Resolution of the Cytochrome P-450-containing ω-Hydroxylation System of Liver Microsomes into Three Components

CYP3A isoforms are responsible for the biotransformation of a wide variety of exogenous chemicals and endogenous steroids in human tissues. Two members of the CYP3A subfamily display developmentally regulated expression in the liver; CYP3A7 is expressed in the fetal liver, whereas CYP3A4 is the major cyrochrome P-450 isoform present in the adult liver. To gain insight into the descriptive ontogenesis of CYP3A isoforms during the neonatal period, we have developed several approaches to explore a neonatal liver bank. Although CYP3A4 and CYP3A7 are structurally closely related, they differ in their capacity to carry out monooxygenase reactions. We have cloned CYP3A4 and CYP3A7 and established stable transfectants in Ad293 cells to investigate their substrate specificities. The 16alpha hydroxylation of dehydroepiandrosterone is catalyzed by both proteins, but CYP3A7 has a higher affinity and maximal velocity than CYP3A4. Conversely, the conversion of testosterone into its 6beta derivative is essentially supported by CYP3A4. We used these two probes to determine the ontogenic evolution at the protein level; CYP3A7 was very active in the fetal liver and its activity was maximal during the first week following birth before to progressively decline and reached a very low level in adult livers. Conversely, the activity of CYP3A4 was extremely weak in the fetus and began to raise after birth to reach 30-40% of the adult activity after one month. CYP3A4 RNA accumulation displays a similar pattern of evolution; when probed with an oligonucleotide, its concentration increased rapidly after birth to reach a plateau as soon as the first week of age. These data supports the assumption that CYP3A4 expression is transcriptionally activated during the first week after birth and is accompanied by a simultaneous decrease of CYP3A7 expression, in such a way that the overall CYP3A protein content and the level of pentoxyresorufin dealkylase catalyzed by the two proteins remain nearly constant.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1997.00625.x

Expression of CYP3A in the human liver--evidence that the shift between CYP3A7 and CYP3A4 occurs immediately after birth.

Sex-related differences in drug metabolism

Objectives
The effect of aging on drug metabolism in humans has not yet been completely described.

Methods
Two hundred twenty-six patients with equal histopathologic conditions were investigated. The cytochrome P450 contents in the liver biopsy samples, the plasma antipyrine clearance rates after oral administration and, as an independent control of vitality, serum testosterone levels were determined.

Results
Cytochrome P450 content in subjects from 20 to 29 years of age was 7.2 ± 2.6 nmol · gm−1, increased during the fourth decade (+ 7.2%, p = NS), declined after 40 years (−16%, p < 0.01) to a level that remained unaltered up to 69 years, and declined further after 70 years (−32%, p < 0.001). The antipyrine (phenazone) clearance rate in young subjects was 46.4 ± 18.5 ml · min−1, remained unaltered during the fourth decade, and declined after 40 years by a rate of 0.34 ml · min−1 per year toward old age (−29%, p < 0.001). The half-life in young subjects was 9.5 ± 2.0 hours and increased after 30 years toward old age (+26%, p < 0.001). The volume of antipyrine distribution, 0.46 ± 0.12 L · kg−1 in young subjects, decreased after 30 years (−11%). In line with the testosterone content, the decrease in drug metabolism was equal in both sexes.

Conclusion
This study shows a reduction of in vitro and in vivo drug metabolism with age in humans. The data suggest that at least three age groups—young, middle-aged, and elderly—should be included in the evaluation of the pharmacokinetics of a new drug. The reduction of drug metabolism (−30%) after 70 years of age indicates that care is needed in the prescription of drugs for elderly subjects.

Clinical Pharmacology & Therapeutics (1997) 61, 331–339; doi:

Age and cytochrome P450-linked drug metabolism in humans: An analysis of 226 subjects with equal histopathologic conditions

Induction of Liver Growth by Xenobiotic Compounds and Other Stimuli

Binding and transport characteristics for uptake of taurocholic acid by isolated rat liver cells were studied. 1. An adsorption of taurocholate to the cell surface is terminated in less than 15 s. A Ks of 0.55 mM and a total binding capacity of 3.8 nmol/mg cell protein is determined. 2. The rate of uptake of taurocholate follows Michaelis-Menten kinetics with Km = 19 muM and V = 1.7 nmol/mg protein min. 3. There is a broad pH optimum for uptake between pH 6.5 -- 8.0. 4. The activation energy amounts to 29 kcal/mol. At high taurocholate concentration an unusual upward bend is observed in the Arrhenius plot. 5. Taurocholate uptake is competitively inhibited by taurochenodeoxycholate (Ki = 9 muM). It is noncompetitively inhibited by bromosulfophthalein (Ki = 3 muM). 6. At physiological taurocholate concentrations a 200-fold intracellular accumulation of taurocholate is observed. 7. Uptake is inhibited by about 75% by either antimycin A, carbonylcyanide m-chlorophenyl-hydrazone, ouabain. 8. Replacement of extracellular Na+ by either K+ or sucrose results in a 75% decrease of uptake. 9. It is concluded that taurocholate uptake is a carrier-mediated process, and suggested that the energy for intracellular accumulation is made available by cotransport of Na+.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1975.tb02199.x

Uptake of taurocholic acid into isolated rat-liver cells.

Publisher Summary The chapter describes the methods for the elevation of hepatic microsomal mixed function oxidase levels and cytochrome P-450. Many substances have been shown to elevate the content of the mixed function oxidase activity of liver microsomes; these range from barbiturates to tranquilizers, insecticides, and polycyclic hydrocarbons such as 3,4-benzpyrene and 3-methylcholanthrene. The increase in oxidative activity is associated with an increase in the microsomal content of cytochrome P-450. The time necessary to achieve maximal levels of microsomal mixed function oxidase activities varies with the inducer compound used. Methylcholanthrene or benzpyrene treatment exerts a maximal effect within 24 hours. After one injection of DDT, the maximal activity is achieved after 1 or 2 weeks. When phenobarbital is used, maximal enzyme activity toward all substrates is reached after 3-5 days of treatment. Three procedures, which increase the level of liver microsomal mixed function oxidase activity and cytochrome P-450, are described. One method uses Phenobarbital as the inducer; a second uses 3-methyleholanthrene or 3,4-benzpyrene; and the third method employs DDT. The chapter also discusses the preparation of microsomes.

[109] Methods for the elevation of hepatic microsomal mixed function oxidase levels and cytochrome P-450

The influence of phenobarbital on the turnover of hepatic microsomal cytochrome b5 and cytochrome P-450 hemes in the rat.

Heme moieties of microsomal rat liver cytochromes P-450 and b-5 were labeled with.14C-Aminolevulini cacid. The half life of the b-5 heme radioactivity was found to be 45 hrs, that of the P-450 heme radioactivity was 22 hrs.

Helmut Greim

Papers

Uptake of taurocholic acid into isolated rat-liver cells.

[109] Methods for the elevation of hepatic microsomal mixed function oxidase levels and cytochrome P-450

The influence of phenobarbital on the turnover of hepatic microsomal cytochrome b5 and cytochrome P-450 hemes in the rat.

Synthesesteigerung und Abbauhemmung bei der Vermehrung der mikrosomalen Cytochrome P-450 und b-5 durch Phenobarbital

On the problem of possible other forms of cytochrome P450 in liver microsomes