Measurement of Xenobiotic Metabolising Enzyme Activities in Primary Monolayer Cultures of Immature Rainbow Trout Hepatocytes at Two Acclimatisation Temperatures
Restricted accessResearch articleFirst published online September, 1996
Measurement of Xenobiotic Metabolising Enzyme Activities in Primary Monolayer Cultures of Immature Rainbow Trout Hepatocytes at Two Acclimatisation Temperatures
Rainbow trout hepatocytes with a high viability were isolated by two-step collagenase perfusion through the portal vein. The yield was 1-2.5 x 106 cells/g body weight. Culture conditions were defined for providing enhanced attachment and long-term cell survival at 7 ± 0.5°C and 15 ± 0.5°C, respectively. The hepatocytes, attached to Primaria™ plastic and cultured in Leibowitz L-15 medium with 9% fetal calf serum, were maintained as monolayers for 6–7 days. The activities in hepatocytes from immature trout of the biotransformation enzymes ethoxyresorufin-O-deethylase (EROD), aldrine epoxidase (AEPOX), NADPH-cytochrome c reductase (NCR), glutathione-S-transferase (GST) and UDP-glucuronosyltransferase (UDPGT), were all stable during the culture period. Differences in enzyme stability and activity (particularly the activity of EROD) between hepatocytes from different fish were observed at both temperatures. The temperature did not influence the activities of EROD or NCR, whereas AEPOX showed metabolic compensation. Both GST and UDPGT exhibited inverse temperature compensation. Hepatocyte monolayers, cultured from immature trout, may provide a useful system in pharmacological and toxicological research for investigating drug metabolism.
AnderssonB., BerggrenM., and MoldeusP. (1978). Conjugation of various drugs in isolated hepatocytes.Drug Metabolism and Disposition6, 611–616.
2.
FryJ.R., and BridgesJ.W. (1979). Use of primary hepatocyte cultures in biochemical toxicology.Reviews in Biochemical Toxicology1, 201–247.
3.
MommsenT.P., and SuarezR.K. (1984). Control of gluconeogenesis in rainbow trout hepatocytes: role of pyruvate branchpoint and phosphoenol-pyruvate cycle.Molecular Physiology6, 9–18.
4.
MommsenT.P., WalshP.J., & MoonT.W. (1985). Gluconeogenesis in hepatocytes and kidney of Atlantic salmon.Molecular Physiology8, 89–100.
5.
MaitreJ-L., ValotaireY., & Guguen-GuillouzoC. (1986). Estradiol-17β stimulation of vitellogenin synthesis in primary culture of male rainbow trout hepatocytes.In Vitro Cellular and Developmental Biology22, 337–343.
6.
ShullL.R., KirschD.G., LohseC.L., and WisniewskiJ.A. (1987). Application of isolated hepatocytes to studies of drug metabolism in large food animals.Xenobiotica17, 345–363.
7.
GrantM.H., BurkeM.D., HawksworthG.M., DuthieS.J., EngesetJ., and PetrieJ.C. (1987). Human adult hepatocytes in primary monolayer culture.Biochemical Pharmacology36, 2311–2316.
8.
MommsenT.P., WalshP.J., PerryS.F., and MoonT.W. (1988). Interactive effects of catecholamines and hypercapnia on glucose production in isolated trout hepatocytes.General and Comparative Endocrinology69, 1–11.
9.
HyllnerS.J., AnderssonT., HauxC., & OlssonP-E. (1989). Cortisol induction of methalloth-ionein in primary culture of rainbow trout hepatocytes.Journal of Cellular Physiology139, 24–28.
10.
HoogenboomL.A.P., PastoorF.J.H., ClousW.E., HesseS.E., and KuiperH.A. (1989). The use of porcine hepatocytes for biotransformation studies of veterinary drugs.Xenobiotica19, 1207–1219.
11.
BaksiS.M., and FrazierJ.M. (1990). Isolated fish hepatocytes: model systems for toxicology research.Aquatic Toxicology16, 229–256.
12.
Oppen-BerntsenD.O., JensenE.G., and WaltherB.T. (1992). Hepatocytes from rainbow trout (Oncorhynchus mykiss) synthesise eggshell zr-proteins in vitro after estradiol-17β treatment.Journal of Endocrinology135, 293–302.
13.
KorsgaardB., and MommsenT.P. (1993). Gluconeogenesis in hepatocytes of immature rainbow trout (Oncorhynchus mykiss): control by estradiol.General and Comparative Endocrinology89, 17–27.
14.
AnderssonT. (1984). Oxidative and conjugative metabolism in isolated rainbow trout liver cells.Marine Environmental Research14, 442–443.
15.
AnderssonT., & FörlinL. (1985). Spectral properties of substrate-cytochrome P450 interaction and catalytic activity of xenobiotic metabolising enzymes in isolated rainbow trout liver cells.Biochemical Pharmacology34, 1407–1413.
16.
GrantM.H., MelvinM.A.L., ShawP., MelvinW.T., and BurkeM.D. (1985). Studies on the maintenance of cytochrome P450 and b5, mono-oxygenases and cytochrome reductases in primary cultures of rat hepatocytes.FEBS Letters190, 99–103.
17.
BlaauboerB.J., van HolsteijnI., van GraftM., and PaineA. (1985). The concentration of cytochrome P450 in human hepatocyte culture.Biochemical Pharmacology34, 2405–2408.
18.
DevauxA., PesonenM., MonodG., and AnderssonT. (1992). Glucocorticoid-mediated potentiation of P450 induction in primary culture of rainbow trout hepatocytes.Biochemical Pharmacology43, 898–901.
19.
KlaunigJ.E. (1984). Establishment of fish hepatocyte cultures for use in in vitro carcinogenicity studies.National Cancer Institute Monographs65, 163–173.
20.
MoonT.W., WalshP.J., and MommsenT.P. (1985). Fish hepatocytes: a model metabolic system.Canadian Journal of Fisheries and Aquatic Sciences42, 1772–1782.
21.
KennedyC.J., GillK.A., and WalshP.C. (1991). Temperature acclimatisation of xenobiotic metabolising enzymes in cultured hepatocytes and whole liver of the gulf toadfish (Opsanus beta).Canadian Journal of Fisheries and Aquatic Sciences48, 1212–1219.
22.
WalshP.J., MoonT.W., and MommsenT.P. (1985). Interactive effects of acute changes in temperature and pH on metabolism in hepatocytes from the sea raven (Hemitripterus americanus).Physiological Zoology48, 727–735.
23.
JankowskyH.D., HotoppW., and VsianskyP. (1981). Effects of assay and acclimatisation temperatures on incorporation of amino acids into protein of isolated hepatocytes from the European eel (Anguilla anguilla).Journal of Thermal Biology6, 201–208.
24.
CollodiP., MirandaC.L., ZhaoX., BuhlerD.R., and BarnesD.W. (1994). Induction of zebrafish (Brachydanio rerio) P450 invivo and in cell culture.Xenobiotica24, 487–493.
25.
KlaunigA.E., RuchR.J., and GoldblattP.J. (1985). Trout hepatocyte culture: isolation and primary culture.In Vitro Cellular and Developmental Biology21, 221–228.
26.
VaillantC., MonodG., ValotaireY., & RiviereJ-L. (1989). Mise en evidence et induction du cytochrome P450 et d'activites monooxygénases dans une culture primaire d'hépatocytes de truite arc-en-ciel (Salmo gairdneri).Comptes Rendus de l'Académie des Sciences, Paris308(III), 83–88.
27.
PesonenM., and AnderssonT. (1991). Characterisation and induction of xenobiotic metabolising enzyme activities in a primary culture of rainbow trout hepatocytes.Xenobiotica21, 461–471.
28.
HazelJ.R., and ProsserC.L. (1974). Molecular mechanisms of temperature compensation in poikilotherms.Physiological Reviews54, 620–673.
29.
KoivusaariU. (1983). Thermal acclimatisation of hepatic polysubstrate monooxygenase and UDP glucuronosyltransferase of mature rainbow trout (Salmo gairdneri).Journal of Experimental Zoology227, 35–42.
30.
HochachkaP.W., and SomeroG.N. (1984). Biochemical Adaption, 558 pp. Princeton, NJ, USA: Princeton University Press.
31.
Kloepper-SamsP.J., and StegemanJ.J. (1992). Effects of temperature acclimatisation on the expression of hepatic cytochrome P4501A mRNA and protein in the fish Fundulus heteroclitus.Archives of Biochemistry and Biophysics299, 38–46.
32.
MommsenT.P. (1986). Comparative gluconeogenesis in hepatocytes from salmonid fishes.Canadian Journal of Zoology64, 1110–1115.
33.
DonohueM., BaldwinL.A., LeonardD.A., KosteckiP.T., and CalabreseE.J. (1993). Effect of hypolipidemic drugs Gemfibrozil, Ciprofibrate, and Clofibric Acid on peroxisomal β-oxidation in primary cultures of rainbow trout hepatocytes.Ecotoxicology and Environmental Safety26, 127–132.
34.
MichellA.M., BridgesJ.W., and ElcombeC.R. (1984). Factors influencing peroxisome proliferation in cultured rat hepatocytes.Archwes of Toxicology55, 238–246.
35.
LakeB.G., GrayT.J.B., HodderK.D., BeamandJ.A., StubberfieldC.R., and GangolliS.D. (1986). Effects of peroxisome proliferators in vivo and in primary hepatocyte cultures from Chinese and Syrian hamsters.Archives of Toxicology, Suppl. 9, 167.
36.
BerryM.N., and FriendD.S. (1969). High-yield preparation of isolated rat parenchymal cells. A biochemical and fine structural study.Journal of Cell Biology43, 506–520.
37.
SeglenP.O. (1976). Preparation of isolated rat liver cells. In Methods of Cell Biology, Vol. 13 (ed. PrescottD.M.), pp. 29–93. New York: Academic Press.
38.
KlotzA.V., StegemanJ.J., and WalshC. (1984). An alternative 7-ethoxyresorufin O-deethylase activity assay: a continuous visible spectrophotometric method for measurement of cytochrome P450 monooxygenase activity.Analytical Biochemistry140, 138–145.
39.
MoldenkeA.F., and TerriereL.C. (1981). Cytochrome P450 in insects. III. Increase in substrate binding by microsomes from phenobarbital-induced house flies.Pesticide Biochemistry and Physiology16, 222–230.
40.
DallnerG., SiekevitzP., and PaladeG.E. (1966). Biogenesis of endoplasmic reticulum membranes. II. Synthesis of constitutive microsomal enzymes in developing rat hepatocytes.Journal of Cell Biology30, 97–117.
41.
HabigW.H., PabstM.J., and JacobyW.B. (1974). Glutathione S-transferases, the first enzymatic step in mercapturic formation.Journal of Biological Chemistry249, 7130–7139.
42.
AnderssonT., PesonenM., and JohanssonC. (1985). Differential induction of cytochrome P450–dependent monooxygenase, epoxide hydrolase, glutathione transferase and glucuronosyl-transferase activities in the liver of the rainbow trout by β-naphthoflavone or Clophen A50.Biochemical Pharmacology34, 3309–3314.
43.
BradfordM.M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.Analytical Biochemistry72, 248–254.
FlouriotG., VaillantC., SalbertG., PelisseroC., GuiraudJ.M., and ValotaireY. (1993). Monolayer and aggregate cultures of rainbow trout hepatocytes: long-term and stable liver-specific expression in aggregates.Journal of Cell Science105, 407–416.
46.
BraunbeckT., and StorchV. (1992). Senescence of hepatocytes isolated from rainbow trout (Oncorhynchus mykiss) in primary culture.Protoplasma170, 138–159.
47.
DevauxA., MonodG., MasfaraudJ-F., & VaillantC. (1991). Use of a new coumarin analog in the study of cytochrome P450-dependent monooxygenase activities in primary culture of rainbow trout hepatocytes (Oncorhynchus mykiss).Toxicology312, 63–69.
48.
PesonenM., GoksoyrA., and AnderssonT. (1992). Expression of P4501A1 in a primary culture of rainbow trout hepatocytes exposed to β-naphthoflavone or 2,3,7,8-tetrachlorodibenzo-p-dioxin.Archives of Biochemistry and Biophysics292, 228–233.
49.
DickensM., and PetersonR.E. (1980). Effects of a hormone-supplemented medium on cytochrome P450 content and monooxygenase activities of rat hepatocytes in primary culture.Biochemical Pharmacology29, 1231–1238.
50.
LakeB.G., and PaineA.J. (1982). The effect of hepatocyte culture on cytochrome P450 linked drug metabolizing enzymes.Biochemical Pharmacology31, 2141–2144.
51.
WortelboerH.M., de KruifC.A., van lerselA.A.J., FalkeH.E., NoordhoekJ., and BlaauboerB.J. (1990). The isoenzyme pattern of cytochrome P450 in rat hepatocytes in primary culture, comparing different enzyme activities in microsomal incubations and in intact monolayers.Biochemical Pharmacology40, 2525–2534.
52.
JensenE.G., SkaareJ.U., EgaasE., & GoksøyrA. (1991). Response of xenobiotic metabolising enzymes in rainbow trout (Oncorhynchus mykiss) to endosulfan, detected by enzyme activities and immunochemical methods.Aquatic Toxicology21, 81–92.
53.
SkaareJ.U., JensenE.G., GoksøyrA., and EgaasE. (1991). Response of xenobiotic metabolising enzymes of rainbow trout (Oncorhynchus mykiss) to the mono-ortho substituted polychlorinated PCB congener 2,3,4,4’,5-pentachlorqbiphenyl, PCB-118, detected by enzyme activities and immunochemical methods.Archives of Environmental Contamination and Toxicology20, 349–352.
54.
KoivusaariU., and AnderssonT. (1984). Partial temperature compensation of hepatic biotransformation enzymes in juvenile rainbow trout (Salmo gairdneri) during water warming in spring.Comparative Biochemical Physiology77B, 223–227.
55.
AnderssonT., and KoivusaariU. (1985). Influence of environmental temperature on the induction of xenobiotic metabolism by β-naphthoflavone in rainbow trout (Salmo gairdneri).Toxicology and Applied Pharmacology80, 43–59.
56.
AnderssonT., and KoivusaariU. (1986). Oxidative and conjugative metabolism of xenobiotics in isolated liver cells from thermally acclimatised rainbow trout.Aquatic Toxicology8, 85–92.
57.
BlanckJ., Lindström-SeppaP., ÅgrenJJ., HänninenO., ReinH., and RuckpaulK. (1989). Temperature compensation of hepatic microsomal cytochrome P450 activity in rainbow trout. I. Thermodynamic regulation during water cooling in autumn.Comparative Biochemistry and Physiology93C, 55–60.
58.
KobanM. (1986). Can cultured teleost hepatocytes show temperature acclimatisation?American Journal of Physiology250, 211–220.
