Verificación de los supuestos del modelo de cox

During the course of this study we have set up culture conditions that permit primary rat hepatocytes to express several genes encoding phase I and phase II drug- metabolising enzymes, namely CYP2B1/2, P450 reductase, FM Ol, and GSTs 1-1/2-2, and 3-3/4-4. Levels o f expression of these genes closely resembled those observed in rat liver. Moreover, as is the case in liver cytosolic extracts, GST7-7 expression was undetectable in these cells. Similar to the situation in vivo, in primary hepatocytes cultured under these conditions, phénobarbital was shown to strongly stimulate the accumulation of CYP2B1/2 mRNAs and proteins. To a lesser extent, PB was also shown to promote the accumulation of P450 reductase, GSTl-1/2-2, and GST3-3/4-4 proteins. During the course of this study we have also shown that in this culture system picrotoxin strongly induces CYP2B1/2 mRNAs, with a kinetics that closely resembles that observed using phénobarbital as the inducer. Picrotoxin was found to induce also the expression of other drug-metabolising enzymes according to a pattern that closely resembles that observed using phénobarbital. These observations strongly suggest that picrotoxin and phénobarbital might induce the expression o f drug-metabolising enzymes, in general, and o f CYP2B1/2 mRNAs, in particular, via a similar mechanism, possibly mediated by. an intracellular receptor protein. Altogether these data are a strong indication of the overall ability o f this cell system to constitute a valid in vitro model for the study of drug metabolism, at least on a short term basis. The chemically-defined culture conditions that we employed are relatively simple, fi*ee fi*om complicating factors such as Matrigel or co­ culture (discussed in section 1.3). In addition, we have shown that cells cultured under these conditions can be transfected with high efficiency and that luciferase reporter gene constructs are active in these cells. Therefore, this cell system can be readily employed not only for metabolic studies, but also for studies aimed at understanding the regulation o f the expression o f genes encoding drug-metabolising enzymes. The major problem associated with the primary hepatocyte cell system is the fact that cells need to be isolated each time a new experiment is started. In our efforts to overcome this problem,

we have shown that the culture conditions developed for primary hepatocytes can be employed for the culture of FAZA 967 rat hepatoma cell line. These conditions were found to support the expression of several drug-metabolising enzymes in a manner that was similar to that observed in primary hepatocytes. Although in these cells the fold- induction o f CYP2B1/2 mRNAs by phénobarbital and picrotoxin was comparable to the fold induction observed in primary hepatocytes, expression levels for the two mRNAs was considerably lower. It is doubtful, therefore, whether this cell system can be used for the study o f the metabolism of drugs by CYP2B1/2. Nonetheless, the culture conditions developed in the course o f this study were found to support the expression of GST 1-1/2- 2, FM O l, and P450 reductase in a manner that is comparable to that observed in primary hepatocytes. In addition, these cells expressed GST7-7, and the expression of P450 reductase and GSTl-1/2-2, and GST7-7 was found to be inducible by phénobarbital. Hence, considering the great advantages o f an established cell line over a primary hepatocyte system, the FAZA 967 system developed in the course o f this study should represent an ideal in vitro model for the study of the drug-metabolising enzymes mentioned above.

Finally, we have established conditions that allow primary mouse hepatocytes, isolated from a transgenic mouse harbouring a temperature-sensitive mutant o f the SV40 large T antigen, to divide and survive in culture for longer than a month. These cells could be passaged and were found to secrete albumin. Kerstin Kramer has continued this work in Dr. Ian Phillips' laboratory at Queen Mary and Westfield College, London, and found that these cells could express high levels o f CYP1A2 mRNA when cultured in the presence o f P-naphtoflavone. She has now passaged the cells over 30 times and has cloned nine cell lines. Characterisation o f these clones is now in progress.

In conclusion, the three cell systems developed in the course o f this project have the potential to contribute collectively to a substantially better understanding o f drug- metabolism. Considering that an in vitro system can never represent more than an

approximation of what occurs in the liver, it is reasonable to use a system that is simple to assemble, to use, and whose results can be readily interpreted. The primary hepatocyte system developed in this study offers this advantage over cell systems developed in the past. In addition, the expression of the phase I and phase II enzymes investigated compares very favourably with previously developed cell systems, at least during four days in culture. Longer culture periods were not investigated and it could be possible that the culture conditions developed in this project might allow cells to survive in culture for longer. In this case this system could also be used for long-term drug- metabolism studies. It would be interesting also to investigate drug-metabolism as a function o f cell division in the immortalised cell lines developed. It is possible to conditionally regulate whether these cells divide or not in culture. Therefore, it should be possible to mimic in part what occurs in the liver during regeneration, following damage. In fact, the mechanisms involved in the regulation of liver regeneration are largely unknown and this cell system could be employed in studies aimed at elucidating these mechanisms.