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ABSTRACT:Cultures of primary hepatocytes and hepatoma cell line HepG2 are frequently used in in vitro models for human biotransformation studies. In this study, we characterized and compared the capacity of these model systems to indicate the presence of different classes of promutagens. Genotoxic sensitivity, enzyme activity, and gene expression were monitored in response to treatment with food promutagens benzo[a]pyrene, dimethylnitrosamine (DMN), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). DNA damage could be detected reliably with the comet assay in primary human hepatocytes, which were maintained in sandwich culture. All three promutagens caused DNA damage in primary cells, but in HepG2 no genotoxic effects of DMN and PhIP could be detected. We supposed that the lack of specific enzymes accounts for their inability to process these promutagens. Therefore, we quantified the expression of a broad range of genes coding for drug-metabolizing enzymes with real-time reverse transcription-polymerase chain reaction. The genes code for cytochromes P450 and, in addition, for a series of important phase II enzymes. The expression level of these genes in human hepatocytes was similar to those previously reported for human liver samples. On the other hand, expression levels in HepG2 differed significantly from that in human. Activity and expression, especially of phase I enzymes, were demonstrated to be extremely low in HepG2 cells. Up-regulation of specific genes by test substances was similar in both cell types. In conclusion, human hepatocytes are the preferred model for biotransformation in human liver, whereas HepG2 cells may be useful to study regulation of drug-metabolizing enzymes.
[reaction--see text] Despite the increasing reactivity from benzene to heptacene, the acene resonance energies per pi electron are nearly constant. The reactivities (computed activation energies) of the individual acene rings correlate with the reaction energies and depend on the product stabilities. Nucleus-independent chemical shifts (NICS; note the sizes of the red dots, above) indicate that the more reactive inner rings actually are more aromatic than the less reactive outer rings and even more aromatic than benzene itself.
The geometries of four different series of D(6h)-symmetric polybenzenoid hydrocarbons (PBH) up to and including C(222)H(42) have been optimized at the B3LYP/6-31G(d) level of theory. Excluding C(48)H(24) and C(138)H(42), which have D(3d) minima due to 1,5 H...H repulsions between adjacent perimeter rings, optimized geometries are planar D(6h) minima. Nucleus Independent Chemical Shifts (NICS), at the same level, indicate the presence of individual aromatic rings, which correspond to Clar's qualitative sextets rule (Clar, E. TheAromatic Sextet; Wiley: London, 1972). NICS and the Clar valence electron topologies agree perfectly in the molecule plane; however, the NICS values computed in parallel planes further away from the molecular surface converge, indicating the presence of a uniform magnetic shielding field. For each series, PBH total NICS values (i.e., the sum of NICS values for all rings in the PBH) correlate linearly with the number of carbon atoms, indicating constant magnetic field development within a series. The C-C lengths depend on their proximity to the more olefinic rich molecular perimeters. However, the large PBH (> or =C(48)H(24)) internal C-C distances converge to approximately 1.426 A. In agreement with Clar's rule, HF/6-31G(d)//B3LYP/6-31G(d) vertical ionization potentials and B3LYP/6-31G(d) HOMO-LUMO gaps are largest within the "fully benzenoid" series, where all carbon atoms are members of a single sextet. The largest members of the four series studied are predicted to exhibit semiconducting properties.
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