Hepatocyte suspensions

Green et al. (1986) compared the metabolism of amphetamine in isolated hepatocyte suspensions from rat, dog, squirrel, monkey, and human livers. The metabolite profile of hepatocytes from each species corresponded to the profile of urinary metabolites identified previously. These results indicate that species-specific differences in the metabolic activation of compounds seen in vivo can be reproduced in vitro by the utilization of primary hepatocyte cultures. 

Simmerman, H.J., Kendler, J., Libber, S. and Lukacs, L. (1974). Hepatocyte suspensions as a model for demonstration of drug hepatoxicity. Biochem. Pharmacol. 23 2187-2189. 

Glende EA Jr, Lee PY. 1985. Isopropanol and chlordecone potentiation of carbon tetrachloride liver injury Retention of potentiating action in hepatocyte suspensions prepared from rats given isopropanol or chlordecone. Exp Mol Pathol 42(2) 167-174... 

Typical experimental procedures are as follows The test drug candidate is incubated with pooled human liver microsomes (e.g., 1 mg protein/mL) that were previously preincubated with ABT (1 or 2 mM) for 30 minutes at (37 1)°C in the presence of an NADPH-generating system. Incubations of the drug candidate in the absence of ABT serve as controls. For hepatocytes, suspensions of freshly isolated or cryopreserved hepatocytes (lx 106 cells/ mL) are preincubated with 100-pM ABT for 30 minutes in 0.25 mL of Krebs-Henseleit buffer or Waymouth s medium (without phenol red) supplemented with FBS (4.5%), insulin (5.6 pg/mL), glutamine (3.6 mM), sodium pyruvate (4.5 mM), and dexamethasone (0.9 pM) at the final concentrations indicated. After the preincubation, the drug candidate is added to the incubation and the rate of metabolism of the drug candidate is compared in hepatocytes or microsomes with and without ABT treatment. A marked difference in metabolism caused by ABT is evidence that CYP plays a prominent role in the metabolism of the drug candidate. 

Thaw the frozen hepatocytes quickly by gentle shaking in a 37 °C water bath. The hepatocytes should thaw but not become warm. Transfer the hepatocyte suspension into an ice-cold Erlenmeyer flask immediately after thawing and dilute DMSO gradually by the addition of ice-cold not carbogen-equilibrated suspension buffer, 0.5-, 1-, 2- and 3-fold of the volume of the thawed hepatocyte suspension. Suspension buffer should be added drop wise and hepatocytes should be on ice for 3 min before the next dilution step takes place. After centrifugation at 50 x g for 5 min at 4°C and resuspension in 10 ml suspension buffer, the hepatocytes can be purified by Percoll centrifugation. 

Hepatocyte suspension is added to the precoated dishes at a density of 2 x 106 cells/60 mm dish. Ap-... 

Bontemps, F., van den Berghe, G. Metabolism of exogenous S-adeno-sylmethionine in isolated rat hepatocyte suspensions methylation of plasma-membrane phospholipids without intracellular uptake. Bio-... 

Sample preparation Add 2 volumes acetone to the hepatocyte suspension, centrifuge at 13000 g for 5 min. Remove the clear supernatant and evaporate it to dryness in a centrifugal evaporator, reconstitute the residue in 1 mL MeOH, sonicate, centrifuge at 13000 g for 5 min, inject an aliquot. 

The second caveat is that, in order to accurately predict hepatic clearance, the correct in vitro system must be chosen. If the candidate drug is primarily oxidatively metabolized, then liver microsomes will be sufficient. However, if the potential for non-microsomal biotransformation exists, then a different in vitro system, such as hepatocyte suspensions, should be used. In the illustration above, it turned out, as far as clearance of Compound X is concerned, man is specifically like a rat, and unlike a dog. 

Bujfers Tris-HCl and potassium phosphate buffer (or phosphate-buffered saline, PBS) are used for the microsomal stability assays, while PBS is often the choice for the metabolism in hepatocyte suspensions. 

Studies on metabolic stability using hepatocyte suspensions are not feasible for automation/HTS, but these studies do provide rather complete profiles of hepatic biotransformation without the supplements of cofactors and cosubstrates. The use of S9 in metabolic stability studies can be evaluated in a manner similar to that used for the microsomal assays, but with the possible addition of a broader panel of cofactors or cosubstrates. These include NADPH for CYP/FMO-mediated reactions, NADH for xanthine oxidoreductase and quinone oxidoreductase 2, NADPH-dependent reductions by carbonyl reductases, and NADPH/NADH-dependent reductions catalyzed by aldo-keto reductases, uridine 5 -diphosphate... 

Again, Vi would preferably be detected, for each metabolic pathway, in HLM or hepatocyte suspensions, if appropriate. 

Fig. 2. Influence of anoxia on the concentration of uric acid in isolated hepatocyte suspensions. Same experiment as in Fig. 1.

Fig. 1. depicts the sequence of events taking place after addition of 0.5 mM-adeno ne in hepatocyte suspensions that had been preincubated with [ C]adenine in order to label their adenine nucleotide pool (Van den Berghe et al., 1980). Concentrations of the adenine nucleotides in the hepatocytes and of adenosine and allantoin in the cell suspension are shown on the left, whereas the radioactivity in these compounds is displayed on the right. Cells incubated without adenosine are compared to cells incubated with 0.5 inM-adenosine, in the absence and in the presence... 

The formation of radioactive adenosine from labelled adenine nucleotides, revealed by the addition of the unlabelled nucleoside to the hepatocyte suspension, as well as the reassessment of the activity of the cytoplasmic 5 -nucleotidase in physiological conditions, raised the possibility that adenosine may be continuously formed from adenine nucleotides in basal conditions but not contribute to the production of allantoin because of its reutilization by adenosine kinase. This futile cycle would explain our previous observation that the production of allantoin by isolated hepatocytes is not influenced by coformycin at a concentration that inhibits selectively adenosine deaminase (Van den Berghe et al., 1980). The arrest of the reutilization of adenosine would cause an irreversible loss of adenine nucleotides under the form of this nucleoside, that would be further degraded to allantoin. 

The mechanism of inhibition of HMGCoA R by 25-hydroxycholesterol is still controversial some authors reported that the sterol inhibits the enzyme synthesis but it was also suggested that the effect i modulated by the phosphorylation-dephosphorylatio mechanism. In our experiments the hepatocyte suspensions (4x10 cells) were incubated for one... 

In our study, all three PAHs incorporated adipocytes in a similar manner. Our results are similar to those found by Victor, who obtained a complete incorporation of the 2,3,7,8-TCDD in hepatocytes suspension within 2 minutes [14]. These data reinforce the concept that adipose tissue may act as a reservoir for many different lipophilic exogenous chemicals including PAHs. 

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