Xenobiotic induction of P-450 PB-4 (IIB1) and P-450c (IA1) and associated monooxygenase activities in primary cultures of adult rat hepatocytes.

1. The long-term maintenance of metabolism of representative drugs and steroid hormone substrates by cytochromes P-450, and their inducibility, was investigated in primary cultures of adult rat hepatocytes. Collagenase-isolated cells were seeded on collagen-coated tissue culture dishes and cultured in Chee's essential media in the presence or absence of phenobarbital (PB, 0.75 mM, 96 h or continuously) and 3-methylcholanthrene (MC, 5 microM, 48 h) for up to 45 days. 2. Hepatic P-450-dependent metabolism of diazepam to its primary oxidized metabolite was inducible by PB both in vivo (monitored in isolated liver microsomes) and in cultured cells (up to 100% and 400% increases in the formation of temazepam and nordiazepam, respectively, after 25 days in culture). Hepatocyte microsomal androstenedione 16 beta-hydroxylase activity was also induced by PB treatment of the hepatocytes (350-650% increase in 20-day-old cells). 3. Western blot analysis revealed that immunoreactive P-450 form PB-4 (IIB1), which catalysed the N-demethylation of diazepam to yield nordiazepam as well as androstenedione 16 beta-hydroxylation when assayed in a purified enzyme system, was substantially elevated following PB treatment of the cultured cells. Similarly, MC induced 7-ethoxycoumarin O-deethylase activity (up to 2000% increase from 5 to 45 days) as well as immunoreactive P-450c (IA1) in the hepatocyte cultures. 4. These studies demonstrate that cytochrome P-450 activities can be maintained, and also induced, after extended periods of time in hepatocytes cultured using a simple collagen mixture as substrate and a commercially available tissue culture media. This culture system should provide an important tool for further studies of P-450-dependent xenobiotic metabolism in a well-defined, liver-derived cellular system.

Mammalian hepatocytes as a foundation for treatment in human liver failure.

Technological advances in the separation and culture of mammalian hepatocytes have facilitated the use of these cells as the foundation for either hepatocyte transplantation or hepatocyte-seeded hollow fiber liver assist devices (LAD). To fully appreciate the practical applications of these tissue engineering solutions, it is necessary to understand the types of human liver failure as well as the corresponding animal models. The most immediate application of this type of technology is the treatment of hepatic encephalopathy (HE), an acute and highly fatal complication of fulminant hepatic failure. Although the pathogenesis of HE is unknown, failure of the detoxification function of the liver is accepted as playing an important role in this disorder. Consequently, the assaying and preservation of P450 activity in the grafted cells or in the LAD must be among the main targets of this research. This review explores the problems in hepatocyte transplantation and culture that deserve special consideration and emphasizes the conditions contributing to the in vitro maintenance of phenotypic expression of these cells.

Changes in ribosomal RNA, poly(A)+ RNA, and collagen alpha 2(I) mRNA synthesis and processing during hypertonic treatment of cultured chick embryo cells.

The effect of hypertonic conditions on RNA synthesis in cultured chick embryo cells was examined. The appearance of newly synthesized 28 S, 18 S, and 4 S and 5 S RNA into the cytoplasm was found to be decreased by hypertonic conditions. The appearance of newly synthesized poly(A)+ RNA into the cytoplasm was also found to be depressed. To examine the behavior of a specific mRNA, nuclear and cytoplasmic levels of procollagen alpha 2(I) mRNA were measured during high salt treatment. While nuclear levels of this mRNA were found to increase, those of the cytoplasm fell markedly. S1 nuclease digestion studies of an intron flanked by two exons revealed that the pro alpha 2(I) collagen nuclear RNA that accumulated under hypertonic conditions was spliced. The nuclear accumulation of mRNA appears therefore to be due to a hypertonic block of nuclear-cytoplasmic transport, and not to an inhibition of RNA splicing.

A highly bent fragment of Crithidia fasciculata kinetoplast DNA.

Kinetoplast DNA minicircles from Crithidia fasciculata contain a single major region of bent helix. Restriction fragments containing this bent helix have electrophoretic behavior on polyacrylamide gels which is much more anomalous than that of previously studied bent fragments. Therefore, the C. fasciculata fragments probably have a more extreme curvature. Sequencing part of a cloned minicircle revealed an unusual structure for the bent region. In a sequence of 200 bases, the bent region contains 18 runs of 4-6 As with 16 of these runs in the same strand. In some parts of this sequence the A runs are regularly spaced with a periodicity of about 10 base pairs. This spacing is nearly in phase with the twist of the DNA helix. This same sequence arrangement has been observed in other bent fragments, but the number of A runs is much greater in this C. fasciculata sequence. It is likely that there are small bends associated with each A run which, because of their periodic spacing, add up to produce substantial curvature in this molecule. In addition to having highly anomalous electrophoretic behavior, the fragment has unusual circular dichroism spectra. Its spectrum in the absence of ethanol is that of B DNA, but ethanol in the concentration range of 51-71% (w/w) induces changes to forms which are different from those of any well characterized DNA structure. The C. fasciculata bent helix is neither cleaved by S1 nuclease nor modified by bromoacetaldehyde under conditions in which other unusual DNA structures (such as cruciforms or B-Z junctions) are susceptible to attack by these reagents. Finally, a two-dimensional agarose gel analysis of a family of topoisomers of a plasmid containing the bent helix revealed no supercoil-induced relaxation.