Dorothea Sesardic

The specificity of inhibition of debrisoquine 4-hydroxylase activity by quinidine and quinine in the rat is the inverse of that in man

The kinetics of inhibition of debrisoquine 4-hydroxylase activity by quinidine and quinine in rat and human liver microsomes have been compared. Quinidine is a potent inhibitor of debrisoquine 4-hydroxylase activity of human liver (IC50: 3.6 microM). However, its stereoisomer, quinine, is some 60 times less potent (IC50:223 microM). Both compounds are able to inhibit greater than 95% of 4-hydroxylase activity. In rat liver microsomes quinine is approximately 50 times more potent an inhibitor (IC50:2.4 microM) than quinidine (IC50:137 microM). Again, 4-hydroxylase activity is inhibited by greater than 95%. Inhibition of debrisoquine 4-hydroxylase activity by both quinine and quinidine in human and rat liver is competitive. Values of Ki for quinidine in human and rat were 0.6 microM and 50 microM, whereas with quinidine the Ki values were 13 microM and 1.7 microM, respectively. The data in this paper are consistent with 4-hydroxylation of debrisoquine in both rat and human liver catalysed by a specific form of cytochrome P-450. Although both quinidine and quinine are competitive inhibitors of debrisoquine 4-hydroxylase activity in rat and man, their potency is reversed. This suggests that the nature of the active site of cytochrome P-450dbl differs between the two species, and indicates that data on the specificity of this isoenzyme in the rat should be extrapolated to man with extreme caution.

Immunocytochemical localization of cytochrome P-450 in hepatic and extra-hepatic tissues of the rat with a monoclonal antibody against cytochrome P-450 c

The cellular distribution of cytochrome P-450 has been studied in the liver and a number of extrahepatic tissues in the rat by immunocytochemistry, using an antibody raised against cytochrome P-450 form c. Immunoreactive cytochrome P-450, most probably form c, was found in the proximal tubules of the kidney, in the Clara cells of the lung, and in the olfactory epithelium and Bowmans glands of the olfactory tissue, in addition to its location in the liver. Immunoreactive cytochrome P-450 was not found in the small intestine, the testes or the adrenal gland, although these organs are known to contain isoenzymes of cytochrome P-450. The use of antibody titration enabled the effects of phenobarbitone, beta-naphthoflavone and clofibrate on the content and distribution of immunoreactive cytochrome P-450 to be studied in both the liver and in the other organs discussed. Phenobarbitone induces epitope-specific cytochrome P-450 in the centrilobular cells of the liver but has no effect in any of the other tissues studied. Clofibrate is without effect on the levels of immunoreactive cytochrome P-450 in any of the tissues studied. In contrast, beta-naphthoflavone induces immunoreactive cytochrome P-450 in the periportal region of the liver, and also in the Clara cells of the lung, in the enterocytes of the small intestine and in the proximal tubules of the kidney. Of all of the tissues studied, in which immunoreactive cytochrome P-450 could be detected, only the olfactory epithelium failed to undergo enzyme induction following treatment with beta-naphthoflavone.

The inducibility and catalytic activity of cytochromes P450c (P450IA1) and P450d (P450IA2) in rat tissues

The metabolism of phenacetin is primarily by cytochrome P450-dependent O-deethylation to paracetamol (POD activity). In untreated rats, microsomal POD activity is detectable in both the liver and lung, but not in the small intestine or the kidney. POD activity is highly induced in both hepatic and extrahepatic tissues of the rat following treatment with polycyclic aromatic hydrocarbons such as 3-methylcholanthrene (MC). Only cytochrome P450c (P450IA1) is inducible in rat extrahepatic tissues by MC or isosafrole, whereas in the liver both cytochromes P450c and P450d (P450IA2) are inducible by these compounds. Specific antibodies to cytochromes P450c and P450d were used to study the expression and function of these two related isoenzymes in rat liver and extrahepatic tissues before and after induction with MC. Whereas cytochrome P450d is responsible for all of the high affinity POD activity in hepatic microsomal fractions of both untreated and MC treated rats, this activity is mediated only by P450c in microsomal fractions from extrahepatic tissues following MC treatment. POD activity of microsomal fractions from lung of untreated rats was not mediated by either cytochrome P450c or P450d.

High affinity phenacetin O-deethylase is catalysed specifically by cytochrome P450d (P450IA2) in the liver of the rat

Phenacetin is metabolized primarily by O-deethylation to paracetamol (POD activity), a reaction catalysed by cytochrome P450. The high affinity component of POD activity is inducible in rat liver by treatment of the animals with polycyclic aromatic hydrocarbons. Following treatment with hydrocarbons such as 3-methylcholanthrene (MC) and isosafrole (ISF) both cytochromes P450c (P450IA1) and P450d (P450IA2) are also induced in rat liver. Studies with the reconstituted enzymes have shown that both forms of P450 catalyse phenacetin O-deethylation at rates that exceeded that of the high affinity component of activity of hepatic microsomal preparations from 3-methylcholanthrene-treated rats (at 4 microM phenacetin: P450c, 440 +/- 40 pmol/nmol/min; P450d, 1030 +/- 10 pmol/nmol/min; microsomal fraction, 163 pmol/mg/min). Specific inhibitory antibodies (both monoclonal and monospecific polyclonal) were used to define the specificity of microsomal POD activity. These studies have shown that hepatic high affinity POD activity is exclusively catalysed by cytochrome P450d in both untreated rats and in rats pretreated with MC.

Immunohistochemical localization of cytochrome P450b/e in hepatic and extrahepatic tissues of the rat

The cellular distribution of cytochrome P450b/e has been studied in the liver and a number of extrahepatic tissues in the Wistar rat by immunocytochemistry, using two monoclonal antibodies, 10/1 and 1/4, raised against the major phenobarbitone-inducible form of cytochrome P450. The specificity of these antibodies was verified by a number of techniques. In Western blotting, both antibodies recognised a single band of Mr 52,000 in liver microsomes from rats pre-treated with phenobarbitone, acetone and isosafrole, which co-migrated with a purified preparation of cytochrome P450b. In untreated rats, a weak specific immunostain was visible across the whole of the liver lobule, with stronger staining in a few hepatocytes around the central vein. Immunoreactive cytochrome P450b/e was also found in the Clara cells of the lung and in the enterocytes of the small intestine, with maximal staining at the tips of the villi. No immunoreactive cytochrome P450b/e was detected in kidney, testis or pancreas. Phenobarbitone treatment resulted in a strong, specific immunostain of all hepatic centrilobular cells, antibody titration indicating that induction of cytochrome P450b/e had occurred. Marked induction was also found in the enterocytes of the small intestine, a strong immunostain being apparent in cells along the length of the villus, from crypt to tip. No induction was apparent in lung, kidney, testis or pancreas. Immunoquantification of cytochrome P450b/e, by densitometric scanning of dot blots probed with a monoclonal antibody, 10/1, confirmed these observations. Thus, there are very marked, specific inter- and intra-tissue differences in both the expression and inducibility of cytochrome P450b/e in the rat.

Antibodies to a synthetic peptide that react specifically with a common surface region on two hydrocarbon-inducible isoenzymes of cytochrome P-450 in the rat

An antibody that reacts with two hydrocarbon-inducible isoenzymes of rat cytochrome P-450 (c and d) in the rat was produced by immunising with a synthetic peptide, Leu-Ile-Ser-Lys-Phe-Gln-Lys-Leu-Met, which has the same primary structure as that of a region of both of these isoenzymes. There was no crossreactivity with hydrocarbon-inducible isoenzymes in liver microsomes from rabbit, mouse or in man. Nor was there any crossreactivity detected with liver microsomes from uninduced rats, or rats induced with phenobarbitone or isonicotinic acid hydrazide. This is consistent with the primary structure of these isoenzymes in the regions aligned with amino acids 174-182 (the immunising peptide) in rat isoenzyme c and demonstrates the ability to produce antibodies of defined specificity against isoenzymes of cytochrome P-450 by using synthetic peptide. As the antibody preparation is able to bind to isoenzymes c and d in their native conformations, either as partially purified enzymes, or in microsomes, it is suggested that this region is present on the surface of these cytochromes P-450.

A monoclonal antibody raised to rat liver cytochrome P-448 (form c) which recognises an epitope common to many other forms of cytochrome P-450

A murine monoclonal antibody has been raised against a partially purified preparation of hepatic cytochrome P-448 (form c) from beta-naphthoflavone-treated rats. The monoclonal origin of the antibody was established by limiting dilution culture and isoelectricfocusing. The antibody has been designated 3/4/2. It reacts with apparently homogeneous cytochrome P-448 from rat liver in solid phase assay. It also cross reacts with a number of other cytochromes P-450, from rat and rabbit. In addition, a positive reaction was obtained with microsomal fractions from a variety of species, including man. None of the species tested was negative. The antibody does not react appreciably with purified haemoproteins other than cytochromes P-450. Antibody 3/4/2 is not inhibitory, either in reconstituted systems or with intact microsomal fraction. However, evidence was obtained that the antibody does cause some perturbation of the tertiary structure of the apoprotein at or near the haem.

Identification of the epitope of a monoclonal antibody which binds to several cytochromes P450 in the CYP1A subfamily

The monoclonal antibody, 3/4/2, which was raised against purified rat cytochrome P450 isoenzyme 1A1 (CYP1A1) binds to cytochromes P4501A in many species. It was shown by immunoblotting that the antibody binds to CYP1A1 in microsomal fractions prepared from rat, mouse, rabbit, hamster and human. The antibody also binds to cytochrome P450 isoenzyme 1A2 in microsomal fractions prepared from rabbit and human, but not rat or mouse. Using purified isoenzymes in an enzyme-linked immunosorbent assay it was found that the affinity of binding to the two rabbit hydrocarbon-inducible isoenzymes is reduced compared with that for rat CYP1A1. Binding is not affected by denaturation of the antigens. The effects of chemical and enzymatic treatments on rat CYP1A1 showed that the epitope contains a trypsin-sensitive site that includes arginine, but lacks lysine. The epitope does not contain methionine, cysteine, aspartic acid or glutamic acid residues. In addition, digestion of the protein with cyanogen bromide produces a fragment of Mr 20,000 which contains the antibody binding site. By comparing the cross-reactivity of the antibody with the primary structures of CYP1A1 and 1A2 from the rat, mouse, rabbit and human, and by considering the results of the chemical and enzymatic treatments, it was possible to deduce the likely location and structure of the binding site of 3/4/2 on members of the CYP1A subfamily. It is concluded that the epitope for this antibody is Phe-Arg-His-Ser-Ser-Phe, which lies at positions 380-385 in rat CYP1A1. Further, it is predicted from a model of the tertiary structure of eukaryotic cytochrome P450 that a part of this binding site lies within a helix in the native protein.