Isolation of a new human fetal liver cytochrome P450 cDNA clone: Evidence for expression of a limited number of forms of cytochrome P450 in human fetal livers

Komori, Masaharu; Nishio, Kanako; Fujitani, Tomomichi; Ohi, Hiroshi; Kitada, Munehiro; Mima, Satoaki; Itahashi, Koshiro; Kamataki, Tetsuya

doi:10.1016/0003-9861(89)90213-0

Cited by 57 publications

(17 citation statements)

References 24 publications

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“…In vitro maturation is probably related to addition of fetal calf serum and hormones, specially corticosteroids, to the culture medium. In contrast to P-450 IIC8/ 9/10 and P-450 IIE1, at least some forms of P-450 IIIA already well expressed in fetal hepatocytes [28] indicating that these gene families are independently regulated.…”

Section: Discussionmentioning

confidence: 96%

“…However, studies with specific oligonucleotide probes indicated that the P-450 IIIA3 mRNA of Molowa et al [27] was not found in very high abundance relative to P-450 IIIA4 mRNA in any of the livers examined [25]. The cDNA clone of Komori et al [28] (P-450 IIIA6) is highly similar but was isolated from a fetal liver cDNA library; the occurrence of this mRNA in adult liver is unknown (as is the occurrence of P-450 IIIA4 in fetal liver). The cDNA clone for P-450 IIIA5 has been alluded to [2] and published only very recently [26]; this gene appears to be expressed in only a few individuals and, since the molecular mass of its product is slightly different, we have not utilized specific probes to distinguish it from P-450 IIIA4.…”

Section: Preparation Qf Rnicrosornes and Irnrnunoblot Analysismentioning

confidence: 99%

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Expression of cytochrome P‐450 enzymes in cultured human hepatocytes

Morel

Beaune

Ratanasavanh

et al. 1990

European Journal of Biochemistry

149

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Hepatocytes from adult and newborn humans were put into primary culture and exposed to phenobarbital, 3-methylcholanthrene, or rifampicin, three well-known inducers of cytochrome P-450 in animals. The expression of four cytochrome P-450 enzymes (or groups of enzymes, namely P-450 IIIA, P-450 IIC8/9/10, P-450 IIE1, and P-450 IA2) was investigated. These enzymes were found to remain expressed during the period of culture studied. Treatment with the inducers for three days resulted in different responses, depending upon the inducer and the enzyme. Phenobarbital and rifampicin increased P-450 IIC8/9/10 mRNA transcripts and the corresponding protein, while 3-methylcholanthrene was ineffective. Both P-450 IIIA mRNA and protein were strongly induced by rifampicin. All of the hepatocytes were found to synthesize P-450 IIIA in response to rifampicin, as shown by immunoperoxidase staining. P-450 IIIA expression was not affected by phenobarbital and was decreased by 3-methylcholanthrene. P-450s IA2 and IIEl decreased to 25 -50% of the initial level during these cultures. P-450 IA2 and ethoxyresorufin 0-deethylase activity (which is a monooxygenase activity related to P-450 IA family) were increased only by 3-methylcholanthrene and did not respond to the other inducers. P-450 IIEl was not induced by any of these compounds. P-450 IIC8/9/10 and P-450 IIIA mRNA levels were also measured in human hepatocytes from one newborn. P-450 IIC8/9/10 was barely expressed in freshly isolated cells but increased dramatically with time in culture. P-450 IIIA transcripts were abundant in both freshly isolated and cultured cells derived from a newborn.These results clearly demonstrate that human hepatocytes continue to express cytochrome P-450 enzymes and respond to inducers in culture. This model system provides a useful approach for investigating the effects of drugs on maturation and expression of drug-metabolizing enzymes in human liver.The cytochrome P-450 (P-450) monooxygenase system (EC 1.14.14.1; for nomenclature see [I] and [2]) consists of a large family of enzymes responsible for the oxidation of drugs and environmental pollutants, as well as many endogenous compounds. More than 20 distinct forms of P-450 have been purified from rat liver [3, 41 (and references therein). These distinct enzymes are encoded by different genes and several forms are inducible with agents such as phenobarbital, 3-methylcholanthrene, polyhalogenated biphenyl mixtures, or pregnenolone 16a-carbonitrile (PCN) [5]. While much information has been gathered concerning P-450 in experimental animals, the application of this knowledge to human beings has lagged [l, 6,7]. It is now well established that P-450 forms may contribute to inter-individual variations among humans in biological responses to drugs and other chemicals [8]. Genetic polymorphisms have been recognized in drug oxidations in humans [9] and possible relationships to cancers and other lesions have been proposed [lo-131. Furthermore, induction of P-450 may be clinically important since in some c...

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Section: Discussionmentioning

confidence: 96%

Section: Preparation Qf Rnicrosornes and Irnrnunoblot Analysismentioning

confidence: 99%

Expression of cytochrome P‐450 enzymes in cultured human hepatocytes

Morel

Beaune

Ratanasavanh

et al. 1990

European Journal of Biochemistry

149

View full text Add to dashboard Cite

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“…Another crucial element is the maturity of the enzymatic system implicated in its metabolism. To date, only few data have reported the onset of P-450 isoenzymes in the human liver: these studies concluded on the presence of CYP3A protein in the fetal liver as early as the 14th week of gestation [2,[19][20][21][22][23] and the absence of proteins of the CYP2C subfamily, explaining the very low activity towards benzopyrene and mephenytoin [2, The CYP2D subfamily is a third group of P-450 genes, composed of three genes in the human species, but only one is functional 1241. We have accumulated evidence indicating that, during ontogenesis, the surge in CYP2D6 protein is mainly postnatal since its concentration rises in newborns 24 h after birth.…”

Section: Discussionmentioning

confidence: 99%

Expression of CYP2D6 in developing human liver

Tréluyer

Jacqz-Aigrain

Álvarez

et al. 1991

European Journal of Biochemistry

181

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The CYP2D6 protein is a polymorphic isoenzyme involved in the biotransformation of several drugs including the probe drug dextromethorphan. The rise in the protein concentration, immunochemically determined with a specific antibody, was shown to occur within the first week following birth, whatever the gestational age at birth.In fetuses, the concentration of hepatic CYP2D6 protein was very low or undetectable in 70% of samples tested. In the remaining 30%, its concentration was comparable to that of newborns aged 1 -7 days. This early rise was associated with spontaneous abortion in 70% of positive samples, whereas in fetuses with an intermediate CYP2D6 protein concentration, 80% were from induced abortions.The rise in CYP2D6 protein was associated with the developmental onset of dextromethorphan O-demethylation, but not N-demethylation, even if activity was lower in fetal than in neonatal and in adult liver microsomes.Lastly, the CYP2D6 RNA is detectable earlier than the protein and exhibits a peak of hepatic accumulation in newborns, before declining in adulthood. A positive correlation between RNA accumulation and protein concentration can be demonstrated only in the adult. This suggests that regulation is primarily at the transcriptional level, but cannot rule out the participation of post-transcriptional events in the regulation process throughout ontogenesis.Monooxygenase activities involved in the biotransformation of endogenous and exogenous hydrophobic molecules are mainly supported by cytochrome P-450 isoenzymes. To date, more than 30 isoenzymes of cytochrome P-450 have been identified, purified and/or cloned from the human liver (for review see [l]). These P-450s exhibit different profiles for their developmental evolution. In fetal liver, only one P-450 has been repeatedly detected: a CYP3A protein is present at a level roughly similar to that of the adult liver when determined by Western blotting [2] and is able to catalyze enzymatic activities like benzphetamine demethylation, aldrin epoxidation and dehydroepiandrosterone 16P-hydroxylation as the adult isoenzyme does. CYP2C8, one of the major P-450 isozymes immunologically detected in adult liver, is totally absent from fetal liver. The absence of this P-450 is responsible for the very low or negligible activity towards various substrates including mephenytoin, benzopyrene, p-nitroanisole [2, 31. To date, little or no data are available for other P-450 isoenzymes.We have focused our attention on CYP2D6, a gene coding for a polymorphic isoenzyme involved in the biotransformation of many drugs like P-blockers, tricyclic antidepressants, antitussives drugs, etc. In the adult Caucasian population, approximately 7% of individuals (poor vs extensive metabolizers) are defective in the active enzyme able to carry Enzymes. P-450, cytochrome P-450 (EC 1.14.14.1); isocitrate dehydrogenase (EC 1 .I .I .42). out the reaction [4]. This deficiency leads to an altered profile of urinary metabolites and consequently the ratio metabolite/ parent drug is markedly lo...

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“…Using these cDNA clones as probes, we examined the possibility of whether human fetal livers contained the same molecular forms of cytochrome P-450. The results of Northern blot analysis showed that fetal livers expressed limited forms of cytochrome P-450 (Komori et al 1989a). No hybridization bands were detectable when the cDNA coding for human adult P-45011C were applied as a probe.…”

Section: Mutagen-producing Capacity Of Human Fetal Liversmentioning

confidence: 98%