Anders Åström

Characterization of the induction of drug-metabolizing enzymes by 2-acetylaminofluorene

Changes in hepatic drug-metabolizing enzymes after intraperitoneal treatment of rats with 2-acetylaminofluorene have been investigated. This treatment was found to increase microsomal epoxide hydrolase to 762%, cytochrome P-450 to 143%, NADPH-cytochrome c reductase to 160%, cytochrome b5 to 171%, cytoplasmic DT-diaphorase to 229% and soluble glutathione S-transferase activities to 200-250% of control values. These increases were time- and dose-dependent, being maximal after injection of 50 mg 2-acetylaminofluorene/kg body wt. once daily for 5 days. Enzyme markers for the plasma membrane, mitochondria, lysosomes and the soluble cytoplasm were not affected by treatment with 2-acetylaminofluorene. The present study indicates that this induction is different from that obtained with phenobarbital and 3-methylcholanthrene and more closely resembles that seen with trans-stilbene oxide.

Induction of cytochrome P-450 and related drug-metabolizing activities in the livers of different rodent species by 2-acetylaminofluorene or by 3-methylcholanthrene.

In general, large differences in the control levels of different cytochrome P-450-catalyzed activities (aminopyrine N-demethylase, benzo(a)pyrene monooxygenase, ethoxyresorufin O-deethylase, ethoxycoumarin O-deethylase and total 2-acetylaminofluorene metabolism and metabolite pattern) and in the inducibility of these activities in different rodent species (rat, hamster, guinea pig and mouse) and sexes were observed. For all the activities measured the lowest levels were observed in untreated rats. With a few minor exceptions, the only species tested in which cytochrome P-450-catalyzed activities were induced by treatment with 2-acetylaminofluorene was the rat. A larger number of the species tested were susceptible to induction by 3-methylcholanthrene. However, this xenobiotic proved also to induce most potently in the rat. There are relatively large differences between the male and female rat both in terms of control cytochrome P-450-catalyzed activities and in the inducibility of these activities by 2-acetylaminofluorene and 3-methylcholanthrene. In general, both of these xenobiotics proved to be more potent inducers in the female than in the male. Thus, it is quite clear that in quantitative terms the hepatic microsomal cytochrome P-450-catalyzed activities and their inducibility by 2-acetylaminofluorene or 3-methylcholanthrene in the male Sprague-Dawley rat are not representative for other rodent species or even for the female of the same species.

Induction of liver microsomal epoxide hydrolase, UDP-glucuronyl transferase and cytosolic glutathione transferase in different rodent species by 2-acetylaminofluorene or 3-methylcholanthrene

Control activities vary 12-fold for microsomal epoxide hydrolase, two-fold for UDP-glucuronyl transferase and five-fold for cytosolic glutathione (GSH) transferase among the different rodents (rat, hamster, guinea-pig, mouse) examined. For all three enzymes the activities in rat liver are towards the lower values. In these rodents, except for a 100% increase in microsomal epoxide hydrolase in guinea-pig liver, 2-acetylaminofluorene induces the three phase 2 enzymes only in rat. Treatment with 3-methylcholanthrene also produces the largest effects on these three enzyme activities in rat liver; exceptions are its failure to induce microsomal epoxide hydrolase in female rat and the large induction of cytosolic GSH transferase in hamster liver. Quantitatively, hepatic microsomal epoxide hydrolase, UDP-glucuronyl transferase and cytosolic GSH transferase activities, and their inducibility by 2-acetylaminofluorene or 3-methylcholanthrene, in male Sprague-Dawley rats are not representative for other rodent species or even, in all cases, for female rat.

Preparation and characterization of subcellular fractions from the liver of the northern pike, Esox lucius

The present study was designed to prepare and characterize subcellular fractions from the liver of the Northern pike (Esox lucius), with special emphasis on the preparation of microsomal fractions suitable for studying xenobiotic metabolism. The purity of the different fractions obtained by differential centrifugation, as well as the recovery of different organelles, was determined using both enzyme markers and morphological examination with the electron microscope. Attempts were also made to increase the recovery of fragments of the endoplasmic reticulum in the microsomal fraction. Finally, the subcellular distribution of several drug-metabolizing enzymes (cytochrome P-450, benzpyrene monoxygenase, epoxide hydrolase and glutathione transferases) were determined. With the exception of the subcellular distribution of epoxide hydrolase, the results obtained here resemble closely those reported fo rat liver and the microsomal fraction prepared is highly suitable for further studies of drug metabolism in pike liver.

Identification of the forms of cytochrome P-450 induced in rat liver by 2-acetylaminofluorene using immunoblotting and partial purification.

The amounts of 5 different forms of cytochrome P-450 in liver microsomes from rats treated with 2-acetylaminofluorene were determined and compared with the corresponding patterns in microsomes from control, 3-methylcholanthrene- and phenobarbital-treated animals. 2-Acetylaminofluorene was found to increase the amount of cytochromes P-450b + e 10-fold and of cytochrome P-450d 3-fold, while there was a 54% increase in the level of cytochrome P-450 PB/PCN-E. Cytochrome P-450c was increased from a level too low to detect (less than 0.001 pmol/mg protein) to 0.019 pmol/mg protein. These findings were also confirmed by partial purification of cytochromes P-450b + e and c after 2-acetylaminofluorene treatment.

Characterization of the microsomal cytochrome p-450 species induced in rat liver by trans-stilbene oxide

trans-Stilbene oxide differs from the classical inducers of drug-metabolizing enzymes, phenobarbital and 3-methylcholanthrene, in that it induces the so-called phase II activities, epoxide hydrolase and glutathione S-transferase, to a much larger extent than it induces cytochrome P-450. Nonetheless, the level of cytochrome P-450 in liver microsomes from rats treated with trans-stilbene oxide is increased significantly to twice the control value. The existence of a number of different isozymes of cytochrome P-450 has now been clearly demonstrated and in the present study we have posed the question. What form(s) of cytochrome P-450 is induced by trans-stilbene oxide? A number of criteria including substrate specificity, pattern of benzo(a)pyrene metabolism, sensitivity to inhibitors, substrate binding spectra, ethylisocyanide binding spectra, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and crossed immunoelectrophoresis were used to answer this question. It seems clear that trans-stilbene oxide induces the same form(s) of cytochrome P-450 as phenobarbital.

Subcellular and organ distribution of cholesterol epoxide hydrolase in the rat

The subcellular and organ distributions of microsomal epoxide hydrolases measured with cis-stilbene oxide and cholesterol 5,6 alpha-epoxide as substrates have been investigated. These two enzyme activities were found to have essentially the same subcellular distribution, with the highest total and specific activities localized in rough and smooth endoplasmic reticulum. Among the tissues studied (i.e., liver, kidney, lung, testis, spleen, brain and intestinal epithelium), the highest specific activities were recovered in liver microsomes, where the activities were at least 5-fold greater than in any of the other microsomal preparations.