxycoumarin and 7-hydroxycoumarin by human liver slices was found to proceed at similar rates for up to 24 h. This was demonstrated using five separate human liver preparations. 4. Human liver slices also metabolized mono-chlorobenzene and o-, m- and p-dichlorobenzene to aqueous soluble metabolites. There was a time-dependent increase in the appearance of aqueous soluble metabolites present in the incubation medium. Metabolites were not retained by the liver slices. 5. A cold-storage transit buffer has been described and used to maintain the levels of drug metabolism in both rat and human tissue for periods of up to 6 h. 6. The use of human liver slices in dynamic organ culture as a suitable method for the direct assessment of integrated hepatic drug metabolism is proposed.     

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

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

Structural and functional integrity of precision-cut liver slices in xenobiotic metabolism: a comparison of the dynamic organ and multiwell plate culture procedures

1. Objectives were two-fold: (1) to compare the viability of precision-cut liver slices in two culture systems, namely the dynamic organ and the multiwell plate; and (2) to evaluate whether increasing the number of slices per incubation results in a proportional increase in the extent of metabolism. 2. With both culturing systems, the major products of 7-ethoxycoumarin metabolism were the sulphate and glucuronide conjugates of 7-hydroxycoumarin with very low levels of the free compound. When the multiwell plate procedure was used, metabolism increased linearly for at least 10 h, whereas it tended to plateau after 6 h in the dynamic organ culture system. At preincubations > 10 h, significantly more metabolism of 7-ethoxycoumarin was seen in the slices cultured using the multiwell system compared with the dynamic organ system. 3. Morphological evaluation employing light and electron microscopy revealed that liver slices incubated using the multiwell system were structurally better preserved compared with those incubated using the dynamic organ system. 4. Using the multiwell system, increasing the number of slices per incubation from one to two resulted in only a modest increase in the metabolism of 7-ethoxycoumarin. The rate of metabolism of this substrate was much higher with one liver slice when expressed per mg homogenate protein. 5. It is concluded that (1) the multiwell plate culture system for culturing slices is superior to the dynamic organ system in studying the metabolism of xenobiotics following long-term incubations, (2) increasing the number of slices per incubation does not result in a corresponding increase in the rate of metabolism, and (3) in both culture systems optimal viability appears to be within 24 h of incubation.     

Prediction of whole-body metabolic clearance of drugs through the combined use of slices from rat liver, lung, kidney, small intestine and colon

1: The aim was to investigate whether precision-cut rat tissue slices could be used to predict metabolic drug clearance in vivo. To obtain a complete picture, slices not only from liver, but also from lung, kidney, small intestine and colon were included. 2: The metabolic clearances of 7-ethoxycoumarin, 7-hydroxycoumarin, testosterone, methyltestosterone and warfarin were determined by measuring the disappearance of these compounds during incubation with slices prepared from liver, lung, kidney, small intestine and colon. 3: The total in vitro metabolic clearance was determined by adding the individual in vitro organ clearances from the slices. Prediction based on the in vitro clearance was within an order of magnitude to the corresponding in vivo values. Interestingly, the relative contribution of extrahepatic metabolic clearance of the studied compounds to total clearance was remarkably high, ranging from 35 to 72% of the total metabolic clearance. 4: It is concluded that the model of multi-organ precision-cut slices is a useful in vitro tool for prediction of in vivo metabolic clearance. In addition, it provides information about the relative contribution of the liver, lung, kidney, small intestine and colon to the total metabolic clearance.     

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

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

Prediction of whole-body metabolic clearance of drugs through the combined use of slices from rat liver,lung, kidney,small intestine and colon

1.?The aim was to investigate whether precision-cut rat tissue slices could be used to predict metabolic drug clearance in vivo. To obtain a complete picture, slices not only from liver, but also from lung, kidney, small intestine and colon were included.

2.?The metabolic clearances of 7-ethoxycoumarin, 7-hydroxycoumarin, testosterone, methyltestosterone and warfarin were determined by measuring the disappearance of these compounds during incubation with slices prepared from liver, lung, kidney, small intestine and colon.

3.?The total in vitro metabolic clearance was determined by adding the individual in vitro organ clearances from the slices. Prediction based on the in vitro clearance was within an order of magnitude to the corresponding in vivo values. Interestingly, the relative contribution of extrahepatic metabolic clearance of the studied compounds to total clearance was remarkably high, ranging from 35 to 72% of the total metabolic clearance.

4.?It is concluded that the model of multi-organ precision-cut slices is a useful in vitro tool for prediction of in vivo metabolic clearance. In addition, it provides information about the relative contribution of the liver, lung, kidney, small intestine and colon to the total metabolic clearance.     

Morphology and cytochrome P450 isoforms expression in precision-cut rat liver slices

Precision-cut liver slices are a widely accepted in vitro system for the examination of drug metabolism, enzyme induction, or hepatotoxic effects of xenobiotics. The maintenance of the distinct lobular expression and induction pattern of phase I biotransformation enzymes, however, has not been examined systematically so far. Thus, in the present study, both longitudinal and transversal sections of male rat liver slices were investigated morphologically, as well as immunohistochemically for the expression of different cytochrome P450 (CYP) isoforms after prolonged incubation or after exposure to typical inducers. Histopathological examinations revealed an increasing vacuolization of the periportal hepatocytes mainly in the middle of the slices from 6 h of incubation on, paralleled by a loss of glycogen in the respective cells. After 24 h, mainly in the center of the slices, necroses of cells occurred. After 48 h of incubation, typically a central band of coagulative necrosis flanked by superficial layers of viable cells was observed. Freshly prepared slices displayed a CYP subtypes expression as normal liver specimen, a very low centrilobular CYP 1A1 immunostaining, but a strong CYP 2B1 and 3A2 expression predominantly in the central and intermediate lobular zones. From 2 h on, the immunostaining for CYP 2B1 and 3A2 was to some extent reduced. After 24 h of incubation with beta-naphthoflavone, the CYP 1A1 and 2B1 expression was induced mainly in the viable cells around central veins, around some portal fields with bigger vessels and in the cell layers close to the slice surface. At the same sites, phenobarbital led to an increased CYP 2B1 and 3A2 expression and dexamethasone to an elevated CYP 3A2 immunostaining. These results show, that an in vitro induction of phase I enzymes in precision-cut liver slices can be demonstrated also immunohistochemically.     

Induction by xenobiotics of phase I and phase II enzyme activities in the human keratinocyte cell line NCTC 2544

This study analyses the expression and induction of several drug-metabolising enzyme activities involved in either phase I or phase II biotransformations in NCTC 2544 human keratinocytes. The phase I activities 7-ethoxycoumarin O-deethylase (ECOD), 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depenthylase (PROD) were easily detectable in basal conditions. During incubations lasting up to 144 h in the presence of the classical cytochrome P450 inducers β-naphthoflavone (BNF), 3-methylcholanthrene (MC) and phenobarbital (PB), a considerable and significant increase in all the three activities was observed. PROD activity was induced up to 4.5-fold after 96 h in the presence of PB. The MC-induced ECOD and EROD activities were also dose-dependently inhibited by -naphothflavone, which was given to the cells during the incubation with CYP 1A1 inducers. Also the PB-induced PROD activity was decreased by the simultaneous addition of the CYP 2B inhibitor metyrapone. Both cytochrome P450 inhibitors were used at non-cytotoxic concentrations. The phase II enzymes glutathione S-transferase, aldehyde dehydrogenase and quinone reductase were all highly expressed and inducible by MC. The exposure (24 h) of the cells to four hair dyes used in cosmetic formulations resulted in a marked increase in ECOD activity. All data give sustained evidence for the suitability of NCTC 2544 cell line to skin toxicology studies.     

Liver slices in dynamic organ culture. I. An alternative in vitro technique for the study of rat hepatic drug metabolism

1. Precision-cut liver slices in dynamic organ culture, a novel in vitro technique, is described and applied to the study of hepatic drug metabolism in the rat. 2. These slices catalysed the oxidative O-deethylation of the substrate, 7-ethoxycoumarin, over 6 h incubation. In addition, the direct conjugation of 7-hydroxycoumarin with either sulphate or glucuronic acid was maintained over 6 h. 3. The formation of 7-hydroxycoumarin and the presence of the sulphate and glucuronide conjugates in slices exposed to 7-ethoxycoumarin demonstrated integrated phase I and phase II drug metabolizing activities in this system. 4. Minor modifications of the incubation system allowed for the metabolism of four volatile chlorinated benzenes: monochlorobenzene 1,2-, 1,3-, and 1,4-dichlorobenzenes to aqueous soluble metabolites. 5. The use of liver slices in dynamic organ culture as an alternative preparation for the study of xenobiotic metabolism is discussed.     

A comparison of xenobiotic metabolism in cells isolated from rat liver and small intestinal mucosa.

The metabolism of several foreign compounds has been investigated in viable isolated liver and intestinal mucosal cells. Glucuronic acid and sulphate conjugation was observed in both cell preparations with respect to phenol. Conjugation of 1-naphthol was also observed in isolated intestinal cells. Acetanilide was the only metabolic product of aniline observed with intestinal cells. N-Acetylation was also found to be the major pathway of aniline metabolism in liver cells but p-hydroxylation followed by O-conjugation were also important reactions. Intestinal cells thus appear to be generally effective in conjugation reactions. However, in contrast to hepatocytes the intestinal cells were unable to form glycine conjugates from benzoic acid. The metabolism of 7-ethoxycoumarin and 7-hydroxycoumarin was investigated in isolated intestinal cells from control, 3-methylcholanthrene and phenobarbitone pretreated rats. Glucuronic acid and sulphate conjugation rates of 7-hydroxycoumarin appeared to be unaffected by these pretreatments whereas increases in 7-ethoxycoumarin O-deethylation were observed.     

Metabolism of nicotine and induction of CYP1A forms in precision-cut rat liver and lung slices.

The aim of this study was to investigate xenobiotic metabolism and induction of cytochrome P450 (CYP) forms in precision-cut rat liver and lung slices, employing nicotine as a model compound. Freshly cut rat liver and lung slices metabolised nicotine to the major metabolite cotinine. Observed Km values for cotinine formation in liver and lung slices were 323 and 41.7 microM, respectively, with corresponding V(max) values of 47.2 and 3.21 pmol/min/mg protein, respectively. Rat liver and lung slices were cultured for 48 h with Aroclor 1254, benzo(a)pyrene, nicotine and cotinine. Both Aroclor 1254 and benzo(a)pyrene produced a marked induction of CYP1A-dependent 7-ethoxyresorufin O-deethylase activity in both liver and lung slices. However, while nicotine induced 7-ethoxyresorufin O-deethylase activity in lung slices, but not in liver slices, cotinine did not induce enzyme activity in either liver or lung slices. Overall, while higher rates of nicotine metabolism were observed in rat liver slices, nicotine-induced CYP1A form induction was observed in lung slices. These results demonstrate the usefulness of precision-cut tissue slices for studying tissue differences in xenobiotic metabolism and CYP form induction.     

In vitro induction of cytochrome P450 2B1- and 3A1-mRNA and enzyme immunostaining in cryopreserved precision-cut rat liver slices

With the exception of cytochrome P450 (CYP) 1A1 and its mRNA, in vitro induction of other CYP forms has not been demonstrated in cryopreserved liver slices until now. Therefore precision-cut rat liver slices were cultured after cryopreservation and thawing in William's medium E for up to 24 h in the presence of inducers to demonstrate CYP2B1- and CYP3A1-mRNA induction. CYP-mRNA expression was determined by competitive RT-PCR. Exposure to 100 microM phenobarbital caused a more than 20-fold increase in CYP2B1-mRNA expression within 24 h, reaching concentrations comparable with those of PB-exposed fresh rat liver slices. Exposure to 1 microM pregnenolone 16 alpha-carbonitrile enhanced CYP3A1-mRNA expression by more than 30-fold within 24 h. This is in the same range, although with higher variability, as detected with fresh liver slices. In spite of considerable variability among the thawed slices, the induction factors are high enough for a sensitive detection of an induction at mRNA level. Additionally, immunostaining of respective CYP-forms was performed in sections of few samples, indicating CYP increase in viable cells of cryopreserved slices.     

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

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

The metabolism of 7-ethoxycoumarin and 7-hydroxycoumarin by rat and hairless mouse skin strips

The metabolism of 7-ethoxycoumarin and 7-hydroxycoumarin was studied in rat and hairless mouse skin strips. These preparations supported de-ethylation, sulphation and glucuronidation reactions. The de-ethylation reaction was inducible in both species by pretreatment with either 5,6-benzoflavone or 3-methylcholanthrene. The hairless mouse strips exhibited a greater basal de-ethylase activity than rat strips, although the latter was the more responsive to inducers. Accompanying the increase in de-ethylation activity was a change in the pattern of metabolites, with a large increase in the percentage of the unconjugated metabolite. When 7-hydroxycoumarin was employed as the primary substrate the glucuronide was the major metabolite formed by strips from both species. The glucuronidation and sulphation activities were unchanged by 3-methylcholanthrene pretreatment.     

Time- and concentration-dependent induction of CYP1A1 and CYP1A2 in precision-cut rat liver slices incubated in dynamic organ culture in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin

In a previous 24-h study, precision-cut rat liver slices were validated as a useful in vitro model for assessing the dose-related induction of CYP1A1 and CYP1A2 in rat liver following exposure to 2, 3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Further assessment of the utility of this model was accomplished by initially exposing rat liver slices to medium containing TCDD (0.01 nM) for 24 h and incubating the slices up to an additional 72 h in TCDD-free medium. The slices remained viable throughout the incubation period with an intracellular potassium content varying from 45.2 +/- 2.3 micromol/g at 48 h to 50.0 +/- 1.6 micromol/g at 72 h. In TCDD-exposed slices, CYP1A1 protein and its respective enzymatic activity, the O-deethylation of ethoxyresorufin (EROD), significantly increased with time over the 96-h incubation period, with EROD activity increasing from 63.6 +/- 14.2 at 24 h to 905 +/- 291 pmol/mg/min at 96 h. Under identical incubation conditions, but in the absence of TCDD, the EROD activity for the control liver slices ranged from 14. 3 +/- 4.3 to 44.9 +/- 11.9 pmol/min/mg. Conversely, the level of CYP1A2 protein and its respective activity (acetanilide hydroxylation) transiently decreased from 24 to 96 h with no significant differences observed between the control (0 nM TCDD) and treatment group (0.01 nM TCDD). The concentration-effect relationship at 96 h was characterized by incubating rat liver slices for the initial 24 h in medium containing TCDD at concentrations ranging from 0.1 pM to 10 nM. Induction of CYP1A1 protein and EROD activity was observed for all treatment groups with the 10 nM TCDD treatment group displaying greater than 100-fold induction compared to control (0 nM TCDD). Immunohistochemical localization of CYP1A1 protein within liver slices supported the time- and concentration-dependent induction of EROD activity by TCDD. The induction of CYP1A1 was initially observed to be centrilobular, with increased expression due to both elevated CYP1A1 within cells and the recruitment of additional cells expressing CYP1A1 throughout the entire liver slice. Additionally, the immunohistochemical analysis of the liver slices demonstrated the conservation of tissue architecture following up to 96 h of incubation in dynamic organ culture and provided further evidence for maintenance of tissue viability. In comparison to CYP1A1, the induction of CYP1A2 at 96 h was a less sensitive response, with significant induction of CYP1A2 protein and its respective activity occurring at a medium concentration of 0.1 nM TCDD (686 pg/g liver). In general, increasing the incubation period from 24 to 96 h markedly increased TCDD-induced expression of CYP1A1 and minimally enhanced CYP1A2 expression. Moreover, extending the incubation period to 96 h resulted in in vitro induction profiles for CYP1A1 and CYP1A2 that were qualitatively and quantitatively similar to that previously observed following in vivo exposure to TCDD (Drahushuk et al., Toxicol. Appl. Pharmacol. 140, 393-403, 1996).