Liver-cell microsomal enzymes

The enzyme induction properties of PBDEs have been less studied than for other structurally similar chemicals, but the existing information suggests that they can be classified as mixed-type inducers of hepatic microsomal monooxygenases (Damerud et al. 2001 de Wit 2002 Hardy 2002b). Few studies have examined the structure-induction relationships for PBDEs. Chen et al. (2001) examined the ability of 12 PBDE congeners and 3 commercial mixtures to induce EROD activity in chick and rat hepatocytes, in liver cell lines from rainbow trout, rat, and human, and in a human intestinal cell line. The number of... 

Amine oxidation. As well as the microsomal enzymes involved in the oxidation of amines, there are a number of other amine oxidase enzymes, which have a different subcellular distribution. The most important are the monoamine oxidases and the diamine oxidases. The monoamine oxidases are located in the mitochondria within the cell and are found in the liver and also other organs such as the heart and central nervous system and in vascular tissue. They are a group of flavoprotein enzymes with overlapping substrate specificities. Although primarily of importance in the metabolism of endogenous compounds such as 5-hydroxy try pt-amine, they may be involved in the metabolism of foreign compounds. 

The most important enzyme involved in bio transformation is cytochrome P-450, which catalyzes many phase 1 reactions. This enzyme is located primarily in the SER (microsomal fraction) of the cell and is especially abundant in liver cells. Cytochrome P-450 primarily catalyzes oxidation reactions and consists of many isoforms (isozymes). These isoenzymes have overlapping substrate specificities. The most important subfamily in humans is CYP3A4, although there is considerable variation in CYP3A4 expression between individuals. 

Phase I enzymes are located primarily in the endoplasmic reticulum of cells. These enzymes are membrane bound within a lipoprotein matrix and are referred to as microsomal enzymes. This is in reference to the subcellular fraction isolated by differential centrifugation of a liver homoge-... 

Pharmaceutical sponsors now frequently conduct in vitro studies in the preclinical phase of drug development programs to assess the contribution of CYP or other enzymes to the metabolic elimination of an investigational drug and the ability of an investigational drug to inhibit specific metabolic pathways. The utility of these studies has been enhanced by the availability of specific enzyme preparations, microsomal preparations, and liver cell preparations, together with... 

The transfer of labeled amino acids from aminoacyl sRNA to purified rat-liver ribonucleoprotein particles has been shown to require GTP, and a soluble portion (pH 5 Supernatant) of the cell. An enzyme fraction, aminoacyl transferase (or polymerase) I, purified from the pH 5 Supernatant was found to catalyze the transfer of amino acid to protein with microsomes, but not with the more purified ribonucleoprotein particles (ribosomes). When transferase I was supplemented with glutathione and a microsomal extract, microsomal aminoacyl transferase (or polymerase) H, transferring activity was restored. Since the pH 5 Supernatant was active in catalyzing the transfer of amino acids from sRNA to ribosomal protein, it was concluded that both transferring activities were present in this crude fraction. Resolution of the two activities from the pH 5 Supernatant fraction was obtained by salt-fractionation procedures. Neither enzyme fraction was active when incubated individually or with glutathione, but together in the presence of... 

Adaptive Enzyme Theory. The aliesterases are largely found in the microsomes of rat liver cells (44). Recently Hart and Fouts (51,52, 67-69) have presented evidence that in vivo administration of chlordan or chemically related DDT stimulates the activity of hepatic microsomal drug-metabolizing enzymes, as evidenced by proliferation of smooth-surfaced endoplasmic reticulum (SER) which was first noted with phenobarbital. Several reviews of hepatic drug metabolism... 

If liver cells (hepatocytes) are isolated and grown in culture, drugs are exposed to a similar array of enzymes. Because CYP enzymes are bound to the internal membrane fraction of hepatocytes, the liver can be homogenized and a preparation of vesicles of the hepatocyte endoplasmic reticulum called microsomes can be incubated with drug molecules. This preparation suffers because many of the soluble Phase II conjugation enzymes that are found in the cellular cytoplasm are lost. An alternative method for measuring microsomal metabolism involves isolation of the so-called S9 fraction , which includes the cytosolic soluble conjugation enzymes. 

Principle After the test substance has been demethylated by the microsomal enzyme system of the SER (cytochrome P 450), the labelled carbon atoms are catabolized to C02. The velocity of demethylation can be measured by determining the C02 value in expired air. Catabolization of aminopyrine is enhanced with elevated activity of the cytochrome P 450 and reduced accordingly due to the loss of liver cell volume. The metabolization is independent of perfusion. Exposure to radiation is minimal. 

Vitamin K is critical to (he formation of clotting factors VII. IX. and X. These factors are glycoproteins that h.avc y-vaiboxyglutamic acid residues at the N-terminal end of (he ptptide chain. The enzyme involved in forming an active prothrombin is a vitamin K-dcpendcn( carboxyla.se located in the microsomal fraction of liver cells. It has been sug-... 

Fig. 12. Paraffin sections of rat liver, periodic acid Schiff stain X22. A, normal liver of control animal killed 24 h after injection of 1 ml of seasame oil i.p. B, extensive centrolobular necrosis of parenchymal cells in rat killed 24 h after administration of high dose of bromobenzene (0.2 ml i.p.). C, Liver 24 h after lower dose of bromobenzene (0.03 ml i.p.). The centrolobular areas exhibit some small patches of round cell infiltration and decreased glycogen staining in the cytoplasm after hepatocytes but little necrosis. D, Extensive centrolobular necrosis after administration of the same low dose of bromobenzene (0.03 ml i.p.) to a rat treated with pheno-barbital (20 mg/kg) for 3 days in order to induce hepatic microsomal enzymes. After Brodie et al...

Microsomal oxidative reactions constitute the most prominent phase I biotransformation pathway for a wide variety of structurally unrelated drugs (Table 1.4). Some drugs (e.g. amphetamine, diazepam, propranolol, lignocaine) simultaneously undergo more than one type of microsomal-mediated oxidative reaction. Microsomal enzymes are located primarily in liver cells, where they are associated with the smooth-surface (without ribosomes) endoplasmic reticulum (Fouts, 1961). Lipid solubility is a prerequisite for drug access to the... 

Elevated activities of GGT are found in the sera of patients with alcoholic hepatitis and in the majority of sera from people who are heavy drinkers. Increased concentrations of the enzyme are also found in serum of subjects receiving anticonvulsivant drugs such as phenytoin and phe-nobarbital. Such an increase of GGT activity in serum may reflect induction of new enzyme activity by the action of the alcohol and drugs and/or their toxic effects on microsomal structures in liver cells. 

These studies can be done with liver microsomes, hepatocytes, and recombinant preparations of CYP enzymes. Microsomes are membrane fragments prepared from homogenized liver, while hepatocytes are liver cells that have been collected from collagenase-perfused liver. These are then cocultured with collagen, and fibroblasts... 

On the other hand, it is also the case that more compounds are identified that are metabolized extensively in the intact liver, but only trace amounts of metabolites are produced in hepatocytes or microsome preparations. When this situation occurs, it is not difficult to conjure up a host of possible explanations. For example, the lack of metabolism in hepatocytes, but not in whole liver, may be due to metabolism in other liver cells such as Kupffer cells or cells of the biliary tree. The inability to remove metaboUtes via the bile may cause feedback inhibition/toxicity [5]. The cells may have lost key substrates or enzymes, and intermediary metabolism may be depleted despite an intact plasma membrane with high viability measurements. For example, protein synthesis may be zero, but the cell membrane is still intact and able to exclude dyes. 

Salhab AS, James MO. Wang SL, et al. 1987. Formation of benzo( a)pyrene-DNA adducts by microsomal enzymes Comparison of maternal and fetal liver, fetal hematopoietic cells and placenta. 

---