The use of surfactants for control of specific aspects of the VPE growth process is beginning to be studied for both the elemental and III/V semiconductors. The objective is to change the ...characteristics of the material grown epitaxially by the addition of a surfactant during growth. Most reported surfactant effects for semiconductors relate to some detail of the morphology of the growing films. For ordered semiconductor alloys the effects can be much more dramatic, including major changes in the electrical and optical properties. Since the bandgap energy is dependent on the microscopic arrangement of the atoms in an alloy with a fixed composition, the change in order parameter induced by the surfactant translates into a marked change in the bandgap energy. This paper presents the results of a study of the effects of n-type (Te and Si), p-type (Zn), and isoelectronic (Sb) dopants on the ordering process in GaInP grown by OMVPE. All of the dopants studied were found to decrease or eliminate ordering; however, the mechanisms are quite different. The donor Te apparently affects the adatom attachment kinetics at steps on the (001) surface, a surfactant effect. On the other hand the donor Si was found to decrease the degree of order by an entirely different mechanism, attributed to an increase in the Ga and In diffusion coefficients in the bulk. It apparently does not involve the surface. Disordering due to the acceptor Zn was found to occur by the same mechanism. The isoelectronic impurity Sb is found to act as a surfactant and to decrease the order parameter by changing the surface reconstruction, eliminating the 1 10-P dimers that provide the thermodynamic driving force for formation of the CuPt structure during growth.
Precision-cut rat liver slices were prepared with a Krumdieck tissue slicer and cultured in three standard hepatocyte culture media. Rat liver slices cultured in either RPMI 1640 medium or Williams ...Medium E could be maintained in culture for up to 72 hr. In contrast, Leibovitz's L-15 medium was unsatisfactory in that slice viability, assessed either by morphological examination or by measurement of enzyme activities, could not be maintained for periods greater than 24 hr. As a measure of functional viability liver slices were cultured with some known rodent peroxisome proliferators, namely clofibric acid, nafenopin, ciprofibrate and Wy-14,643. The peroxisome proliferators induced both palmitoyl CoA oxidation and carnitine acetyltransferase activities in 48- and 72-hr slice cultures. Ultrastructural examination of liver slices cultured with either ciprofibrate or Wy-14,643 for 72 hr revealed an increase in the number of peroxisomes. These results demonstrate that rat liver slices may be maintained in culture for up to 72 hr, and that they respond in a similar manner to rat primary hepatocyte cultures to some peroxisome proliferators. Precision-cut liver slices may therefore be a useful alternative in vitro system to hepatocyte cultures for screening compounds for effects on enzyme activities and for assessing species differences in response.
The construction of a homology model of the ligand binding domain of the rat peroxisome proliferator-activated receptor-
α (rPPAR
α) based on the crystal structure of the human retinoic acid X ...receptor-
α (hRXR
α) is reported. It is demonstrated that many known peroxisome proliferators are able to occupy the putative ligand binding site of the rPPAR
α, including clofibric acid, ciprofibrate, nafenopin and related compounds. The log. relative potency of several peroxisome proliferators can be quantitatively related (R=0.99) to their binding affinity and lipophilicity as measured by their distribution coefficients (logD
7.4 values) and other QSARs are discussed in the light of receptor–ligand interactions. The molecular modelling of a representative number of peroxisome proliferators within the putative ligand binding site is consistent with experimental information on relative potency and enantioselectivity.
Precision-cut human liver slices obtained from 11 donors were cultured for 72 h in a defined medium (serum free Williams' medium E) supplemented with 0.1 microM insulin and 0.1 microM dexamethasone ...(DEX). Liver slices were treated with 50 microM concentrations of beta -naphthoflavone (BNF), lansoprazole, rifampicin (RIF), DEX and methylclofenapate and 500 microM sodium phenobarbital (NaPB). The relative apoprotein levels of 12 cytochrome P450 (P450) enzymes were determined in liver slice microsomes using a panel of antipeptide antibodies. Treatment with BNF significantly induced mean levels of CYP1A2 apoprotein to 160% of levels in 72-h control (no test compound) human liver slice microsomes. NaPB significantly induced levels of CYP3A4 apoprotein to 255% of control and RIF significantly induced levels of CYP2C19 and CYP3A4 apoproteins to 265 and 330% of control, respectively. In addition, treatment with RIF increased levels of CYP2A6 apoprotein to 205% of control, and treatment with both NaPB and RIF increased levels of CYP2B6 apoprotein to 370 and 615% of control, respectively. However, these increases were not statistically significant, owing to a variable response between liver slice preparations from different subjects, this being apparent for all inducible P450s. In contrast, none of the compounds examined significantly increased levels of CYP2C8, CYP2C9, CYP2D6, CYP2E1, and CYP4A11 apoproteins. Levels of CYP1A1 apoprotein were not detected in any liver slice sample, either before or after treatment with the model inducers. Overall, these results demonstrate the utility of cultured human liver slices for assessing the effects of chemicals on P450 enzymes.
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 μM 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 ratCYP1A2 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 μg 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 μM 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.
1. The metabolism of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomal preparations and in cDNA-expressed human cytochrome ...P450 (CYP) isoforms. 2. Kinetic analysis of the NADPH-dependent metabolism of BFC to HFC in four preparations of pooled human liver microsomes revealed mean (±SEM) Km and Vmax of 8.3±1.3 μM and 454±98 pmol/min/mg protein respectively. 3. The metabolism of BFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing BFC substrate concentrations of 20 and 50 μM (i.e. about two and six times Km respectively). With 20 μM BFC the highest correlations were observed between BFC metabolism and markers of CYP1A2 (r2 = 0.784-0.797) and then with CYP3A (r2 = 0.434-0.547) isoforms, whereas with 50 μM BFC the highest correlations were observed between BFC metabolism and markers of CYP3A (r2 = 0.679-0.837) and then with CYP1A2 (r2 = 0.421-0.427) isoforms. At both BFC substrate concentrations, lower correlations were observed between BFC metabolism and enzymatic markers for CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP4A9/11. 4. Using human β-lymphoblastoid cell microsomes containing cDNA-expressed CYP isoforms, 20 μM BFC was metabolized by CYP1A2 and CYP3A4, with lower rates of metabolism being observed with CYP2C9 and CYP2C19. Kinetic studies with the CYP1A2 and CYP3A4 preparations demonstrated a lower Km with the CYP1A2 preparation, but a higher Vmax with the CYP3A4 preparation. 5. The metabolism of 20 μM BFC in human liver microsomes was inhibited to 37-48% of control by 5-100 μM of the mechanism-based CYP1A2 inhibitor furafylline and to 64-69% of control by 5-100 μM of the mechanism-based CYP3A4 inhibitor roleandomycin. While some inhibition of BFC metabolism was observed in the presence of 100 and 200 μM diethyldithiocarbamate, the addition of 2-50 μM sulphaphenazole, 50-500 μM Smephenytoin and 2-50 μM quinidine had little effect. 6. The metabolism of 20 μM BFC to HFC in human liver microsomes was also inhibited by an antibody to CYP3A4, whereas antibodies to CYP2C8}9 and CYP2D6 had no effect. 7. In summary, by correlation analysis, use of cDNA-expressed CYP isoforms, chemical inhibition and inhibitory antibodies, BFC appears metabolized by a number of CYP isoforms in human liver. BFC metabolism appears to be primarily catalysed by CYP1A2 and CYP3A4, with possibly some contribution by CYP2C9, CYP2C19 and perhaps other CYP isoforms. 8. The results also demonstrate the importance of the selection of an appropriate substrate concentration when conducting reaction phenotyping studies with human hepatic CYP isoforms.
To determine a microsomal scaling factor for human liver suitable for prediction of in vivo drug clearance from in vitro data and to explore the role of inter-liver variability in this factor on the ...reported underprediction from microsomal parameters.
Cytochrome P450 (henceforth P450) content in whole homogenates and microsomes from 38 donor livers was used to determine a microsomal scaling factor. In a subset (n = 20) of these preparations, individual P450 enzymes were examined by Western blotting and selective probe activities were determined.
The scaling factor from 38 livers averaged 40 mg microsomal protein per gram liver with a coefficient of variation of 31%. Western blotting experiments indicated that there was no P450 enzyme-specific trend in the distribution of individual P450 enzymes in liver microsomes relative to whole homogenate. Predictions based on an average scaling factor resulted in a satisfactory prediction of intrinsic clearance of three benzodiazepines similar to that obtained using individual factors for the same livers.
A value for human liver microsomal scaling of 40 mg microsomal protein per gram liver has been established. The reason for underprediction previously reported for 52 different drug substrates was not the use of an incorrect value for the scaling factor.
Hepatic microsomal cytochrome P450 (CYP) forms have a major role in the metabolism of drugs and other chemicals. Primary hepatocyte cultures from humans and experimental animals are a valuable in ...vitro system for studying the effects of chemicals on CYP forms. This chapter describes methods to evaluate CYP form induction in human and rat hepatocytes cultured in a 96-well plate format. The use of a 96-well plate format permits studies to be performed with relatively small numbers of hepatocytes and obviates the need to harvest cells and prepare subcellular fractions prior to the assay of enzyme activities. The induction of CYP1A and CYP3A forms in human and rat hepatocytes can be determined by measurement of 7-ethoxyresorufin O-deethylase and testosterone 6beta-hydroxylase activities, respectively, whereas 7-benzyloxy-4-trifluoromethylcoumarin (BFC) O-debenzylase can be employed to assess both CYP1A and CYP2B form induction in rat hepatocytes. An assay for determining the protein content of hepatocytes cultured in a 96-well plate format is also described.
Peroxisome proliferators are well known to cause liver enlargement in rodents. In this investigation, we have examined the effect of acute (1 week) and chronic (26 week) exposure to the peroxisome ...proliferators methylclofenapate (MCP) and clofibric acid (CA), at 0.05 and 0.5% in the diet respectively, on hepatocyte replication in the Sprague-Dawley rat. Both compounds induced an early increase in hepatocyte replication, with a concomitant increase in peroxisome proliferation as assessed by induction of palmitoyl CoA oxidation. However, after 26 weeks of treatment, there was no difference in the labelling index (LI) of control and CA-treated rat livers, while in MCP-treated rats the LI was 5- to 6-fold above control. Palmitoyl CoA oxidation remained elevated in both treated groups at 26 weeks. Analysis of the slides by a 'zonal' scoring procedure demonstrated that the induced replication was predominantly periportal after 1 week of treatment with either compound. The number of 5-bromo-2'-deoxyuridine (BrdU)-positive hepatocyte nuclei per field in the periportal region increased approximately 4-fold after CA treatment and 7-fold after MCP treatment. There was no significant difference in the number of BrdU-positive nuclei per field in the centrilobular areas of control and treated rats after 1 week. After 26 weeks of treatment, periportal replication was still elevated in the MCP-treated animals (approximately 10-fold above control), but there was no difference in periportal replication between control and CA-treated rats. CA induced a significant reduction in the replication of centrilobular areas at 26 weeks, while there was no effect of MCP. In summary, these results demonstrate that the acute mitogenic effects of MCP and CA are predominantly periportal, and, in the case of MCP, the mitogenicity is sustained up to 26 weeks of treatment.
Abstract High doses of Pyrethrins produce liver and thyroid gland tumours in rats by modes of action involving the induction of hepatic xenobiotic metabolising enzymes. The aim of this study was to ...compare the effects of Pyrethrins with those of the rat liver and thyroid tumour promoter sodium Phenobarbital on some cytochrome P450 (CYP) forms in cultured rat and human hepatocytes. The treatment of female Sprague–Dawley rat and human (both male and female) hepatocytes for 72 h with 0–1000 μM Pyrethrins and 0–1000 μM Phenobarbital did not result in any marked cytotoxicity. In rat hepatocytes both Pyrethrins and Phenobarbital produced an induction of 7-benzyloxy-4-trifluoromethylcoumarin O -debenzylase activity (a CYP1A/2B form marker) and CYP2B1 and CYP2B1/2 mRNA levels. Pyrethrins and Phenobarbital also induced CYP3A-dependent testosterone 6β-hydroxylase activity in rat hepatocytes. In human hepatocytes Pyrethrins and Phenobarbital induced both testosterone 6β-hydroxylase activity and CYP3A4 mRNA levels and also increased CYP2B6 mRNA levels. The effects of Pyrethrins and Phenobarbital were concentration-dependent and exhibited a threshold. These results demonstrate that the effects of Pyrethrins on CYP forms in cultured rat and human hepatocytes are qualitatively similar to those of Phenobarbital. Pyrethrins induce CYP2B and CYP3A forms in cultured rat hepatocytes and can induce CYP3A and CYP2B forms in human hepatocytes. While CYP form induction by Pyrethrins, Phenobarbital and related compounds can be associated with liver and thyroid gland tumour formation in rodents, epidemiological data for Phenobarbital suggests that such effects do not occur in humans.
