Mammalian cell systems

Dunlop [18] proposed a model for sub-lytic effects in plant cells, based on the same principles, but including four properties postulated to be of particular importance in these systems, namely calcium ion flux, osmo-regulation, cell-cell contact/aggregation and stress protein expression. Of these factors, osmo-regulation (and its inter-relationship with the cell wall) and aggregation patterns, in particular, distinguish plant cells from mammalian cell systems. 

Modified mammalian cell systems for the study of the role of transporters and/or metabolism in oral absorption consists of two main components the cell line and the vector bearing a cDNA encoding the protein of interest. The cell line serves two roles first, to support adequate expression of the cDNA and second, to provide a barrier function which is generally critical in assays for transporter function. 

Professor Bruce Ames, a biochemist at the University of California at Berkeley is one of the pioneers of this type of short-term testing. The Ames Test, as it is called, is now widely used, typically as one of several short-term tests that constitute a series of tests, or battery. A battery is thought necessary because no single test is adequate to detect all types of genotoxicity. The Ames Test involves the use of mutant strains of a common bacterium. Salmonella typhimurium, that back-mutate to their normal state in the presence of a mutagenic chemical or metabolite. Many other bacterial and mammalian cell systems have been made available for this type of testing. 

Tubercidin 5 -monophosphate is converted to the " corresponding analogue of NAD when incubated with cell free extracts of S.faecalis (see above). Furthermore, the inhibition of S. faecalis by tubercidin can be prevented by a mixture ot amino acids, nucleosides, ribose-5-phosphate, and pymvate suggesting that the primary action of the antibiotic is due to interference with NAD-dependent reactions [202]. This protective effect is not seen in mammalian cell systems [193]. 

B[/ ]P metabolites have been shown to bind to DNA in culmred human hepatocytes and in human bladder and tracheobronchial explants. The metabolites identified were identical to those produced in other species and differed only in the relative percentages of formation. Human tissues were most active in metabolizing P>[a P and exhibited at least a threefold higher covalent binding of metabolites to DNA than hamsters, dogs, monkeys, or rats. In addition, P>[a P has been tested extensively in several bacterial and mammalian cell systems and has been chosen as a positive control for the validation of some of these systems. ... 

Biphenyl was not mutagenic in bacterial assays but was positive in vitro in mammalian cell systems in the presence of metabolic activation. ... 

Methyl chloride was mutagenic to bacteria and genotoxic in a number of mammalian cell systems in vitro. " It gave positive results in the dominant lethal test in rats in vivo ... 

Goeger, D.E., Anderson, K.E. Hsie, A.W. (1998) Coumarin chemoprotection against afla-toxin B1-induced gene mutation in a mammalian cell system a species difference in mutagen activation and protection with chick embryo and rat liver S9. Environ, mol. Mutag., 32, 64-74... 

The level of enzyme needed can influence the choice of preparation used for the study. Microsomal preparations from cell cultures allow the use of higher concentrations of active enzyme per unit volume than use of whole cells or cell lysates. The use of whole, viable cells allows the use of longer incubation times but at a lower enzyme concentration per unit volume. In addition, adequate oxygen transfer and nutrient concentrations are needed to maintain culture viability. These requirements impose limitations on cell concentration. In addition, microsomes cannot be efficiently prepared from all cultured cell types. We have found that standard microsome preparation procedures as used for human or rodent liver were unsuitable for isolating active enzymes from human lymphoblasts, and this appears to be a general property of cultured cell lines. Specific catalytic activities in microsomes were lower than for whole cell lysates. This loss of activity appears to happen in other mammalian cell systems which has led to the common use of whole cell lysates.With human lymphoblasts, shortening the length of... 

Fig. 1. The testosterone 6j8-hydroxylase activity (expressed as turnover number) for cDNA-expressed CYP3A4 using yeast, bacteria and mammalian cell systems. Yeast-based expression with coexpressed human cytochrome P450 reductase and human cytochrome or native, yeast cytochrome P450 reductase and cytochrome bs (Peyronneau et al., 1992). E. co/i-GSH E. co/i-based expression where the enzyme was purified and reconstituted with and without the addition of glutathione (Gillam et al., 1993). Human lymphoblast-based expression with endogeneous cytochrome P450 reductase (Crespi et al., 1991a) and with coexpression of cytochrome P450 reductase cDNA (C. Crespi, unpublished observation). Vaccinia virus (vv)/HepG2 cell expression data from Buters et al. (1994).

In this section the properties and significance of individual cytochrome P450 forms and the state of the art for cDNA expression of each form are discussed. The focus of this discussion is on human cell expression of the human P450 forms. However, for many of these forms data for only one or two human cell expression systems are available. Where appropriate and where data are available, some expression data from other mammalian cell and non-mammalian cell systems are discussed. 

One difficulty in framing this discussion is a lack of commonality in units for the expression systems. For example, the same substrate may not have been examined in all systems or activity may be expressed per mg total cell lysate protein, per mg cytosol-free cell membrane protein, per mg microsomal protein or per million cells. In this section, activity levels will be compared in the units originally reported. The following values, as determined in the human lymphoblast system, may be used to compare among the alternative methods of enzyme preparation cytosol-free membranes provide about a 2-fold enrichment in activity, microsomes provide 5-fold enrichment in activity and there are about 7 million cells per mg total protein. These ratios may differ somewhat for other mammalian cell systems but they are unlikely to be off by more than 2-fold. 

While many CYP2C proteins have been expressed using vv/HepG2 cells (Rettie et al., 1992), yeast (Romkes et al., 1991 Kaminsky et al., 1993 Lopez-Garcia et al., 1993) and E. coli (Sandhu et al., 1993), human CYP2C proteins have not been reported to have been stably expressed in any mammalian cell systems. 

Introduction of Chromosomal Aberrations by a LH20 Fraction of Drinking Water Concentrate. Because in previous experiments, analysis of XAD-4/8 concentrates of drinking water in in vitro mammalian cell systems was hampered by the toxicity of the concentrates for the cells, an attempt was made to investigate whether the LH20 fractions obtained could be analyzed for the induction of chromosomal aberrations in CHO cells (Table I). 

Table I shows that a combination of LH20 drinking water fractions II and III (Figure 6) was able to induce chromosomal aberrations in the CHO cells. This experiment was repeated several times with LH20 concentrates throughout the year at the same location three out of five drinking water concentrates showed increased levels of chromosomal aberrations compared to the control, whereas no toxic effects were observed (Table I). The negative results are not shown. The results indicate that the LH20 fractionation procedure may (in part) be suitable for testing drinking water concentrates for genotoxic effects in mammalian cell systems.

Gillies, S D (1992) Design of expression vectors and mammalian cell systems suitable for engineered antibodies, in Antibody Engineering A Practical Guide (Borrebaeck, C. A K., ed.), Freeman, New York. 

Figure 2-7. Chromosomal changes caused by toxic chemicals on the mammalian cell system in in vitro.

Six human sugar transporters with different tissue distributions, substrate kinetics, and specificities have been identified. A number of facilitated amino acid transporters have also been identified in mammalian cells. System L, which transports neutral amino acids, such as leucine and phenylalanine, is probably the best known of these. 

Cook JA Mitchell JB (1989) Viability measurements in mammalian cell systems. Analytical Biochemistry 179 1-7. 

The potential for mutagenic effects was determined in bacterial, fungal, and mammalian cell systems in vitro. Although chrysene produced negative results in Escherichia coli and Saccharomyces, it produced positive results in S. typhimurium in TAIOO. In mammalian cells, chrysene produced positive effects in Syrian hamster embryo cells in vitro. Administration of chrysene to Chinese hamsters by intraperitoneal injection also produced increased sister chromatid exchanges in bone marrow cells. Increased aberrations were also noted in phase II oocytes collected from NMRI mice treated orally with chrysene. Chrysene induced aryl hydrocarbon hydroxylase in cultured human lymphocytes. 

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