Mutagenicity towards Salmonella typhimurium of some known genotoxic agents, activated by isolated hepatocytes of monkey (Macaca fascicularis). Comparison with isolated human hepatocytes.

This paper describes some striking differences between isolated human and monkey hepatocytes in their capacity to activate some known genotoxic agents into products mutagenic towards Salmonella typhimurium. Isolated monkey hepatocytes, in contrast to human hepatocytes, appeared to activate benzidine (BZ), N-acetylbenzidine (MABZ), N,N'-diacetylbenzidine (DABZ), 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) poorly. With monkey hepatocytes BZ was slightly more mutagenic than DABZ, whereas with human hepatocytes DABZ was more active than BZ. N-Nitrosodimethylamine (DMN) and N-nitrosodiethylamine (DEN) were also found to be poorly mutagenic when activated by monkey hepatocytes, unlike the human hepatocytes. However, the polycyclic arylhydrocarbons benzo[a]pyrene (B[a]P) and 7,12-dimethylbenzanthracene (7,12-DMBA) were highly active in the presence of monkey hepatocytes, unlike the human hepatocytes. A metabolic study showed that monkey liver preparations seem to possess a higher monooxygenase activity towards B[a]P than human liver preparations.

Activation of mutagens by hepatocytes and liver 9000 X g supernatant from human origin in the Salmonella typhimurium mutagenicity assay. Comparison with rat liver preparations.

The mutagenicity of 10 known genotoxic compounds, of several chemical classes, was measured in Salmonella typhimurium mutagenicity assays comprising isolated human hepatocytes or human liver 9000 X g supernatant (S9) from 4 different individuals, as activating system. The mutagenic activity of several compounds as determined with the Salmonella/hepatocyte suspension assay showed obvious differences when compared with the values obtained in the Salmonella/S9 plate assay. For instance, the mutagenic activity of BZ, DMN and DEN appeared to be much higher in the hepatocyte assay than in the S9 assay. However, 2-AF and 2-AAF were activated more effectively into mutagens in the S9 assay than in the hepatocyte assay. 2-AF was slightly more mutagenic than 2-AAF in the hepatocyte assay, whereas it was far more mutagenic than 2-AAF in the S9 assay. DMN was found more mutagenic than DEN in the hepatocyte assay, whereas in the S9 assay DEN appeared to be slightly more mutagenic. Furthermore, great interindividual differences in the metabolic activation of certain compounds, e.g. BZ and DMN, were observed in the hepatocyte suspension assay, whereas these variations were less evident in the S9 plate assay. Comparison of the mutagenicity data obtained with the human liver preparations, with those obtained with rat liver preparations, showed great interspecies differences in the capacity to activate certain chemicals into mutagens. The use of human liver preparations, in particular isolated human hepatocytes, may be of great value in studies on inter- and intraspecies variations in metabolic activation of genotoxic agents.

Mutagenicity and DNA-excision repair induced by isoniazid after metabolic activation by isolated human and rat hepatocytes.

The mutagenic potency of isoniazid (INH) (a widely used antitubercular drug) towards Salmonella typhimurium strain hisG46 was studied in the Salmonella/hepatocyte suspension-assay, comprising isolated human or rat hepatocytes as metabolic system. The potency of INH to induce DNA-excision repair in these hepatocytes was also measured. With rat hepatocytes, INH appeared to be only weakly mutagenic and did not induce significant increases in hepatocellular DNA-excision repair. With isolated hepatocytes of two human subjects, INH appeared also only weakly mutagenic. However, with hepatocytes of two other human subjects, INH was found to be highly mutagenic. Comparable results were obtained for the induction of hepatocellular DNA-excision repair.

Cryopreservation of adult human hepatocytes. The influence of deep freezing storage on the viability, cell seeding, survival, fine structures and albumin synthesis in primary cultures.

Isolated and cultured human hepatocytes provide a useful model for studies of the liver cell function in man. In vitro studies using human hepatocytes are scarce, due to the limited availability and the lack of suitable methods for storage. In this study, we report the effect of deep freezing storage on the viability, fine structures and albumin synthesis of human adult hepatocytes in classical culture conditions. Hepatocytes were isolated using collagenase perfusion (9 isolations). The cell yield was 4-37 X 10(8) with a viability of 60-87%. Cryopreservation was performed in medium containing 10% DMSO and 20% fetal calf serum using a Cryoson BV-4 programmable freezer (0 degree C for 5 min, followed by a freezing rate of 1.5 degrees C/min for 20 min and 7 degrees C/min for 10 min). The cells were stored for 25-275 days in the liquid nitrogen vapor phase (-150 degrees C). Within 16 h about 80% of viable cells from freshly isolated hepatocytes whereas after cryopreservation, 55% of viable cells as determined by Trypan Blue exclusion before the cryopreservation attached to plastic and survived. Electron microscopy showed well developed tight junctions, structures similar to bile canaliculi. Cell polarity was evident. However, 'bleb' formation, more lipid droplets and lysosomes were found in cryopreserved hepatocytes during a short period after thawing. At the 3rd week, cells detached and died. These changes were associated with increased secretion of lactate dehydrogenase, whereas the albumin secretion dropped (from 10 to 4 micrograms/micrograms DNA), regardless of whether hepatocytes were cultured from fresh preparations or after cryopreservation. These findings suggest the cryopreservation is a useful technique to preserve hepatocytes for in vitro studies. Nevertheless, an improved method is necessary to increase the efficiency of cell seeding after cryopreservation.

Metabolic activation of N-acetylbenzidine and N,N'-diacetylbenzidine to mutagens, using isolated hepatocytes and the 9000 X g liver supernatant from rat, hamster and guinea pig.

The metabolic activation of MABZ and DABZ, forming products mutagenic towards Salmonella typhimurium TA1538, was studied with isolated hepatocytes from rat, hamster and guinea pig and the S9 fraction (9000 X g supernatant) prepared from these hepatocytes. Special attention was given to the influence of acetyl-CoA, the cofactor for N-acetylation, on the mutagenicity of these arylamides. The rat and guinea pig S9 preparation activated MABZ as well as DABZ to a much higher degree than the intact hepatocytes of these animal species. Addition of acetyl-CoA to the S9 preparation decreased the mutagenicity of MABZ and DABZ. On the contrary, for the hamster the mutagenicity of MABZ and DABZ appeared to be lower with the S9 preparation than with intact hepatocytes. Addition of acetyl-CoA to the S9 here increased the mutagenic activity of these arylamides. In the presence of intact hepatocytes obvious interspecies differences were observed in the activation of MABZ and DABZ. DABZ was far more effectively activated by hamster hepatocytes than by rat hepatocytes. This was not found with MABZ. Both substrates were poorly activated by guinea pig hepatocytes.

Influence of ethanol induction on the metabolic activation of genotoxic agents by isolated rat hepatocytes.

The effects of ethanol-feeding to rats, over a 6-week period, on the activation of genotoxic compounds of different chemical classes, requiring metabolic conversion to exert their mutagenic activity, were studied in isolated rat hepatocytes. The influence of such treatment on cytochrome P-450 content and N-acetylation in isolated hepatocytes was also investigated. Benzidine (BZ), dimethylnitrosamine (DMN), diethylnitrosamine (DEN), isoniazid (INH) and cyclophosphamide (CP) were more effectively activated to products mutagenic towards Salmonella typhimurium by hepatocytes from ethanol-pretreated rats than by hepatocytes from controls. The mutagenic potency of 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) was not influenced by ethanol pretreatment. Ethanol consumption was found to be associated with increased cytochrome P-450 content and enhanced N-acetylation in the isolated hepatocytes. Our results support the hypothesis that an alteration of the hepatic drug-metabolizing system may be responsible for the ethanol-induced increase in susceptibility to certain genotoxic compounds.

Mutagenicity of five arylamines after metabolic activation with isolated dog and human hepatocytes.

The mutagenicity of benzidine (BZ) N-acetylbenzidine (MABZ), N,N'-diacetylbenzidine (DABZ), 4-aminobiphenyl (4-AB) and 2-aminoanthracene (2-AA) towards Salmonella typhimurium strain TA1538 was measured in the presence of isolated hepatocytes from dog and man. The influence of paraoxon, an inhibitor of the deacetylation reaction, on the mutagenicity of these compounds was also investigated. Obvious interspecies differences in the mutagenic activation of benzidine and its acetylated-derivatives were seen. However, with liver cell preparations from both species it was found that MABZ and DABZ were more mutagenic than BZ itself. 4-AB appeared to be weakly mutagenic in the presence of human hepatocytes but non-mutagenic with dog hepatocytes. 2-AA was highly mutagenic in both species. When human hepatocytes were used as the metabolic factor, the mutagenicity of all arylamines decreased in the presence of paraoxon. With dog hepatocytes, however, the mutagenicity of all arylamines except DABZ was enhanced in the presence of paraoxon.

Mutagenicity of benzidine and 4-aminobiphenyl after metabolic activation with isolated hepatocytes and liver 9000 X g supernatant from rat, hamster and guinea pig.

The mutagenicity of benzidine and 4-aminobiphenyl towards Salmonella typhimurium strain TA1538 was measured in the presence of isolated hepatocytes from rat, hamster and guinea pig. The mutagenic potency of these compounds was also assayed with S9 (9000 X g supernatant) prepared from disrupted hepatocytes. The influence of acetyl coenzyme A, the cofactor for the acetylation reaction, on the mutagenicity of these aryl amines was investigated. For all 3 animal species it was found that the mutagenicity of benzidine is higher with intact hepatocytes than with S9 prepared from disrupted hepatocytes. Addition of acetyl coenzyme A to the S9 fraction increased the mutagenicity of benzidine. In contrast to benzidine, the mutagenicity of 4-aminobiphenyl appeared to be lower with hepatocytes than with S9. Addition of acetyl coenzyme A to the S9 fraction decreased the mutagenicity of 4-aminobiphenyl. The mutagenic potency of 4-aminobiphenyl was almost equal in the presence of the liver preparations from the 3 different species, whereas obvious species differences were seen with benzidine.

Disappearance of free SH-groups in hemoglobin of man, rat and rabbit after exposure to alkylating agents in vitro.

The effect of alkylating agents on the content of free SH-groups in human, rat and rabbit hemoglobin was examined. Treatment of blood in vitro with iodoacetamide, styrene oxide and methyl methanesulfonate resulted in a dose-dependent decrease of free SH-groups in hemoglobin. The free SH-groups of rat hemoglobin were more susceptible to the alkylating agents tested than those of human and rabbit hemoglobin. The method is based on the isolation of hemoglobin and a subsequent measurement of the free SH-content of the hemoglobin by means of a spectrophotometric procedure. This procedure may be useful to examine differences in susceptibility of free SH-groups in hemoglobin, e.g. of different animal species, and on the other hand, to study the potencies of different electrophilic agents to alkylate these nucleophilic centers.

The influence of glucocorticoid on the fibrinogen messenger RNA content of rat liver in vivo and in hepatocyte suspension culture.

The plasma concentration of fibrinogen, one of the major acute-phase proteins produced by the liver, increases during the acute-phase response as a result of enhanced synthesis in liver. Since adrenal-cortical hormones have been thought to have a key role in the regulation of the fibrinogen synthesis, fibrinogen-polypeptide mRNA sequences were determined in the present study, by using a specific complementary-DNA probe, in RNA fractions obtained from rat hepatocytes exposed to glucocorticoids in vitro (hepatocyte suspension cultures) and in vivo. Maximal induction of the fibrinogen-polypeptide mRNA (to 400% of the control value) was found in vitro at 0.1 microM-dexamethasone after 9 h of incubation. The same magnitude of induction was obtained with 20 microM-cortisol or 60 microM-corticosterone. In contrast with the findings in vitro, no induction of the fibrinogen-polypeptide mRNA was observed in the liver at various times after injection of different doses of glucocorticoids into rats. These results suggest that more complex regulatory mechanisms are involved and that glucocorticoids are not the sole regulatory factors in vivo in the enhanced synthesis of fibrinogen during the acute-phase response.

Mutagenicity testing with the Salmonella/hepatocyte and the Salmonella/microsome assays. A comparative study with some known genotoxic compounds.

The applicability of isolated intact hepatocytes as a metabolic factor in bacterial mutagenicity screening was studied. Mutagenic activities of 12 known premutagenic compounds were determined in a Salmonella typhimurium test system comprising hepatocytes and were compared with mutagenicity data obtained with the commonly used Salmonella/microsome plate assay. In a qualitative sense the results obtained with the two systems were, in general, equivalent. However, some specific differences were found depending on the bacterial strain used. For instance, dimethylnitrosamine was only mutagenic for Salmonella strain TA1535 in the hepatocyte suspension system. On the other hand, benzo[a]pyrene was hardly mutagenic towards TA100 with hepatocytes in contrast with the clear-cut effects in the microsome plate assay. In a quantitative respect, for benzidine, 2-acetylaminofluorene, 2-aminoanthracene and dimethylnitrosamine, obviously divergent mutagenic values were recorded with the different procedures. These differences were found to be connected with the presence of intact hepatocytes. This appeared from a comparison between mutagenicities with intact hepatocytes and with S9 prepared from disrupted hepatocytes. The results support previous recommendations that tests with intact cell metabolism should be included in a battery for screening of carcinogens in vitro.

Mutagenic effects of benzo[a]pyrene after metabolic activation by hepatic 9000 g supernatants or intact hepatocytes.

The activating capacities of isolated rat hepatocytes and 9000 g supernatant from these cells with respect to the mutagenic effect of benzo[a]pyrene on Salmonella typhimurium TA100 were investigated. No mutagenicity of benzo[a]pyrene was found with the cell-mediated assay, unless the hepatocytes were disrupted after pre-incubation with benzo[a]pyrene or the intracellular glutathione content was reduced. It is suggested that a retention of active metabolites and an effective detoxication may account for the absence of mutagenic response.