Up-regulation of CYP1A/B in rat lung and liver, and human liver precision-cut slices by a series of polycyclic aromatic hydrocarbons; association with the Ah locus and importance of molecular size.

Exposure of precision-cut rat liver slices to six structurally diverse polycyclic aromatic hydrocarbons, namely benzo[a]pyrene, benzo[b]fluoranthene, dibenzo[a,h]anthracene, dibenzo[a,l]pyrene, fluoranthene and 1-methylphenanthrene, led to induction of ethoxyresorufin O-deethylase, CYP1A apoprotein and CYP1A1 mRNA levels, but to a markedly different extent. In liver slices, constitutive CYP1A1 mRNA levels were higher, as well as being markedly more inducible by PAHs, compared with CYP1B1, a similar profile to that observed in human liver slices following exposure to the PAHs. Increase in ethoxyresorufin O-deethylase and in CYP1A1 apoprotein levels was also observed when precision-cut rat lung slices were incubated with the same PAHs, the order of induction potency being similar to that observed in liver slices. Under the same conditions of exposure, CYP1B1 apoprotein levels were elevated in the lung. Up-regulation of CYP1A1 by the six PAHs correlated with their affinity for the Ah receptor, determined using the chemical-activated luciferase expression (CALUX) assay. It may be concluded that (a) precision-cut liver and lung slices may be used to assess the CYP1 induction potential of chemicals at the activity, apoprotein and mRNA levels; (b) rat is a promising surrogate animal for human in studies to evaluate CYP1 induction potential; (c) CYP1A1 is far more inducible than CYP1B1 in both rat liver and lung; (d) CYP1 up-regulation by PAHs is related to their affinity for the Ah receptor, and finally (e) computer analysis revealed that the ratio of molecular length/width is an important determinant of CYP1 induction potency among equiplanar PAHs.     

Differential regulation of the human CYP3A4 promoter in transgenic mice and rats.

Previously we described a transgenic mouse model [FVB/NTg(CYP3A4-luc)Xen] using a reporter construct consisting of 13 kilobases of the human CYP3A4 promoter driving the firefly luciferase gene in the inbred FVB/N mouse strain. Here we report regulation of the same CYP3A4-luc reporter gene in a transgenic outbred mouse strain (CD-1) and in a transgenic rat (Sprague-Dawley). Basal reporter expression and responses to several xenobiotics in the transgenic CD-1 mice [CD-1/Crl-Tg(CYP3A4-luc)Xen] were similar to those in the transgenic FVB/N mice. In both mouse backgrounds, the basal levels of the reporter were higher in male compared with female, and in the FVB/N strain there was greater induction for all drugs in male compared with female; however, in the CD-1 background this gender difference for induction was not obvious. In contrast with transgenic mice, transgenic rats [SD/Tac-Tg(CYP3A4-luc)Xen] expressed the luciferase reporter at higher basal levels in female compared with male rats. Responses to some compounds were much greater in rats than in mice, and the kinetics of induction was different with peak induction occurring later in the rat compared with the mouse. Our results suggest that the human CYP3A4 promoter is regulated differently in transgenic mice and rats in some aspects.     

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.     

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.     

A Cyp1a2-luciferase transgenic CD-1 mouse model: responses to aryl hydrocarbons similar to the humanized AhR mice.

Here we describe a transgenic mouse model [Crl:CD-1(ICR)BR-Tg(Cyp1a2-luc)Xen] using luciferase as a reporter for Cyp1a2 gene regulation. An 8.4-kilobase mouse Cyp1a2 promoter driving the firefly luciferase gene was microinjected into single-cell-stage CD-1 mouse embryos. A transgenic mouse line was selected based on basal and induced levels of the transgene in mouse liver by an in vivo bioluminescent imaging method. The basal levels of the luciferase reporter in liver were expressed much higher than other tissues, which correlated well with the endogenous Cyp1a2 mRNA tissue distribution. Male signals were about 23-fold higher than females in liver. However, the Cyp1a2 mRNA showed no gender difference. When mice were challenged with xenobiotics, the liver luciferase signal was induced to various degrees. At the doses we used, the relative effects were phenobarbital > 2,3,7,8-tetrachlorodibenzo-p-dioxin > 3-methylcholanthrene > benzo[a]pyrene and beta-naphthoflavone. Induction of the Cyp1a2-luc reporter was generally consistent with the endogenous Cyp1a2 mRNA. However, phenobarbital induction was unexpectedly higher, while beta-naphthoflavone induction of the reporter was much lower than that of the endogenous Cyp1a2 gene. Induction of the Cyp1a2-luc transgene by aryl hydrocarbons (Ah) in the CD-1 background was much less than that found in the Ah responsive C57BL/6 mice, while being similar to the nonresponsive DBA/2 strain. Sequence analysis of the CD-1 Ah receptor (AhR) cDNA clones demonstrated that consensus sequence was identical to some of the Ah-responsive strains such as BALB/C and CBA/J mice. The 104-kD AhR protein was not detectable in CD-1 mice, while the 97-kD AhR was detected in the C57BL/6 mice by Western blot using an AhR antibody. Low expression of the AhR in CD-1 mice could be in part responsible for low responsiveness to Ah compounds. The findings demonstrated the outbred CD-1 mouse is a low-responsive strain, and the Cyp1a2-luc transgenic CD-1 mice can be used for studying the regulation of the mouse Cyp1a2 gene in an Ah low-responsive strain in real time using the bioluminescent imaging approach.     

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).     

Tissue specific induction of cytochrome P450 (CYP) 1A1 and 1B1 in rat liver and lung following in vitro (tissue slice) and in vivo exposure to benzo(a)pyrene.

Cytochrome P-450s (CYPs) detoxify a wide variety of xenobiotics and environmental contaminants, but can also bioactivate carcinogenic polycyclic aromatic hydrocarbons, such as benzo(a)pyrene (BaP), to DNA-reactive species. The primary CYPs involved in the metabolism and bioactivation of BaP are CYP1A1 and CYP1B1. Furthermore, BaP can induce expression of CYP1A1 and CYP1B1 via the aryl hydrocarbon receptor. Induction of CYP1A1 and CYP1B1 by BaP in target (lung) and non-target (liver) tissues was investigated utilizing precision-cut rat liver and lung slices exposed to BaP in vitro. Tissue slices were also prepared from rats pretreated in vivo with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to induce expression of CYP1A1 and CYP1B1. In addition, in vivo exposure studies were performed with BaP to characterize and validate the use of the in vitro tissue slice model. In vitro exposure of liver and lung slices to BaP resulted in a concentration-dependent increase in CYP1A1 and CYP1B1 mRNA and protein levels, which correlated directly with the exposure-related increase in BaP-DNA adduct levels observed previously in the tissue slices [Harrigan, J.A., Vezina, C.M., McGarrigle, B.P., Ersing, N., Box, H.C., Maccubbin, A.E., Olson, J.R., 2004. DNA adduct formation in precision-cut rat liver and lung slices exposed to benzo(a)pyrene. Toxicological Sciences 77, 307-314]. Pretreatment of animals in vivo with TCDD produced a marked induction of CYP1A1 and CYP1B1 expression in the tissue slices, which was similar to the levels of CYP1A1 and CYP1B1 mRNA achieved in liver and lung following in vivo treatment with BaP. Following in vitro exposure to BaP, the levels of CYP1A1 were greater in the lung than the liver, while following all exposures (in vitro and in vivo), the levels of CYP1B1 mRNA were greater in lung tissue compared to liver. The higher expression of CYP1A1 and CYP1B1 in the lung was associated with higher levels of BaP-DNA adducts in the lung slices (Harrigan et al.'s work) and together, these results may contribute to the tissue specificity of BaP-mediated carcinogenesis.     

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.     

DNA adduct formation in precision-cut rat liver and lung slices exposed to benzo[a]pyrene.

Chemical-DNA adducts provide an integrated measure of exposure, absorption, bioactivation, detoxification, and DNA repair following exposure to a genotoxic agent. Benzo[a]pyrene (BaP), a prototypical polycyclic aromatic hydrocarbon (PAH), can be bioactivated by cytochrome P-450s (CYPs) and epoxide hydrolase to genotoxic metabolites which form covalent adducts with DNA. In this study, we utilized precision-cut rat liver and lung slices exposed to BaP to investigate tissue-specific differences in chemical absorption and formation of DNA adducts. To investigate the contribution of bioactivating CYPs (such as CYP1A1 and CYP1B1) on the formation of BaP-DNA adducts, animals were also pretreated in vivo with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) prior to in vitro incubation of tissue slices with BaP. Furthermore, the tissue distribution of BaP and BaP-DNA adduct levels from in vivo studies were compared with those from the in vitro tissue slice experiments. The results indicate a time- and concentration-dependent increase in tissue-associated BaP following exposure of rat liver and lung tissue slices to BaP in vitro, with generally higher levels of BaP retained in lung tissue. Furthermore, rat liver and lung slices metabolized BaP to reactive intermediates that formed covalent adducts with DNA. Total BaP-DNA adducts increased with concentration and incubation time. Adduct levels (fmol adduct/microg DNA) in lung slices were greater than liver at all doses. Liver slices contained one major and two minor adducts, while lung slices contained two major and 3 minor adducts. The tissue-specific qualitative profile of these adducts in tissue slices was similar to that observed from in vivo studies, further validating the use of this model. Pretreatment of animals with TCDD prior to in vitro incubation with BaP potentiated the levels of DNA adduct formation. TCDD pretreatment altered the adduct distribution in lung but not in liver slices. Together, the results suggest that tissue-specific qualitative and quantitative differences in BaP-DNA adducts could contribute to the lung being a target tissue for BaP carcinogenesis. Furthermore, the results validate the use of precision-cut tissue slices incubated in dynamic organ culture as a useful model for the study of chemical-DNA adduct formation.     

Induction of phase I and II drug metabolism in rat small intestine and colon in vitro.

The aim of this study was to evaluate drug metabolism in rat small intestinal and colon precision-cut slices during 24 h of incubation and the applicability of these slices for enzyme induction studies. Various parameters were evaluated: intracellular levels of ATP (general viability marker), alkaline phosphatase activity (specific epithelial marker), villin expression (specific epithelial marker), and metabolic rates of 7-ethoxycoumarin (CYP1A), testosterone (CYP3A and CYP2B), and 7-hydroxycoumarin (glucuronide and sulfate conjugation) conversions. ATP and villin remained constant up to, respectively, 5 and 8 h in small intestine and up to 24 h in colon. The metabolic rate remained constant in small intestinal slices up to 8 h and decreased afterward to 24 to 92%, depending on the substrate studied. The inducibility of metabolism in small intestinal and colon slices was tested with several inducers at various concentrations and incubation times. The following inducers were used: 3-methylcholanthrene, beta-naphthoflavone, indirubin, and tert-butylhydroquinone (aryl hydrocarbon receptor ligands), dexamethasone (glucocorticoid receptor/pregnane X receptor ligand) and phenobarbital (constitutive androstane receptor ligand). After incubation with inducers, metabolic rates were evaluated with 7-ethoxycoumarin and testosterone (phase I) and 7-hydroxycoumarin (phase II) as substrate. All inducers elevated the metabolic rates consistent with the available published in vivo induction data. Induction of enzyme activity was already detectable after 5 h (small intestine) and after 8 h (colon) for 3-methylcholanthrene and beta-naphthoflavone and was clearly detectable for all tested inducers after 24 h (up to 20-fold compared with noninduced controls). In conclusion, small intestinal and colon precision-cut slices are useful for metabolism and enzyme induction studies.