Sprague-Dawley Hepatocytes Research Articles

Regulation of mdr1b gene expression in Fischer, Wistar and Sprague-Dawley rats in vivo and in vitro.

Administration of 2-acetylaminofluorene (2-AAF) to rats increased mdr1b expression in Fischer, Wistar and Sprague-Dawley rat livers; however, the response was much smaller in Sprague-Dawley livers. To investigate the basis of this difference we further examined the regulation of the mdr1b gene in hepatocytes isolated from Fischer, Sprague-Dawley and Wistar rats. A time-dependent increase in basal expression of mdr1b but not mdr2 was observed in hepatocytes isolated from all three strains of rats. After 4 days in culture, a larger increase in mdr1b mRNA levels was observed in Fischer and Wistar rat hepatocytes (3.5- and 4.6-fold respectively) than Sprague-Dawley hepatocytes (2-fold). Treatment of primary hepatocytes with 2-AAF caused an induction of mdr1b expression that varied among the three strains. Notably, Sprague-Dawley hepatocytes were not responsive to 2-AAF. In contrast to the parent compound, the electrophilic metabolites N-hydroxy-2-acetylaminofluorene and N-acetoxy-2-acetylaminofluorene caused a dose-dependent induction of mdr1b expression in both Fischer and Sprague-Dawley hepatocytes, indicating that differences in the metabolic activation of 2-AAF between the strains may account for the differences in mdr1b by 2-AAF. Hepatocytes isolated from all three strains of rats showed an equivalent induction of mdr1b after treatment with cycloheximide. Nuclear run-on assays demonstrated that the increases in mdr1b expression with time in culture and after xenobiotic treatment were due to increased transcription.

Cryopreservation and long-term storage of primary rat hepatocytes: effects on substrate-specific cytochrome P450-dependent activities and unscheduled DNA synthesis.

The effects of cryopreservation and long-term storage on substrate-specific cytochrome P450-dependent activities and unscheduled DNA synthesis were studied in freshly isolated and cryopreserved hepatocytes derived from adult male Fischer 344 and Sprague-Dawley rats. Primary rat hepatocytes were isolated via an in situ collagenase perfusion technique, cryopreserved at -196 degrees C, and thawed at 5 weeks and 104 and 156 weeks post-freezing. In Fischer 344 and Sprague-Dawley rats, cryopreserved hepatocytes were equivalent or similar to freshly isolated hepatocytes in substrate-specific activities for 7-ethoxyresorufin-O-deethylase and dimethylnitrosamine-N-demethylase and unscheduled DNA synthesis responses. No significant differences in activities toward 7-ethoxyresorufin-O-deethylase and dimethylnitrosamine-N-demethylase, the substrate-specific activities for cytochromes P4501A1 and P4501A2 and cytochrome P4502E1, respectively, were observed between freshly isolated and cryopreserved hepatocytes. Similar unscheduled DNA synthesis responses, a measure of DNA damage and repair, were observed after exposure to the genotoxic carcinogens 2-acetylamino-fluorene, 7,12-dimethylbenz[a]anthracene, and dimethylnitrosamine; although some decreases were also observed in Fischer 344 hepatocytes after 104 weeks and Sprague-Dawley hepatocytes after 156 weeks in the highest concentrations tested. These results suggest that cryopreserved hepatocytes, stored for extended periods of time in liquid nitrogen, are metabolically equivalent to freshly isolated hepatocytes in their ability to activate precarcinogens.

The Concentration and Temporal Relationships of Acetaminophen-Induced Changes in Intracellular and Extracellular Total Glutathione in Freshly Isolated Hepatocytes from Untreated and 3-Methylcholanthrene Pretreated Sprague-Dawley and Fischer Rats

Fischer rats are more sensitive to acetaminophen-induced hepatotoxicity than Sprague-Dawley rats, however, the mechanisms for this enhanced sensitivity remain unclear. The susceptibility to hepatotoxicity is determined largely by the balance between acetaminophen toxification and detoxification. Since glutathione plays a critical role in the detoxification process, it would be of interest to compare the effects of acetaminophen on hepatic glutathione homeostasis in the Sprague-Dawley and Fischer rat, and relate these effects to cytotoxicity. To this end, we measured the sequential changes of intracellular and extracellular total glutathione in freshly isolated hepatocytes from untreated and 3-methylcholanthrene pretreated Fischer and Sprague-Dawley rats, both in the absence (basal) and presence of acetaminophen. In the basal state, the intracellular total glutathione content was significantly (P less than 0.01) increased in hepatocytes from untreated Fischer rats. Nevertheless, the sequential release of total glutathione into the medium and the sequential depletion of intracellular total glutathione were quantitatively similar in hepatocytes from untreated Fischer and Sprague-Dawley rats. Following exposure to acetaminophen, there was a striking dose and time associated depletion of intracellular total glutathione in untreated hepatocytes from both rat strains, and quantitatively the depletion was similar in untreated hepatocytes from both rat strains. This degree of depletion of intracellular total glutathione was not associated with acetaminophen-induced cytotoxicity in Sprague-Dawley hepatocytes, whereas significant (P less than 0.05) cytotoxicity was demonstrated in Fischer hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Strain differences in in vitro rat hepatocyte unscheduled DNA synthesis (UDS): Effect of UV is independent of strain while increased sensitivity is apparent using Fischer-344 instead of Sprague-Dawley rats

The unscheduled DNA synthesis (UDS) assay measures DNA repair following in vitro treatment of rat primary hepatocytes. This report compares the UDS response of primary hepatocytes from 2 widely used rat strains, the Fischer-344 (F344) and Sprague-Dawley (SD) strains. Ultraviolet (UV) light and 5 known genotoxic chemicals were evaluated in each strain in parallel experiments. The chemicals tested were 2-acetylaminofluorene (2-AAF), 4-aminobiphenyl (4-AB), benzidine, dimethylnitrosamine (DMN) and N-propyl- N′-nitro- N-nitrosoguanidine (PNNG). Four of these compounds (2-AAF, 4-AB, benzidine and DMN) require metabolic activation. Benzidine and PNNG were both negative using SD rat hepatocytes, but were weakly positive using F344 rat hepatocytes. In the first of 2 experiments, 4-AB was inconclusive in SD hepatocytes, but strongly positive in F344 cells. In the second experiment, 4-AB was positive in hepatocytes from both strains. 2-AAF was more strongly positive in F344 cells than in SD cells. DMN and UV light induced positive dose responses with little or no differences between strains. It is concluded that hepatocytes from F344 rats may be more sensitive, qualitatively and quantitatively, than hepatocytes from SD rats as indicators of UDS. This difference is not due to intrinsic differences in DNA repair mechanisms but is probably due to differences in drug-metabolizing enzymes between these strain. Thus, for routine screening, F344 rats are preferable for measurement of the in vitro UDS-inducing potential of compounds.

Encapsulated Hepatocytes for Controlling Hyperbilirubinemia in Gunn Rats

Hepatocytes encapsulated in an alginate-polylysine-membrane were used to lower bilirubin levels in hyperbilirubinemia. The animal model was the Gunn rat. The microencapsulated hepatocytes were implanted intraperitoneally. 15 x 16(6) microencapsulated hepatocytes from Wistar rats lowered the bilirubin from 14 mg/100 ml to 6 mg/100 ml after 20 days. 15 x 10(6) microencapsulated hepatocytes from Sprague-Dawley rats lowered the bilirubin levels from 8 mg/100 ml to 5 mg/100 ml. They were as effective as free Sprague-Dawley hepatocytes. 12 x 10(6) Guinea pig hepatocytes, whether encapsulated or free, lowered the bilirubin from 9.36 mg/100 ml to 4.37 mg/100 ml.

Hepatocytes immobilised by microencapsulation in artificial cells: effects on hyperbilirubinemia in Gunn rats.

The possibility of using hepatocytes encapsulated in a calcium-alginate-polylysine matrix to lower bilirubin levels in hyperbilirubinemia was investigated. The animal model was the Gunn rat. The microencapsulated hepatocytes were injected intraperitoneally. 15 X 10(6) microencapsulated hepatocytes from Wistar rats, lowered the bilirubin from 14 mg/100 ml to 6 mg/100 ml after 20 days. The bilirubin is still depressed after 90 days. After encapsulation, Sprague-Dawley hepatocytes were as effective as free hepatocytes in lowering bilirubin levels in Gunn rats. After 68 days, the free Sprague-Dawley hepatocytes were not rejected.