Lymphoblasts, human

Teratogenic effects have been noted with 2- and 4-aminophenol in the hamster, but 3-aminophenol was without effect in the hamster and rat (129,130). 4-Aminophenol is known to inhibit DNA synthesis and alter DNA stmcture in human lymphoblasts (131,132) and is mutagenic in mouse micronuclei tests (133). The aminophenols have been shown to be genotoxic, as evidenced by the induction of sister chromatid exchanges (134,135), but they also exert a protective effect against DNA interaction with other noxious chemicals (136). After assessment of available data a recent report stated that the aminophenols were safe as cosmetic ingredients in their present uses and concentrations (137). 

Crespi CL, Ryan CG, Seixas GM, et al. 1985. Tests for mutagenic activity using mutation assays at two loci in the human lymphoblast cell lines TK6 and AHH-1. In Ashby J, de serres FJ, et al., eds. 

Recio L, Skopek TR. 1988. Mutagenicity of acrylonitrile and its metabolite 2-cyanoethylene oxide in human lymphoblasts in vitro. Mutat Res 206 297-305. 

Wood, C.L., and O Dorisio, M.S. (1985) Covalent cross-linking of vasoactive intestinal polypeptide to its receptors on intact human lymphoblasts./. Biol. Chem. 260, 1243-1247. 

Table 9.4 The effects (% inhibition) of organic solvents on rCYP activities in human lymphoblast cell microsomes [136].

While dEpoB performed similarly to paclitaxel in sensitive tumor xenografts (MX-1 human mammary and HT-29 colon tumor), clearly superior effects of dEpoB were observed against MDR tumors under these slow infusion conditions. Thus, dEpoB (6 h, Q2D, 30 mg/kg x 5 doses, i.v.) demonstrated a foil curative effect when administered to nude mice bearing the resistant human lymphoblastic T-cell leukemia,... 

Santana, P, Pena, L.A, Haimovitz-Friedman, A, Martin, S., Green, D., McLougWin, M., Cordon-Cardo, C., Schuchman, E.H., Fuks, Z. and Kolesnick, R., 1996, Acid sphingomyehnase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis. Cell 86 189-199... 

Human lymphoblasts Tk6 Forward mutation No data + Crespi et al. 1985... 

Zhou L, Erickson RR, Hardwick JP, Park SS, Wrighton SA, et al. 1997a. Catalysis of the cysteine conjugation and protein binding of acetaminophen by microsomes from a human lymphoblast line transfected with the cDNAs of various forms of human cytochrome P450. J Pharmacol Exp Ther 281 785-790. 

Human Lymphoblasts Versatile Indicator Cells for Many Forms of Chemically Induced Genetic Damage... 

Figure 4. Toxic and mutagenic response of diploid human lymphoblast line MIT-2 to aflatoxin B,. Open symbols afatoxin + phenobarbital-induced rat liver postmitochondrial supernatant (PMS) closed symbols effect of treatment with either aflatoxin or FMS alone.

Schwartz GK, Arkin H, Holland JF, Ohnuraa T (1991) Protein kinase C activity and multidrug resistance in MOLT-3 human lymphoblastic leukemia cells resistant to trimetrexate. Cancer Res 51 55-61... 

Crespi, C. L., and W. G. Thilly, Assay for Gene Mutation in a Human Lymphoblast Line, AHH-1, Competent for Xenobiotic Metabolism, Mutat. Res., 128, 221-230 (1984). 

Sasaki, J. C., J. Arey, D. A. Eastmond, K K Parks, and A. J. Grosovsky, Genotoxicity Induced in Human Lymphoblasts by Atmospheric Reaction Products of Naphthalene and Phenan-threne, Mutat. Res., 393, 23-35 (1997b). 

Penman, B.W., Hoppe, rv, H., and Thilly, W.G. (1979). Concentration-dependent mutation by alkylating agents in human lymphoblasts and Sal-monella typhimurium N-methyl-N-nitrosourethane and 0 -propiolactone," J. Natl. Cancer Inst. 63,903. 

Thilly, W.G. (1979). Study of mutagenesis in diploid human lymphoblasts, page 341 in Banbury Report 2, Mammalian Cell Mutagenesis The Maturation ofTkst Systems, HsiE, A.W., O Neill, J.P. AND McElheny, V.K., Eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). 

Gibson KM, Lee CF, Chambliss KL, Kamali V, Francois B, Jaeken J, Jakobs C (1991) 4-Hy-droxybutyric aciduria application of a fluorometric assay to the determination of succinic semialdehyde dehydrogenase activity in extracts of cultured human lymphoblasts. Clin Chim Acta 196 219-222... 

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).

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. 

Human CYPlAl has been expressed in several of the heterologous expression systems including yeast, human lymphoblasts, V79 cells and human fibroblasts. The EROD activity in V79 cells expressing CYPlAl is 50pmol/(mg total protein x min) (Schmalix et al., 1993). In human lymphoblasts, EROD activity of 155 pmol/(mg microsomal protein X min) was obtained (Penman et al., 1994). EROD activity in human fibroblasts expressing CYPlAl cDNA is 1.2pmol/(10 cells x min) or approximately... 

CYPlAl has also been expressed in yeast (Eugster et al., 1990 Sengstag et al., 1994) and EROD activities of 223 pmol/(mg min) have been reported. The turnover numbers for EROD in yeast-expressed CYPlAl (1.4/min) were substantially lower than observed for human lymphoblast-expressed CYPlAl (7.6/min). Apparent KmS were quite similar (92 nM and 87 nM in yeast and human lymphoblasts, respectively). Modified CYPlAl has also been expressed in E. coli (Guo et al., 1994). The expression level of CYPlAl in E. coli (per mg membrane protein) was comparable to that obtained with human lymphoblasts (about 30 pmol/mg in both systems). The turnover number for EROD for E. co//-expressed CYPlAl was quite low in isolated membranes, but could be increased to 8/min if the protein was purified and reconstituted. The apparent for E. coli-exptessed CYPlAl EROD activity, 580 nM, was about 6-fold higher than that for the yeast- or human lymphoblast-expressed enzymes. It is not clear whether this difference is due to the base substitutions necessary to obtain expression in E. coli, or some other cause. 

Fig. 2. 7-Elhoxyresorufin O-deethylase activity for cDNA-expressed CYP1A2 in human lymphoblasts (Penman et al., 1994) and V79 cells (Wolfel et al., 1992). Comparison to the mean levels for two human liver microsome panels (Human Liver-1 Murray et al., 1993 Human Liver-2 Yamazaki et al., 1993).

Fig. 3. Coumarin 7-hydroxylase activity for cDNA-expressed CYP2A6 in human lymphoblasts (Crespi et al., 1990b, when microsomes are prepared according to Penman et al., 1993), w/HepG2 cells (Waxman et al., 1991), 3T3 mouse cells (Salonpaa et al., 1993) and lOTl/2 mouse cells (Tiano et al., 1993). Comparison with the mean levels for five panels of human liver microsomes. Liver 1 Murray et al. (1993) Liver 2 Yamazaki et al. (1993) Liver 3 Wrighton et al. (1993b) Liver 4 Yun et al. (1992) Liver 5 Pearce et al. (1992).

Fig 4. Turnover number for cDNA-expressed CYP2A6 and purified/reconstituted human liver CYP2A6. Data from human lymphoblasts ( lymphoblast , Crespi et al., 1991a), vaccinia virus using phosphate buffers ( vv Phosphate , Yamano et al., 1990), vv using Tris buffers ( vv Tris , C. Crespi and F. Gonzalez, unpublished observation) and purified/reconstituted human liver CYP2A6 ( purified , Yun et al., 1992). 

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