The capacity of a primary hepatocyte culture of black catfish (Ictalurus melas) to metabolise toxic compounds was evaluated by using aflatoxin B1 (AFB1) as a xenobiotic model. Hepatocytes were isolated by a two-step liver perfusion procedure and cultured in a serum-free medium on both porous membranes and 96-well microplates coated with Matrigel®. A viability of 95–98% was obtained. The ability to maintain cytochrome P450 activities was examined by measuring ethoxyresorufin-O-deethylase and pentoxyresorufin-O-depentylase activities, at various culture times. The cytotoxic effects of AFB1 on catfish hepatocytes were examined by using a neutral red uptake assay on 96-well microplates. The concentration causing 50% inhibition of uptake (IC50) value was 3μM. The AFB1 metabolites were analysed by HPLC with an ultraviolet diode array and fluorescent detectors. The results obtained show that catfish hepatocytes cultured on Matrigel retain their AFB1 metabolising activities for at least 6 days after seeding. This comparative study, using primary hepatocytes and isolated microsomes from black catfish, trout and mice, confirms that the observed marked differences in toxic response following exposure to AFB1 in these species are likely to be related to their various metabolising capacities.
BaksiS.M., & FrazierJ.M. (1990). Isolated fish hepatocytes: model systems for toxicology research.Aquatic Toxicology16, 229–256.
2.
BlaauboerB.J., BoobisA.R., CastellJ.V., CoeckeS., GroothuisG.M.M., GuillouzoA., HallT.J., HawksworthG.M., LorenzonG., MiltenburgerH.G., RogiersV., SkettP., VillaP., & WiebelF.J. (1994). The practical applicability of hepatocyte cultures in routine testing. ECVAM workshop report 1.ATLA22, 231–241.
3.
SaadB., SchollF.A., ThomasH., SchawalderH., StreitV., WaechterF., & MaierP. (1993). Crude liver membrane fractions and extracellular matrix components as a substrata regulate differentially the preservation and inducibility of cytochrome P450 isoenzymes in cultured rat hepatocytes.European Journal of Biochemistry213, 805–814.
4.
Guguen-GuillouzoC. (1992). Isolation and culture of animal and human hepatocytes. In Culture of Epithelial Cells, Vol. 1 Culture of Specialized Cells (ed. FreshneyR.I.), pp. 197–223. Glasgow: Alan R. Liss.
5.
SidhuJ.S., & OmiecinskiC.J. (1993). Effect of extracellular matrix (ECM or Matrigel matrix) overlay on cytochromes P450 inducibility in primary rat hepatocyte cultures: comparison of non-vs. defined Matrigel.Technical Brief: The cell/line. Collaborative Biomedical Products2, 1–2.
6.
JudahD.J., DaviesR., HayesJ.D., McLellanL.I., & NealG.E. (1994). The primary and secondary metabolism of aflatoxin B1 by rat hepatocytes cultured on Matrigel.Toxicology and Applied Pharmacology125, 27–33.
7.
PersonenM., & AnderssonT. (1991). Characterisation and induction of xenobiotic metabolising enzyme activities in a primary culture of rainbow trout hepatocytes.Xenobiotica21, 461–471.
8.
KobanM. (1986). Can cultured teleost hepatocytes show temperature acclimatisation?American Journal of Physiology250, 211–220.
9.
KobanM., GrahamG., & ProsserC.L. (1989). Induction of heat-shock protein synthesis in teleost hepatocytes: effects of acclimatisation temperature.Physiology and Zoology60, 290–296.
10.
LipskyM.M., SheridanT.R., BennettR.O., & MayE.B. (1986). Comparison of trout hepatocyte culture on different substrates.In Vitro Cellular and Developmental Biology22, 360–362.
11.
FerrarisM., DosanjhM.K., & MarafanteE. (1996). Metabolism of benzo[a]pyrene in fish hepatocytes cultured on microplates.Polycyclic Aromatic Compounds11, 91–98.
12.
EatonD.L., & GallagherE.P. (1994). Mechanisms of aflatoxin carcinogenesis.Annual Review of Pharmacology and Toxicology34, 135–172.
13.
GallagherE.P., & EatonD.L. (1995). In vitro biotransformation of aflatoxin B1 (AFB1) in channel catfish liver.Toxicology and Applied Pharmacology132, 82–90.
14.
KraemerB.L., StaeckerJ.L., SawadaN., SattlerG.L., HsiaM.T.S., & PitotC. (1986). Use of a low-speed, iso-density Percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations.In Vitro Cellular and Developmental Biology22, 201–209.
15.
LeP.T., & MortensenR.F. (1984). Mouse hepatocyte synthesis and induction of the acute phase reactant: serum amyloid p-component.In Vitro20, 505–510.
16.
KlaunigJ.E. (1984). Establishment of fish hepatocyte cultures for use in in vitro carcinogenicity studies.National Cancer Institute Monographs65, 163–173.
17.
JaureguiH.O., HaynerN., DriscollJ., Williams-HollandG., LipskyR.M., & GallettiP. (1981). Trypan blue uptake and lactate dehydrogenase in adult rat hepatocytes — freshly isolated cells: cell suspension and primary monolayer cultures.In Vitro Cellular and Developmental Biology17, 1100–1109.
18.
ZhangS., LipskyM.M., TrumpB.F., & HsuI. (1990). Neutral red (NR) assay for the cell viability and xenobiotic induced cytotoxicity in primary cultures of human and rat hepatocytes.Cell Biology and Toxicology6, 219–232.
19.
SchillerC.D., KlainzA., MynettK., & GescherA. (1992). Assessment of viability of hepatocytes in suspension using the MTT assay.Toxicology in Vitro6, 575–578.
20.
LowryO.H., RosebroughN.J., FarrA.L., & RandallR.J. (1951). Protein measurement with the Folin phenol reagent.Journal of Biological Chemistry193, 265–275.
21.
DonatoM.T., Gómez-LechónM.J., & CastellJ.V. (1993). A microassay for measuring cytochrome P4501A1 and P4502B1 activities in intact human and rat hepatocytes cultured on 96-well plates.Analytical Biochemistry213, 29–33.
22.
GoergerD.E., SheltonD.W., HendricksJ.D., PereiraC., & BaileyG.S. (1988). Comparative effect of dietary butylated hydroxyanisole and β-naphthoflavone on aflatoxin B1 metabolism, DNA adduct formation and carcinogenesis in rainbow trout.Technical Paper No. 8188, pp. 1793–1800. Oregon Agricultural Experiment Station: Corvallis, OR, USA.
23.
JensenE.G., ThaulandR., & SøliN. (1996). Measurement of xenobiotic metabolising enzyme activities in primary monolayer cultures of immature rainbow trout hepatocytes at two acclimatisation temperatures.ATLA24, 727–740.
24.
WortelboerH.M., De KruifC.A., van IerselA.A.J., FalkeH.E., NoordhoekJ., & BlaauboerB.J. (1990). The isoenzyme pattern of cytochrome P450 in rat hepatocytes in primary culture, comparing different enzyme activities in microsomal incubations and intact monolayers.Biochemical Pharmacology40, 2525–2534.
25.
HaaschM.L., KleinowK.M., & LechJ.J. (1988). Induction of cytochrome P450 mRNA in rainbow trout: in vitro translation and immunodetection.Toxicology and Applied Pharmacology94, 246–253.
26.
GeorgeS.G. (1994). Enzymology and molecular biology of Phase II xenobiotic-conjugating enzymes in fish. In Aquatic Toxicology (ed. MalinsD.C., & OstranderG.K.), pp. 51–52. Boca Raton, FL, USA: Lewis.
