Pig hepatocytes as an in vitro model to study the regulation of human CYP3A4: prediction of drug-drug interactions with 17α-ethynylestradiol

doi:10.1016/S0009-2797(97)00077-X

Chemico-Biological Interactions

Volume 107, Issues 1–2, 6 November 1997, Pages 93-108

https://doi.org/10.1016/S0009-2797(97)00077-X Get rights and content

Abstract

The objective of this study was to provide evidence of the validity of pig hepatocytes as a model to study the regulation of human CYP3A4 with special emphasis on drag-drug interactions. Thirteen different drugs were incubated with primary monolayer cultures of pig hepatocytes (n = 4). The study included both drugs reported to cause drug interactions in the clinic with 17α-ethynylestradiol (EE₂), other drugs metabolized by CYP3A4, and drugs not reported to cause any problems. Effect of the drug exposure to pig hepatocytes was determined by immunodetection using a monoclonal human CYP3A4 antibody and measurement of 6β-hydroxylation of testosterone and 2-hydroxylation of 17α-ethynylestradiol (EE₂), both reactions known to be catalyzed by CYP3A4 in humans. Data were compared to data from human hepatocytes and to reported observations of drug-drag interactions in the clinic. The drugs known to be inducers of CYP3A4 in humans significantly increased a CYP isoform in pigs catalyzing 6β-hydroxylation of testosterone and 2-hydroxylation of EE₂, whereas drugs not reported to have clinical interactions with EE₂ had no or only marginal effect. Induction by the drugs known to be inducers of CYP3A4 increased with drug exposure time and the CYP3A4 activity, represented by testosterone 6β-hydroxylation, was highest at 72 h for the investigated induction periods (24, 48 and 72 h), except for dexamethasone where the effect peaked after 24 h. Induction of the 2-hydroxylation of EE₂ correlated well with the increase in 6β-hydroxylation of testosterone (except for sulphinpyranzone) and the increase in the protein level of CYP3A detected by a monoclonal human CYP3A4 antibody, thus confirming the 2-hydroxylation of EE₂ in pigs as being biotransformed by a CYP isoform presumably belonging to the CYP3A subfamily as in humans. In conclusion, these results indicate that pig hepatocytes may be a valuable model to mimic the regulation of human CYP3A4.

References (40)

S.J. Wassner et al.
The adverse effect of anticonvulsant therapy on renal allograft survival
J. Paediatr.
(1976)
D. Janz et al.
Anti-epileptic drugs and failure of oral contraceptives
Lancet
(1974)
M.P. Diamond et al.
Interaction of anticonvulsants and oral contraceptives in epileptic adolescents
Contraception
(1985)
M. Monshouwer et al.
Suppresion of cytochrome P450- and UDP glucuronosyl transferase dependent enzyme activities by pro-inflammatory cytokines and possible role of nitric oxide in primary cultures of pig hepatocytes
Toxicol. Appl. Pharmacol.
(1996)
T. Omura et al.
The carbon monoxide-binding pigment of liver microsomes. II. Solubilization, purification and properties
J. Biol. Chem.
(1964)
G.L. Peterson
A simplification of the protein assay method of Lowry et al. which is more generally applicable
Anal. Biochem.
(1977)
D. Larrey et al.
Effects of erythromycin on hepatic drug-metabolizing enzymes in humans
Biochem. Pharmacol.
(1983)
D. Pessayre et al.
Selfinduction by triacetyloleandomycin of its own transformation into a metabolite forming a stable 456 nm absorbing complex with cytochrome P450
Biochem. Pharmacol.
(1981)
D. Larrey et al.
Formation of inactive cytochrome P-450 Fe(II)-metabolite complexes with several erythromycin derivatives but not with josamycin and midecamycin in rats
Biochem. Pharmacol.
(1983)
B. Saad et al.
Oxygen tension, insulin, and glucagon affect the preservation and induction of cytochrome P450 isoforms in cultured rat hepatocytes
Toxicol. Appl. Pharmacol.
(1994)

A.P. Li et al.

Substrates of human hepatic cytochrome P450 3A4

Toxicology

(1995)

G.T. McInnes et al.

Drug interactions that matter. A critical reappraisal

Drugs

(1988)

Y. Choi et al.

Effect of diphenylhydantoin on Cortisol kinetics in humans

Pharmacol. Exp. Ther.

(1971)

N. Haque et al.

Studies on dexamethasone metabolism in man: effect of diphenylhydantoin

J. Clin. Endocrinol. Metab.

(1972)

D.M. Edwards et al.

Changes in Cortisol metabolism following rifampicin therapy

Lancet

(1974)

M.R. Stjernholm et al.

Effects of diphenylhydantoin, phenobarbital and diazepam on the metabolism of methylprednisolone and its sodium succinate

J. Clin. Endocrinol. Metab.

(1975)

T.J. Wadhwa et al.

Cyclosporin drug interactions: a review

Ther. Drug Monit.

(1987)

D.J. Back et al.

Pharmacokinetic drug interactions with oral contraceptives

Clin. Pharmacokinet.

(1990)

F.P. Guengerich

Oxidation of 17α-ethynylestradiol by human liver cytochrome P-450

Mol. Pharmacol.

(1988)

H.M. Bolt et al.

Interactions of rifampicin treatment with pharmacokinetics and metabolism of ethinyloestradiol in man

Acta endocrinol.

(1977)

Cited by (43)

The utility of the minipig as an animal model in regulatory toxicology
2010, Journal of Pharmacological and Toxicological Methods
In this article we review the value and utility of the minipig as an animal model in regulatory toxicity testing. Our review is based on detailed consideration of the comparative biology of the minipig, and of the practical features of toxicity testing in the minipig. The minipig presents a favourable profile as a non-rodent toxicology model, in terms of the similarity to man and also in terms of applicability to different study types. Studies of general toxicology can be performed in the minipig by oral, cutaneous, parenteral and inhalation routes. For reproductive toxicology studies the minipig offers numerous advantages as a non-rodent model although the lack of placental transfer of macromolecules may limit the role of the minipig in reproductive testing of biotechnology products. For safety pharmacology studies the minipig is an advantageous model, particularly as regards the cardiovascular system. The immune system of the pig is better characterized than that of the dog, making the pig an interesting alternative model to the nonhuman primate for therapeutic approaches based on manipulation of the immune system. Overall, this review leads us to believe that the minipig might be a better non-rodent toxicology model than the dog. At the present time, however, insufficient comparative data is available to permit a rigorous evaluation of the predictivity of the minipig for human drug-induced toxicities and research is urgently needed to provide experimental data for evaluation of the hypothesis that minipig studies may better reflect human drug-induced toxicities than studies performed in traditional non-rodent toxicology models. It would be of particular value to gain a better vision of the potential utility of the minipig as a model for the safety testing of new biologics, where the minipig could potentially replace the use of non-human primates in the testing of some new products.
Interspecies difference in liver-specific functions and biotransformation of testosterone of primary rat, porcine and human hepatocyte in an organotypical sandwich culture
2009, Toxicology Letters
Citation Excerpt :
Monshouwer et al. (1998) explained pig might represent a more appropriate model species for oxidative drug metabolism in humans than rats based on enzymatic activities, apoprotein and mRNA analyses of CYP enzymes. Porcine hepatocytes may be a valuable model to mimic the regulation of human CYP3A4 (Olsen et al., 1997). Froehlich et al. (2005) carried out comparative study of porcine and human hepatocytes and suggested cultured porcine hepatocytes might be a suitable alternative to human hepatocytes for studying metabolism of xenobiotics in vitro.
Interspecies difference is an important issue in toxicology research. We compared the potential in vitro metabolism of human, porcine and rat hepatocytes over 2 weeks in culture in an organotypical culture model which reflects the in vivo situation. All three species show similar LDH-rates. Albumin measurements showed that rat cells are about twice as active as human and porcine hepatocytes. The ethoxyresorufin-O-deethylase (EROD) activity of the rat hepatocytes is with about 14 μU/10⁶ cells distinctly higher than those of porcine and human cells (1.8 and 0.5 μU/10⁶ cells respectively), furthermore, the activity of the rat EROD increases slightly during the prolonged time in culture, whereas those of porcine and human enzymes slightly decrease. Concerning ethoxycoumarin-O-deethylase (ECOD), the enzyme activities are found to be in three different ranges where rat cells show the highest activity with 66 μU/10⁶ cells, porcine hepatocytes exhibit an activity of about 23 μU/10⁶ cells, and human activity is lowest with 0.7 μU/10⁶ cells. All three species show a similar decreasing trend of ECOD during the period of study. Regarding the biotransformation of testosterone, human and porcine liver cells form three major metabolites whereas rat cells form a mixture of all measured metabolites. Hence, in vitro metabolism using porcine hepatocytes would be much more scientific sense than one using rat hepatocytes since the metabolic pathways are much closer to human metabolism.
Expression and induction by rifampicin of CAR- and PXR-regulated CYP2B and CYP3A in liver, kidney and airways of pig
2008, Toxicology
The transcript levels of CYP2B22, 3A22, 3A29, 3A46, CAR, PXR and HNF4α were investigated in liver, kidney and airways from control and rifampicin-treated male pigs. The presence and induction of CYP genes transcription were studied by RT-PCR, real-time PCR, Western blotting and enzymatic activity whereas the expression of receptors was studied by RT-PCR or real-time PCR. Pretreatment with rifampicin resulted in a transcriptional activation, although to different extents, of all the CYP3A genes in liver but not in kidney, lung, bronchi or trachea. In the hepatic microsomes, the induction of CYP3A genes was accompanied by an increase of CYP3As marker activities and of two protein bands immunoreactive with anti-human CYP3A4. The CYP2B22 transcript was found to be markedly induced only in liver and kidney. In parallel, a protein band immunoreactive with anti-rat CYP2B1 was elevated while enhanced CYP2B marker activities were observed in hepatic and renal microsomes. As expected, based on human data, the basal expression of CAR, PXR and HNF4α was found to be high in liver and low in airways and not susceptible to induction by rifampicin. A significant expression of these transcriptional factors was also demonstrated in kidney. Thus, it is likely that rifampicin induced CYP2B22 both in liver and kidney of pig, not via activation of CAR, but via PXR, through a cross-talk mechanism, as previously observed in human liver. Taken together, our results demonstrated a differential expression and regulation of three individual CYP3As, CYP2B22, CAR, PXR and HNF4α genes in liver, kidney and airways of pig.
Effects of mammalian CYP3A inducers on CYP3A-related enzyme activities in grass carp (Ctenopharyngodon idellus): Possible implications for the establishment of a fish CYP3A induction model
2008, Comparative Biochemistry and Physiology - C Toxicology and Pharmacology
Unexpected drug–drug interactions in fish are generally associated with the induction of CYP3A activity and may lead to the formation of drug residues and thus threaten the safety of fishery products. However, little information is available about CYP3A induction in fish. In the present study, we determined the in vivo and in vitro effects of typical mammalian CYP3A inducers (rifampicin, phenobarbital and dexamethasone) on CYP3A-related enzyme activities in a freshwater teleost, the grass carp (Ctenopharyngodon idellus). Our results showed that the response to rifampicin was similar for grass carp liver cell line (GCL), liver microsomes and the primary hepatocytes of grass carp, as indicated by the activity of aminopyrine N-demethylase (APND). When erythromycin N-demethylase (ERND) and 6β-testosterone hydroxylase (6β-TOH) were taken into consideration, the GCL displayed a greater capacity for conducting CYP3A metabolism and induction than the C. idellus kidney cell line (CIK). Using erythromycin and testosterone as substrates, we demonstrated that CYP3A catalysis exhibited non-Michaelis-Menten kinetics in GCL cells, and that V_max/K_m values were significantly increased due to rifampicin-treatment. Overall, this study may have implications for the use of GCL as a CYP3A induction model to identify physiological changes in fish as well as the similarities or differences between fish and mammals.
Acetaminophen-induced hepatotoxicity of gel entrapped rat hepatocytes in hollow fibers
2006, Chemico-Biological Interactions
Citation Excerpt :
As these in vitro models frequently minimize the volume and cost of screening as well as the amount of compounds required, much of the early ADME/Tox evaluation relies on in vitro studies before the initiation of any in vivo animal testing. Monolayer cultures of hepatocytes have been widely used to investigate drug metabolism [2,3], drug–drug interactions [4,5] and mechanism of hepatotoxicities [6,7]. However, it has been generally recognized that primary hepatocytes in monolayer culture are subject to a gradual loss of liver-specific functions, with a special reference to decreased CYP isoforms [8].
An important application of primary hepatocyte cultures is for hepatotoxicity research. In this paper, gel entrapment culture of rat hepatocytes in miniaturized BAL system were evaluated as a potential in vitro model for hepatotoxicity studies in comparison to monolayer cultures. After exposure for 24 and 48 h to acetaminophen (2.5 mM), gel entrapped hepatocytes were more severely damaged than hepatocyte monolayer detected by methyl thiazolyl tetrazolium (MTT) reduction, intracellular glutathione (GSH) content, reactive oxygen species (ROS) levels, urea genesis and albumin synthesis. CYP 2E1 activities detected by 4-nitrocatechol (4-NC) formation were higher in gel entrapped hepatocytes than in hepatocyte monolayers while the addition of CYP 2E1 inhibitor, diethyl-dithiocarbamate (DDC), more significantly reduced acetaminophen-induced toxicity in gel entrapped hepatocytes. In addition, protective effects of GSH, liquorice extract and glycyrrhizic acid against acetaminophen hepatotoxicity were clearly observed in gel entrapped hepatocytes but not in hepatocyte monolayer at an incubation time of 48 h. Overall, gel entrapped hepatocytes showed higher sensitivities to acetaminophen-induced hepatotoxicity than hepatocyte monolayer by a mechanism that higher CYP 2E1 activities of gel entrapped hepatocytes could induce more severe acetaminophen toxicity. This indicates that gel entrapped hepatocytes in hollow fiber system could be a promising model for toxicological study in vitro.
Phase I and II metabolism and carbohydrate metabolism in cultured cryopreserved porcine hepatocytes
2005, Chemico-Biological Interactions
Citation Excerpt :
The pigs were euthanized by use of a captive bolt pistol followed by exsanguination after which the liver was removed and perfused with cold saline. Hepatocytes were isolated according to Olsen et al. [20] with the following modifications. Two liters of buffer I (pH 7.4, containing 142 mM NaCl, 6.7 mM KCl, 10.1 mM hepes and 0.5 mM EGTA) and 2 l of buffer II (as buffer I except EGTA was omitted) was used.
Primary porcine hepatocytes were cryopreserved using freezing boxes or a programmable freezer (PF). Upon thawing and culturing in 12-well plates cryopreserved hepatocytes were compared with their fresh controls on days 1 and 2 after plating. Cryopreserved hepatocytes attached approximately as well as fresh hepatocytes and useful cultures were obtained. In cryopreserved hepatocytes, coumarin 7-hydroxylation, 6β-testosterone hydroxylation and p-nitrophenol glucuronidation were reduced to about 10–40, 35 and 40%, respectively, compared to their fresh counterparts. Glycogen synthesis in cryopreserved hepatocytes was reduced to about 30% on day 1 of culture and about 47% on day 2 of culture compared to the synthesis in fresh hepatocytes. Both fresh and cryopreserved hepatocytes increased the synthesis by twofold in response to stimulation with insulin. Reduced basal levels of glycogen and of glycogen synthesis could be explained by an increased energy demand in cryopreserved hepatocytes needing to repair damages caused by cryopreservation. Glycogenolysis was reduced to about 50% in cryopreserved hepatocytes and gluconeogenesis to about 40% of the glucose production in fresh hepatocytes. In both fresh and cryopreserved hepatocytes the glucose production from glycogenolysis and gluconeogenesis, respectively, was increased fourfold in response to stimulation with glucagon. Overall, the hepatocytes cryopreserved in boxes had a tendency to perform better than hepatocytes cryopreserved in a programmable freezer. In conclusion, the cryopreserved hepatocytes were metabolic active; however, to a lower extent than the fresh hepatocytes, although, the cryopreserved hepatocytes responded as well as the fresh hepatocytes to insulin and glucagon.

View all citing articles on Scopus

View full text

Pig hepatocytes as an in vitro model to study the regulation of human CYP3A4: prediction of drug-drug interactions with 17α-ethynylestradiol

Abstract

J. Paediatr.

Lancet

Contraception

Toxicol. Appl. Pharmacol.

J. Biol. Chem.

Anal. Biochem.

Biochem. Pharmacol.

Biochem. Pharmacol.

Biochem. Pharmacol.

Toxicol. Appl. Pharmacol.

Toxicology

Drug interactions that matter. A critical reappraisal

Drugs

Effect of diphenylhydantoin on Cortisol kinetics in humans

Pharmacol. Exp. Ther.

Studies on dexamethasone metabolism in man: effect of diphenylhydantoin

J. Clin. Endocrinol. Metab.

Changes in Cortisol metabolism following rifampicin therapy

Lancet

Effects of diphenylhydantoin, phenobarbital and diazepam on the metabolism of methylprednisolone and its sodium succinate

J. Clin. Endocrinol. Metab.

Cyclosporin drug interactions: a review

Ther. Drug Monit.

Pharmacokinetic drug interactions with oral contraceptives

Clin. Pharmacokinet.

Oxidation of 17α-ethynylestradiol by human liver cytochrome P-450

Mol. Pharmacol.

Interactions of rifampicin treatment with pharmacokinetics and metabolism of ethinyloestradiol in man

Acta endocrinol.