Langzeitkulturen humaner Hepatozyten als Alternative zur Wirkstofftestung in TierversuchenAnett Ullrich
Taschenbuch
Due to species differences, primary human hepatocytes are still the in vitro system of choice to analyse liver specific processes and functions. Human hepatocytes were cultured several weeks in an optimized, chemical defined and serum-free two-dimensional culture system. The expression of drug-metabolizing cytochrome P450 enzymes and liver-enriched transcription factors were maintained during culture. The anabolic albumin synthesis and catabolic urea production remained on a constant high level. The addition of growth factors H G F and E G F turned out to be very beneficial for maintaining these functions.
The system was used to study the effects of acetaminophen on primary human and rat hepatocytes. Non-invasive determinations of albumin, urea and lactate dehydrogenase concentrations in cell culture supernatants thereby allowed a continuous monitoring for at least two weeks. Acetaminophen in a concentration of 2815 mg/l (18. 6 m M) reduced the urea production by 25 % and the albumin synthesis by approximately 70 % without any effects on cellular viability. After removal of the substance, hepatocellular functions reached the control level within one to three days. The repetitive analyses of acetaminophenmediated effects on cellular metabolism yielded to identical results for multiple cycles every four days. The drug also caused reversible and repetitive ultrastructural modifications, in particular an almost complete replacement of rough endoplasmic reticulum by smooth endoplasmic reticulum and a massive degradation of glycogen stores. The different sensitivity of species towards the active component was documented by comparing incubations with rat hepatocytes. So far the majority of published studies proofed a rapid depletion of cytosolic glutathione, which could only be detected here long delayed. The microarray displayed an increase in expression of stress-induced genes and a slowdown of cellular metabolism already with non-toxic concentrations. [. . . ]
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