Wendy Burgess

Flavin-containing monooxygenase activity in human liver microsomes.

Human liver microsomal flavin-containing monooxygenase activity has been studied using dimethylaniline N-oxidation and thiobenzamide S-oxidation. Except for one subject, the capacity of human liver microsomes to mediate these reactions were markedly increased at pH 8.4 compared to pH 7.4. The mean dimethylaniline N-oxidase activities at pH 7.4 and 8.4 in the four subjects tested were 2.49 +/- 1.13 and 6.59 +/- 4.04 nmol mg-1 min-1, respectively (mean +/- SD, N = 4). The mean thiobenzamide S-oxidase activities at pH 7.4 and 8.4 were 1.39 +/- 0.51 and 2.74 +/- 1.28 nmol mg-1 min-1, respectively. At pH 7.4, an antibody to the human liver NADPH-cytochrome P-450 reductase inhibited dimethylaniline N-oxidation between 4 and 38%. The same antibody had no effect on this reaction at pH 8.4. Except for one subject, a battery of cytochrome P-450 inhibitors also had little effect on this reaction. Further, preincubating human microsomes at 45 degrees C in the absence of NADPH for 4 min destroyed approximately 90% of the dimethylaniline N-oxidase activity. These data collectively suggested that the flavin-containing mono-oxygenase is the major enzyme mediating this reaction in human liver microsomes. In contrast to dimethylaniline N-oxidation, thiobenzamide S-oxidation was significantly inhibited by the anti-reductase at both pH 7.4 and 8.4, respectively. These data indicate that cytochromes P-450 contribute significantly to this reaction in human liver microsomes.

Metabolic differences in colon mucosal cells

The colonic expression of cytochromes P450 from the CYP1A, CYP3A and CYP4B subfamilies has been characterized in rabbit and human tissues using RNA blotting, immunoblotting, immunohistochemistry and hybridization histochemistry. These studies demonstrate negligible expression of the CYP1A subfamily in either rabbit or human colon. The CYP3A6 gene is expressed in rabbit colon although at markedly reduced levels relative to liver and small intestine. Whilst at least two CYP3A genes are expressed at the mRNA level in human colon tissue from some individuals, no expression was demonstrated in others. Where expression was observed, this expression was continuous throughout the length of the colon. In rabbits, CYP4B1 represents a major colon P450 enzyme, expressed at levels in colon comparable to liver and small intestine. In contrast, the human CYP4B1 gene is expressed at low levels in some individuals. These studies highlight individual differences in the expression of cytochrome P450 enzymes of importance in procarcinogen metabolism.

Species-specific expression of CYP4B1 in rabbit and human gastrointestinal tissues.

CYP4B1 is a P450 enzyme displaying tissue and species specific regulation. The rabbit CYP4B1 enzyme exhibits activity towards procarcinogenic aromatic amines. In the present study, CYP4B1 expression has been characterized in rabbit tissues using histological techniques, Northern blotting and Western blotting. Similar analyses were attempted using available human tissues. CYP4B1 mRNA and protein was demonstrated throughout the rabbit small intestine and colon. A unique 1.8 kb transcript, that is smaller than the transcript found in other tissues, was detected in rabbit stomach with a CYP4B1 specific RNA probe. No CYP4B1 protein was detected in this tissue. In rabbit liver, CYP4B1 was induced by phenobarbital primarily in zone 1 hepatocytes (periportal). In humans, CYP4B1 expression was demonstrated at low levels in human colon using in situ hybridization but not in liver or the small intestine. All rabbit gastrointestinal tissues other than stomach possess a high capacity for the activation of 2-aminofluorene compatible with CYP4B1 expression. In contrast, no activity was observed in human gastrointestinal microsomes. The present study therefore shows that CYP4B1 is an abundant P450 in the rabbit gastrointestinal tract and identifies species-specific differences in CYP4B1 expression and function.

Identification and quantitation in human liver of cytochromes P-450 analogous to rabbit cytochromes P-450 forms 4 and 6

1. Polyclonal antibodies raised against rabbit liver cytochrome P-450 isozymes form 4 and 6 have been used to probe human liver microsomes for analogous proteins using the Western blot technique. 2. Anti-Form 4 IgG recognized a protein in human liver microsomes from six subjects of identical molecular weight to purified rabbit liver cytochrome P-450 Form 4. 3. The equivalent content of cytochrome P-450 Form 4 in the same microsomes ranged from 1.1 to 9.1 pmol per mg protein. 4. Anti-Form 6 IgG recognized a protein in human liver microsomes from the same six subjects of slightly higher molecular weight than purified rabbit cytochrome P-450 Form 6. 5. The equivalent content of cytochrome P-450 Form 6 in the above microsomes ranged from 1.6 to 3.8 pmol per mg protein. 6. No significant correlations were observed between equivalent cytochrome P-450 Forms 4 and 6 content and 2-acetylaminofluorene N-hydroxylase, aminopyrine N-demethylase, benzyprene and aniline hydroxylase activities in liver microsomes from the six subjects tested.

Activation of the food-derived mutagen 2-amino-3-methylimidazo[4,5-ƒ]quinoline by human-liver microsomes

The ability of human-liver microsomes to metabolically activate the food-derived heterocyclic amine, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and the model mutagen, 2-aminofluorene (AF), has been investigated using Salmonella typhimurium TA98. In 6 subjects tested the number of revertants produced by 0.1 micrograms IQ per mg microsomal protein varied from 11, 830 +/- 320 to 42, 830 +/- 290 (mean +/- SD). With the same livers and a dose of 10 micrograms AF per plate the number of revertants varied from 15,770 +/- 1600 to 29,380 +/- 810 per mg microsomal protein. Metyrapone and alpha-naphthoflavone caused differential inhibition of the mutagenesis of both IQ and AF indicating the involvement of different forms of cytochrome P450 in the metabolic activation of these amines in human-liver microsomes. In presence of human-liver microsomes IQ produced no detectable increase in mutations at the hypoxanthine phosphoribosyl transferase locus in lymphocytes and caused no increase in micronuclei formation at realistic exposure levels.

Effect of dexamethasone on cytochrome P-450 mediated metabolism of 2-acetylaminofluorene in cultured rat hepatocytes.

The metabolism of 2-acetylaminofluorene (AAF) to its six oxidative metabolites has been used to investigate the effect of dexamethasone on cytochrome P-450 activity in cultured rat hepatocytes. In control hepatocytes the metabolism of AAF to its 1-, 5-, 7-, 9- and N-hydroxylated metabolites rapidly declined in culture over the first 24 hr while 3-hydroxylation remained relatively constant. These activities either remained unchanged or increased slightly during the next 48 hr in culture. The addition of dexamethasone (100 nM) to the culture medium had little effect in arresting the initial decline but by 72 hr the 7-, 5- and 3-hydroxylations increased to values 2.5, 16 and 21 times the respective 24-hr values. The inductive effect of dexamethasone on the 3- and 5-hydroxylations of AAF was maximal at 100 nM whereas the 7-hydroxylation increased linearly as a function of the dexamethasone concentration up to 1 microM. Cortisol and corticosterone and the non-glucocorticoids fluoxymesterone and methyltestosterone induced a pattern of AAF metabolism resembling that in dexamethasone-treated cultures, suggesting that a range of steroids not restricted to glucocorticoids may induce multiple cytochrome P-450 isozymes via related mechanisms. Pregnenolone 16 alpha-carbonitrile induced only the 7-hydroxylation of AAF probably reflecting induction of cytochrome P-450p. While dexamethasone was a strong inducer of the 3- and 5-hydroxylations of AAF in hepatocyte culture, assay of these activities in freshly isolated cells after in vivo treatment with dexamethasone showed a strong induction of 7-hydroxylation but only small effects on 3- and 5-hydroxylations. Indeed the profile of AAF metabolism induced in culture by dexamethasone resembles more closely the profile induced by 3-methylcholanthrene in vivo. These data suggest that factors yet to be identified strongly influence the steroid-induced pattern of cytochrome P-450 gene expression.

Immunochemical and catalytical characterization of the human liver NADPH-cytochrome P450 reductase.

1. The NADPH‐cytochrome P450 reductases (EC 1.6.2.4) from human and rabbit liver have been purified to electrophoretic homogeneity. The human reductase had an apparent monomeric molecular weight of 77 500 and the rabbit enzyme of 76 500.

Heterologous systems for expression of mammalian sulfotransferases

This paper describes the use of both mammalian and bacterial expression systems as tools to study the structural and functional relationships of proteins encoded by cDNAs to both rat and human aryl sulfotransferases. In particular, we describe the use of the mammalian COS cell system for functional expression studies, and the use of Escherichia coli for the expression and purification of a sulfotransferase fusion protein suitable as an antigen for the generation of sulfotransferase antibodies.