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 B1 (AFB1) 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 mM AFB1 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, AFB1 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 AFB1 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 preparations used in this study. In conclusion, these results provide further evidence that PBO is a non-genotoxic agent which does not damage DNA in human liver.     

Effect of some cooked food mutagens on unscheduled DNA synthesis in cultured precision-cut rat, mouse and human liver slices

The present study investigated the rates and perceived effects of past traumatic brain injury (TBI) and substance use in a prison population. Responses to a questionnaire indicated that 86.4% of the 118 respondents had sustained a TBI, with 56.7% reporting more than one, and rates of illicit substance use were higher than the general population. Maori reported 12% more TBI and more substance use than non-Maori. All those with TBI reported difficulties with general memory and socialization on a problem rating scale, but there was no relationship between level of difficulty and severity of TBI, problems with interpersonal relationships, family, and finances were associated with greater substance use.     

Xenobiotic metabolism in rat, dog, and human precision-cut liver slices, freshly isolated hepatocytes, and vitrified precision-cut liver slices

Human, rat, and dog phase I and phase II xenobiotic metabolism in precision-cut liver slices and freshly isolated hepatocytes was compared using a range of substrates. Carbamazepine (50 microM) and styrene (2 mM) were used as probes to study the maintenance of cytochrome P450 and epoxide hydrolase-mediated metabolism in male Sprague-Dawley rat, precision-cut liver slices and hepatocytes. Carbamazepine metabolism in both models resulted in the formation of the bioactive 10,11-epoxide (KM = 766 microM and Vmax = 2.5 pmol/min/mg protein in precision-cut slices). Epoxide formation was higher (2.4-fold) in hepatocytes than slices. Styrene was deactivated to styrene diol at a higher rate in hepatocytes (9.7-fold) than slices. The lower rate of metabolism in slices compared with hepatocytes confirms our previous observations using testosterone, 7-ethoxycoumarin, 1-chloro-2,4-dinitrobenzene and 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone in the rat. Testosterone 6 beta-hydroxylation in human liver slices was similar to cultured hepatocytes, but lower than in freshly isolated hepatocytes. 7-Ethoxycoumarin O-deethylation was higher in freshly isolated human hepatocytes, as was the ratio of glucuronide to 7-hydroxycoumarin. Testosterone hydroxylations, 7-ethoxycoumarin O-deethylation, and 1-chloro-2,4-dinitrobenzene conjugation were also lower in male beagle dog slices, compared with freshly isolated hepatocytes. Attempts at long-term preservation of dog liver slices using vitrification and storage for up to 9 days at -196 degrees C resulted in the retention of phase I and phase II metabolism, although conjugation was lower than in freshly prepared slices. Xenobiotic metabolism in short-term incubations is consistently lower in dog and rat precision-cut slices than in freshly isolated hepatocytes; whereas, in humans, this quantitative difference is partly hidden by the large interindividual variation.     

3,3'-Diindolylmethane induces CYP1A2 in cultured precision-cut human liver slices

1. The effect of 3,3'-diindolylmethane (DIM), an indole derivative derived from cruciferous vegetables, on cytochrome P450 (CYP) isoforms in the CYP1A and CYP3A subfamilies has been studied in 72-h cultured human liver slices. 2. In cultured human liver slices 50 microM DIM induced 7-ethoxyresorufin O-deethylase and to a lesser extent 7-methoxyresorufin O-demethylase activities. 3. Western immunoblotting of liver slice microsomes was performed with antibodies to rat CYP1A2 and human CYP3A4. Compared with control liver slice microsomes (dimethyl sulphoxide-only treated), DIM induced levels of CYP1A2 but had little effect on levels of CYP3A4. The treatment of human liver slices with 2 microg/ml of the polycholorinated biphenyl mixture Aroclor 1254 also resulted in an induction of levels of CYP1A2, but had no effect on CYP3A4. 4. These results demonstrate that DIM induces CYP1A isoforms in cultured human liver slices. Some variability in the magnitude of induction of enzyme activities by DIM was observed in four human liver samples examined. For 7-ethoxyresorufin O-deethylase, the magnitude of induction by 50 microM DIM ranged from 2.3- to 19.3-fold. 5. These results demonstrate that cultured human liver slices can be used to evaluate the effect of chemicals derived from cruciferous and other vegetables on CYP isoforms.     

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.     

Metabolism of [ring-U-14C] agaritine by precision-cut rat, mouse and human liver and lung slices

Agaritine [(beta-N-[gamma-L(+)glutamyl]-4-hydroxymethylphenylhydrazine] is present in the common cultivated mushroom Agaricus bisporus and several agaritine derivatives have been shown to produce tumours in experimental animals. In this investigation the metabolism of [ring-U-14C]agaritine has been studied in precision-cut rat, mouse and human liver slices and in precision-cut rat and mouse lung slices. To confirm the functional viability of the tissue slice preparations, the metabolism of 7-ethoxycoumarin was also studied. Liver and lung slices from all species metabolized 50 microM 7-ethoxycoumarin to 7-hydroxycoumarin, which was conjugated with D-glucuronic acid and sulfate. Incubation of rat, mouse and human liver slices, and rat and mouse lung slices with 25 microM [14C]agaritine resulted in a time-dependent formation of metabolite(s), which bound covalently to tissue slice proteins. Agaritine metabolite covalent binding was greater in mouse liver than in rat and human liver slices and was greater in mouse lung than in rat lung slices. No correlation was observed between agaritine metabolite covalent binding and tissue slice gamma-glutamyltransferase activity. Additional studies with mouse liver slices showed that [14C]agaritine was also metabolized to a number of unknown polar metabolites. These results demonstrate that agaritine can be metabolized by enzymes present in mammalian liver and lung.     

Monooxygenation, cytochrome P450-mRNA expression and other functions in precision-cut rat liver slices

Precision-cut rat liver slices (KRUMDIECK slicer, slice thickness 200-250 microm) were incubated in rollers containing modified William's medium E at 37 degrees C for 2, 24 and 48 hrs. Protein, DNA, potassium and glutathione concentrations did not decrease during 48 hrs. Lactate dehydrogenase (LDH) leakage into the medium was relatively marked during the first 2 hrs of incubation, from the 2nd to the 48th hr LDH leakage was very low. The same is true of the release of thiobarbituric acid-reactive substances. Albumin synthesis and transport into the medium decreased to about 70% after 48 hrs. Cytochrome P450 (CYP)-dependent 7-ethoxycoumarin O-deethylation rate was relatively stable up to 48 hrs, whereas testosterone hydroxylation decreased significantly without alterations of the proportions of the 7 quantified hydroxylated metabolites. After exposure of the slices to beta-naphthoflavone for 6 hrs CYP1A1-mRNA expression, measured by competitive RT-PCR, was increased by a factor of at least 1000. Precision-cut liver slices are a useful tool for the study of various hepatic functions, drug metabolism and its induction in vitro.     

Videomicroscopy of methacholine-induced contraction of individual airways in precision-cut lung slices

Contraction of airways of different size can be studied in viable lung slices by videomicroscopy. However, at present, application of this technique is limited by the heterogeneous responses obtained. We investigated the use of precision-cut lung slices to examine contraction of individual airways. Lung slices of 250 +/- 20 microns were prepared from Wistar rats and cultured in a roller incubator in serum-free minimum essential medium (MEM). Under these conditions, the slices were viable for at least 70 h, as indicated by leakage of lactate dehydrogenase into the supernatant, thymidine incorporation and ciliary beating. The slices were placed in a newly developed incubation chamber and mounted by a nylon thread that was fixed to a platinum wire. The whole chamber was positioned on a microscope stage, and contraction of single airways was followed under a microscope that was coupled to a CCD-camera. Reduction in airway area was taken as an index of bronchoconstriction and was determined by a computer program. Addition of methacholine resulted in a concentration-dependent (concentration producing half the maximal effect (EC50) = 0.64 +/- 0.08 (mean +/- SD) microM; n = 64) contraction of single airways. In the presence of hydrocortisone, the EC50 was about six times greater, i.e. 3.7 +/- 0.9 microM (n = 7), and the effect of the steroid was largely abolished by propanolol (EC50 = 1.1 +/- 0.1 microM; n = 7). Airways with an area smaller than 35,000 microns2 were nearly nine times more sensitive to methacholine (EC50 = 0.1 +/- 0.03 microM; n = 20) than larger ones (EC50 = 87 +/- 0.27 microM; n = 22). We conclude that cultured precision-cut lung slices are a useful model for routine study of contraction of individual airways of various sizes. The measurements were precise and reproducible and showed that smaller airways are more sensitive to methacholine than larger ones.     

Two-dimensional electrophoresis of precision-cut testis slices: toxicologic application

Advances in tissue slice technology and a recent novel application of this technique to reproductive toxicology using bovine testis have demonstrated the remarkable utility of this approach. The objective of the present study was to combine this in vitro toxicity test system with large-scale two-dimensional polyacrylamide gel electrophoresis (2-DE) to detect and study alterations in testicular-slice protein patterns as molecular correlates of 1,3,5-trinitrobenzene (TNB) and 1,3-dinitrobenzene (DNB) toxicity. Previous studies have shown that testicular slices remain viable for > 24 h and, as measured by protein synthesis inhibition, TNB causes dose-related injury. Tissue-slices were prepared from bovine testicles incubated for 2, 4 or 6 h and exposed to either 100 microM, 500 microM or 1 mM DNB or TNB in the incubation medium. Slices were collected, solubilized, and separated by large scale 2-DE. Resulting protein patterns were then examined by image analysis, which revealed coefficients of variation in protein spot abundance comparable to patterns from fresh rodent tissue samples. Furthermore, specific protein alterations indicated dose-related inductions and declines in protein abundance, some progressive over time. The results of this investigation demonstrate the potential toxicologic utility of combining in vitro tissue-slice technology with high-resolution 2-DE protein mapping. The consolidation of these methods offers a novel approach for toxicity screening and testing, reduces experimental cost, and reduces the use of laboratory animals.     

Immunohistochemical localization of cytochrome P450 1A1 in precision-cut rat liver slices after in vitro exposure to beta-naphthoflavone

Mono- and polyclonal antibodies have been used to study the localization and distribution of cytochrome P450 1A1 (CYP1A1) in cultured precision-cut liver slices with various immunohistochemical methods. Neither in non-incubated slices nor in slices incubated in the absence of beta-naphthoflavone (BNF) for 24 hrs was CYP1A1 immunohistochemically detectable. After incubation in the presence of BNF (25 microM), however, CYP1A1 was well visible in parenchymal and biliary epithelial cells. CYP1A1 was not evenly distributed, but was localized predominantly in hepatocyte layers near the surfaces of the slices. This distribution could be due to the preferential uptake of BNF by outer cell layers or due to functional changes of inner cells. Together with results obtained with other methods (e.g. RT-PCR) this investigation also demonstrates that precision-cut liver slices are a useful tool for the detection of in vitro induction of CYP1A1.     

Lack of effect of coumarin in women with lymphedema after treatment for breast cancer

BACKGROUND: Lymphedema of the arms can be a serious consequence of local and regional therapy in women with breast cancer. Coumarin has been reported to be effective for the treatment of women with lymphedema; we undertook a study in which we attempted to replicate those findings. METHODS: We studied 140 women with chronic lymphedema of the ipsilateral arm after treatment for breast cancer. The women received 200 mg of oral coumarin or placebo twice daily for six months and then the other treatment for the following six months. The end points of the study consisted of the volume of the arm (calculated from measurements of hand and arm circumference) and the answers on a questionnaire completed by the patient about symptoms potentially related to lymphedema. RESULTS: The volumes of the arms at 6 and 12 months, were virtually identical, regardless of whether coumarin or placebo was given first. After six months, the average volume of the affected arm increased by 21 ml during placebo treatment and 58 ml during coumarin treatment (P=0.80). In addition, answers to patient-completed questionnaires were similar in the two treatment groups. After six months only 15 percent of the women in the coumarin group and 10 percent of those in the placebo group reported that the study medication had helped a moderate or large amount (P=0.19). Coumarin was well tolerated, except that it resulted in serologic evidence of liver toxicity in 6 percent of the women. CONCLUSIONS: Coumarin is not effective therapy for women who have lymphedema of the arm after treatment for breast cancer.     

The metabolism and DNA binding of the cooked-food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in precision-cut rat liver slices

Precision-cut liver slices prepared from Aroclor 1254 pretreated male rats were used to investigate the metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The acetyltransferase and sulfotransferase inhibitors, pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP), and the cytochrome P450 inhibitor, alpha-naphthoflavone (ANF), were used to modulate PhIP metabolism and DNA and protein adduct formation. PCP and DCNP had similar effects on the formation of some PhIP metabolites. PCP and DCNP decreased the formation of 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate (4'-PhIP-sulfate) and 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP)-glucuronide to 10% and 55% of controls, respectively. 2-Amino-1-methyl-4'-hydroxy-6-phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP) was increased by 50% relative to control levels due to PCP and DCNP treatment. PCP and DCNP had different effects on the formation of other PhIP metabolites. Metabolite formation as percent of control for the uncharacterized metabolite, 'Peak A', was 50% and 100% in incubations with PCP and DCNP, respectively. Formation of 4'-hydroxy-PhIP-glucuronide was decreased to 10% of controls with PCP and increased to 147% of controls with DCNP. PCP and DCNP had no effect on the formation of an unidentified metabolite, 'Peak B'. ANF decreased metabolite formation by 60-95%. None of the enzyme inhibitors had a statistically significant effect on PhIP-DNA binding. Covalent binding of PhIP to protein was slightly decreased in incubations containing DCNP or PCP. The lack of significant changes in covalent binding to either DNA or protein suggests that additional pathways may be important in PhIP bioactivation in rat liver slices. With ANF, there was a significant decrease (35%) in protein binding. These observations on the effects of PCP, DCNP and ANF on PhIP metabolism as well as on covalent binding of PhIP to tissue macromolecules are in close agreement with what was reported earlier in hepatocytes. This indicates that tissue slices from various target tissues for tumorigenesis will be a useful in vitro tool for future studies on heterocyclic amine metabolism. This study provides another important example of the utility of precision-cut tissue slices to investigate xenobiotic metabolism and toxicity.     

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 Brendel-Vitron 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% 02/5% 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 microM 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 slices it is desirable to establish optimal culture conditions     

Validation of precision-cut liver slices in dynamic organ culture as an in vitro model for studying CYP1A1 and CYP1A2 induction

The utilization of precision-cut liver slices in dynamic organ culture as an in vitro model was validated by comparing the induction of the biomarker responses following in vitro (rat liver slice) and in vivo exposure 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 nM TCDD 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 nM and 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 single in vivo exposure of rats to TCDD at doses ranging from 0.002 to 5 microg/kg po. Concentration-effect and dose-response relationships for induction of CYP1A1 and CYP1A2 were similar following in vitro and in vivo exposure to TCDD, although the magnitude of induction was greater for in vivo exposure. The data support the use of liver slices in dynamic organ culture for assessing the relative in vivo potency of a compound to induce CYP1A1 and CYP1A2. Human tissue can also be readily utilized in this in vitro model to predict the biological and toxicological effects of a given in vivo exposure to TCDD.     

Kinetics of DNA repair synthesis in guinea-pig pancreatic slices following in vitro exposure to N-methyl-N-nitrosourethane

In vitro exposure of guinea-pig pancreatic slices to NMUT resulted in an increase in hydroxyurea-insensitive 3H-TdR incorporation into DNA; this represents DNA repair synthesis following NMUT-induced DNA damage. The kinetics of this hydroxyurea-insensitive 3H-TdR incorporation suggest that the NMUT-induced DNA damage is largely repaired within 2 hours.