Aryl hydrocarbon or dioxin receptor: biologic and toxic responses.

1. The AhR represents a ligand-activated transcription factor. Receptor agonists include planar aromatic compounds, a variety of heterocyclic plant constituents, and PCDD/PCDF. The latter lead to persistent activation of the receptor due to their strong binding affinity and long biologic half-life of over 10 years in human blood and fat. Practically every person on earth is exposed to these compounds via the diet (> 90%) and by high concentrations in mother's milk. PCDD/PCDF produced toxic responses in exposed people (primarily chloracne and immunosuppression) in the past. However, the present PCDD/PCDF levels (basal levels) in the general population are below those warranting toxicologic concern. 2. The AhR has been characterized as a helix-loop-helix transcription factor related to the Drosophila developmental genes sim and per. The cytosolic form of the receptor is present as an inactive complex with two subunits of HSP90. After ligand binding HSP90 is released and the receptor enters the nucleus as a heterodimer together with a related protein ARNT. It binds with high affinity to certain enhancer elements in the upstream region of several genes such as cytochrome P4501A1 (CYP1A1). The AhR transcriptionally activates several drug-metabolizing enzymes and proteins involved in growth/differentiation, such as the plasminogen activator inhibitor PAI-2 and IL-1 beta. In addition, it modulates the action of a number of other nuclear transcription factors such as receptors of the steroid hormone receptor superfamily and of cell surface receptors such as EGF. With the exception of CYP1A1 induction, little is known about the mechanism of transcriptional activation of the AhR-controlled genes. Many AhR-modulated biologic responses (such as modulation of the estrogen and EGF receptor) appear to be indirect. 3. Persistent activation of the AhR is probably responsible for toxic responses in experimental animals and humans. They are markedly tissue and species specific. In rodents a wasting syndrome, immunosuppression, teratogenicity, chloracne, and carcinogenicity/tumor promotion have been well studied. There is good evidence for an involvement for the AhR in these responses. However, the chain of events from receptor activation to the diverse toxic endpoints is largely unknown. Alteration of growth and differentiation of epithelial tissues may underlie most of the toxic responses. A lot has already been achieved, mostly by characterizing the AhR and transcriptional activation of CYP1A1. Still more work lies ahead of us, for example, elucidation of the physiologic roles of the AhR and of the chains of events from receptor activation to the various biologic and toxic endpoints.

Increased uridine diphosphate-glucuronyltransferase activity in preneoplastic liver nodules and Morris hepatomas.

In preneoplastic rat liver nodules produced by 2-acetylaminofluorene, certain uridine diphosphate-glucuronyltransferase (UDP-GT) activities, which are ascribed to a distinct enzyme form, were selectively increased (5-fold). This enzyme form, operationally termed UDP-GT1, accepts 1-naphthol,4-methylumbelliferone, and 3-hydroxybenzo(a)pyrene as substrates and is chiefly inducible in liver by 3-methylcholanthrene-type inducers. Glucuronidation of other substrates (morphine, 4-hydroxybiphenyl, chloramphenicol, bilirubin, and estrone) was only slightly enhanced or decreased in nodular tissue. Differentially increased UDP-GT1 activities were also found in Morris hepatomas 9121 and 7777. Rabbit antibodies to rat liver UDP-GT1, purified from 3-methylcholanthrene-treated rats, demonstrated immunological similarity between the enzymes from liver, nodular tissue, and Morris hepatoma 9121. Rocket immunoelectrophoresis ascertained that enhanced enzyme activity in nodular tissue reflected an increased level of enzyme protein. Increased activity of UDP-GT1 together with decreased cytochrome P-450-dependent monooxygenase may contribute to the resistance of preneoplastic hepatocytes to the cytotoxic actions of chemical carcinogens.

Persistently increased expression of a 3-methylcholanthrene-inducible phenol uridine diphosphate-glucuronosyltransferase in rat hepatocyte nodules and hepatocellular carcinomas.

Increased UDP-glucuronosyltransferase in rat hepatocyte nodules and hepatocellular carcinomas produced by feeding 2-acetylaminofluorene or N-nitrosomorpholine was studied using isozyme-selective substrates, antibodies, and DNA probes. UDP-glucuronosyltransferase (UDP-GT) activities toward 4-methylumbelliferone, 1-naphthol, and benzo[a]pyrene-3,6-quinol were reversibly increased by short term feeding of 2-acetylaminofluorene but were persistently increased in hepatocyte nodules and differentiated hepatocellular carcinomas. Immunoblot analysis revealed that short term feeding of 2-acetylaminofluorene increased a Mr 55,000 polypeptide corresponding to the previously characterized UDP-GTI or phenol UDP-GT. However, in some hepatocyte nodules and hepatocellular carcinomas either the Mr 55,000 or a new Mr 53,000 polypeptide was preferentially increased, suggesting heterogeneous UDP-GT forms in liver nodules and carcinomas. Northern blot hybridization with a synthetic DNA probe to phenol UDP-GT demonstrated increased levels of mRNA in liver nodules. The results suggest persistently increased expression of at least two phenol UDP-GT enzyme forms in hepatocyte nodules, which may contribute to the toxin-resistance phenotype frequently observed at cancer prestages.

The influence of environmental and genetic factors on CYP2D6, CYP1A2 and UDP-glucuronosyltransferases in man using sparteine, caffeine, and paracetamol as probes.

The impact of gender, use of oral contraceptive steroids (OCS), coffee consumption and of smoking on the metabolism of sparteine, caffeine, and paracetamol was studied in 194 randomly selected subjects (98 male and 95 female). Thirty-eight of the male volunteers were cigarette smokers, 40 of the female subjects were smokers and/or users of OCS. The metabolic ratio of sparteine oxidation (MRs) showed a trimodal distribution. 7.7% of the subjects had a MRs > 20 and thus were poor metabolizers (PMs). Within the extensive metabolizer (EM) subjects, a distinct subgroup accounting for 11% was observed with 20 > MRs > 1.2. Six of the 15 phenotypical PMs were heterozygous EMs by genotyping. This indicates the existence of one or several CYP2D6 mutations which cannot be identified by the currently employed genotyping methods. In each subgroup, i.e. smokers/OCS and non-smokers/non-OCS, the cumulative frequency distribution of the heterozygous (wt/B) phenotype caused a shift to higher MRs compared with the wild-type homozygotes (wt/wt). Thus, for the in vivo activity of CYP2D6, genetic determinants prevail over environmental factors. Smoking, use of oral contraceptive steroids, caffeine consumption, or gender had no influence on sparteine metabolism. The distribution of the paracetamol glucuronide/paracetamol metabolic ratio appeared to be unimodal although skewed. Glucuronidation capacity was clearly affected by gender, OCS use and smoking. It was higher in male than in female subjects. Male smokers had the highest, and female non-smokers/non-OCS users the lowest metabolic ratio. CYP1A2 activity, as determined by a caffeine metabolic ratio ((AFMU + 1X + 1U)/1, 7U), was multimodally distributed and was clearly increased in smokers. It was significantly correlated to paracetamol glucoronidation in male heavy smokers (r=0.85), suggesting an element of co-regulation of CYP1A2 and of paracetamol conjugating UDP-glucuronosyltransferase isozymes, including UGTI.6.

Ethnic variability in the allelic distribution of human aryl hydrocarbon receptor codon 554 and assessment of variant receptor function in vitro.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcriptional regulator of several genes including the cytochrome P4501 (CYP1) family as well as genes encoding factors involved in cell growth and differentiation. In mice, several polymorphic forms of the AHR are known, some of which have altered affinity for toxic and carcinogenic ligands. Remarkably little genetic variation has been detected in the human AHR gene. In studies on human AHR, Kawajiri et al. (Pharmacogenetics 1995; 5:151-158) reported a variation at codon 554 that results in an amino acid change from arginine to lysine; the frequency of the variant allele in a Japanese population (n = 277) was 0.43. We investigated the Lys554 allele in 386 individuals of various ethnic origins and found the frequency to be: 0.58 in Ivory Coast Africans (n = 58); 0.53 in a mixed African group (n = 20); 0.39 in Caribbean-Africans (n = 55); 0.32 in Canadian Chinese (n = 41); 0.14 in North American Indians (n = 47); 0.12 in French Canadian Caucasians (n = 20); 0.11 in a mixed ethnicity North American group (n = 45); 0.09 in Canadian Inuits (n = 22); and 0.07 in German Caucasians (n = 78). We expressed the human Lys554 allele in an in-vitro transcription-translation system and found that the receptor bearing the R554L substitution had an equivalent ability to that of the wild-type receptor to bind to a dioxin-responsive element following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The Lys554 allele also was equivalent to the wild-type receptor at stimulating CYP1A1 mRNA expression when transfected into TCDD-treated receptor-deficient mouse Hepa-1 cells. It is not yet known if any of the wide variations in allele frequency at codon 554 are related to ethnic differences in susceptibility to adverse effects of environmental chemicals.

The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence.

This review represents an update of the nomenclature system for the UDP glucuronosyltransferase gene superfamily, which is based on divergent evolution. Since the previous review in 1991, sequences of many related UDP glycosyltransferases from lower organisms have appeared in the database, which expand our database considerably. At latest count, in animals, yeast, plants and bacteria there are 110 distinct cDNAs/genes whose protein products all contain a characteristic 'signature sequence' and, thus, are regarded as members of the same superfamily. Comparison of a relatedness tree of proteins leads to the definition of 33 families. It should be emphasized that at least six cloned UDP-GlcNAc N-acetylglucosaminyltransferases are not sufficiently homologous to be included as members of this superfamily and may represent an example of convergent evolution. For naming each gene, it is recommended that the root symbol UGT for human (Ugt for mouse and Drosophila), denoting 'UDP glycosyltransferase,' be followed by an Arabic number representing the family, a letter designating the subfamily, and an Arabic numeral denoting the individual gene within the family or subfamily, e.g. 'human UGT2B4' and 'mouse Ugt2b5'. We recommend the name 'UDP glycosyltransferase' because many of the proteins do not preferentially use UDP glucuronic acid, or their nucleotide sugar preference is unknown. Whereas the gene is italicized, the corresponding cDNA, transcript, protein and enzyme activity should be written with upper-case letters and without italics, e.g. 'human or mouse UGT1A1.' The UGT1 gene (spanning > 500 kb) contains at least 12 promoters/first exons, which can be spliced and joined with common exons 2 through 5, leading to different N-terminal halves but identical C-terminal halves of the gene products; in this scheme each first exon is regarded as a distinct gene (e.g. UGT1A1, UGT1A2, ... UGT1A12). When an orthologous gene between species cannot be identified with certainty, as occurs in the UGT2B subfamily, sequential naming of the genes is being carried out chronologically as they become characterized. We suggest that the Human Gene Nomenclature Guidelines (http://www.gene.acl.ac.uk/nomenclature/guidelines.html++ +) be used for all species other than the mouse and Drosophila. Thirty published human UGT1A1 mutant alleles responsible for clinical hyperbilirubinemias are listed herein, and given numbers following an asterisk (e.g. UGT1A1*30) consistent with the Human Gene Nomenclature Guidelines. It is anticipated that this UGT gene nomenclature system will require updating on a regular basis.

Radiation inactivation analysis of microsomal UDP-glucuronosyltransferases catalysing mono- and diglucuronide formation of 3,6-dihydroxybenzo(a)pyrene and 3,6-dihydroxychrysene.

Indirect evidence has suggested that multiple subunits of microsomal UDP-glucuronosyltransferases (UGTs) are involved in diglucuronide formation of diphenols of polycyclic aromatic hydrocarbons (Bock et al., Mol Pharmacol 42: 613-618, 1992). To substantiate this suggestion functional target sizes of UGTs catalysing these reactions were determined in microsomes in situ by radiation inactivation analysis. Target sizes of UGTs catalysing the glucuronidation of 1-naphthol and 6-hydroxychrysene were found to be 91 +/- 29 and 120 +/- 27 kDa, respectively. However, target sizes for mono- and diglucuronide formation of 3,6-dihydroxybenzo(a)pyrene were 118 +/- 33 and 218 +/- 24 kDa, respectively. Similarly, using 3,6-dihydroxychrysene as substrate target sizes of 109 +/- 21 and 101 +/- 23 kDa were found for 6-O-monoglucuronide and 3-O-monoglucuronide formation and a target size of 192 +/- 34 kDa observed for diglucuronide formation. Based on subunit molecular masses of 50-60 kDa for UGTs, these results suggest that UGTs involved in monoglucuronide formation of phenols may function as dimers. In contrast, UGTs involved in diglucuronide formation of diphenols of polycyclic aromatic hydrocarbons may function as tetramers in microsomes in situ.

Zinc increases EGF-stimulated DNA synthesis in primary mouse hepatocytes. Studies in tumor promoter-treated cell cultures.

To investigate factors influencing cell proliferation, cells are often cultured in serum-free medium. In the present study it is shown that addition of zinc chloride (40 microM) to primary mouse hepatocytes, cultured in Dulbecco's minimal essential medium, markedly enhanced growth factor (EGF)-stimulated [3H]thymidine incorporation into DNA. Treatment of cell cultures with phenobarbital or 3,4,3',4'-tetrachlorobiphenyl (enzyme inducers and tumor promoters in vivo) or with 12-O-tetradecanoylphorbol-13-acetate (the classical skin tumor promoter) further increased EGF-stimulated DNA synthesis. The results emphasize the need to adequately substitute zinc in serum-free cultured cells.

Glucuronide formation of various drugs in liver microsomes and in isolated hepatocytes from phenobarbital- and 3-methylcholanthrene-treated rats.

Various substrates of rat liver microsomal UDP-glucuronosyltransferase were classified in vitro as preferred substrates of either 3-methylcholanthrene- or phenobarbital-inducible enzyme forms. Microsomal UDP-glucuronosyltransferase activities towards a third group of substrates (including oestrone, phenolphthalein, paracetamol and oxazepam) are not markedly altered by treatment with either 3-methylcholanthrene or phenobarbital. Some substrates of the 3-methylcholanthrene- and phenobarbital-inducible enzyme activities were selected to evaluate the importance of multiple enzyme forms for glucuronide formation in the intact cell. The metabolism of these compounds was compared in isolated hepatocytes from untreated controls and from rats treated with 3-methylcholanthrene (MC-hepatocytes) or phenobarbital (PB-hepatocytes). Glucuronidation of 1-naphthol and 3-hydroxybenzo[a]pyrene was chiefly enhanced in MC-hepatocytes (greater than 2-fold), whereas glucuronidation of chloramphenicol and bilirubin was chiefly enhanced in PB-hepatocytes. These observations are in agreement with differential induction of UDP-glucuronosyltransferase activities in vitro suggesting that, besides other factors such as cofactor supply, physiological activators, etc., the levels of the multiple enzyme forms are critically determining glucuronide formation in the intact cell.

N-glucuronide formation of carcinogenic aromatic amines in rat and human liver microsomes.

(1) Sensitive fluorimetric assays were developed for the determination of microsomal UDP-glucuronosyltransferase activities towards 1- and 2-naphthylamine and 4-aminobiphenyl. (2) In rat liver microsomes, enzyme activity towards 1-naphthylamine was orders of magnitude higher than the activities towards 2-naphthylamine, 4-aminobiphenyl or aniline. The differences were less marked with human liver microsomes. (3) Glucuronidation of aniline and 4-aminobiphenyl was not appreciably altered in rat liver microsomes from 3-methylcholanthrene- or phenobarbital-treated rats. UDP-glucuronosyltransferase activities towards 1- and 2-naphthylamine were selectively increased (about 2-fold) by 3-methylcholanthrene-treatment. However the increases were less marked than those observed with representative substrates of the 3-methylcholanthrene-inducible enzyme form. The results suggest that the arylamines investigated are predominantly conjugated by constitutive enzyme forms in rat liver. (4) Arylamine N-glucuronides were found to be susceptible to hydrolysis by E. coli beta-glucuronidase suggesting the release of carcinogenic arylamines in the gut and their enterohepatic circulation.

Immunochemical and functional characterization of UDP-glucuronosyltransferases from rat liver, intestine and kidney.

Glucuronidation of various substrates in hepatic, intestinal and renal microsomes of control, phenobarbital (PB), 3-methylcholanthrene (3MC) and Aroclor-1254 (A1254) pretreated rats was investigated. UDPGT activities tested could be divided in four groups on the basis of their tissue distribution and induction by PB or 3MC in liver microsomes. GT1 activities (1-naphthol, benzo(a)pyrene-3,6-quinol) are induced by 3MC in liver microsomes and are present in all tissues investigated. GT2 activities (morphine, 4-hydroxybipheynl) are induced by PB in liver microsomes and appear to be restricted to the liver and the intestine. UDPGT activity towards bilirubin, although induced by PB, can be detected in hepatic, intestinal and renal microsomes. UDPGT activity towards fenoterol is restricted to the liver and intestine and is not induced by PB, 3MC or A1254. The presence of inducible immunoreactive UDPGT isoenzymes in microsomes of liver, intestine and kidney of control and induced rats was demonstrated by immunoblot analysis using rabbit anti-rat liver-GT1 antibodies. Induction of both 54 and 56 kDa polypeptides in hepatitis, intestinal and renal microsomes by 3MC or A1254 was observed. Purification of UDPGT (1-naphthol as substrate) from intestinal microsomes to apparent homogeneity yielded a polypeptide with an apparent molecular weight of 54-56 kDa. The results indicate that 54 and 56 kDa UDPGT polypeptides are the major A1254 inducible isoenzymes in intestinal and renal microsomes. An increase in immunoreactive protein is correlated with a biochemically measurable increase in glucuronidation capacity for GT1 substrates.

Stereoselective glucuronidation of (R)- and (S)-naproxen by recombinant rat phenol UDP-glucuronosyltransferase (UGT1A1) and its human orthologue.

Recombinant rat phenol UDP-glucuronosyltransferase (UGT1A1) conjugates (R)-naproxen at a much higher rate (> 17-fold) than its (S)-enantiomer, substantiating previous findings on stereoselective glucuronidation of racemic naproxen. In contrast, the recombinant human orthologue conjugated both enantiomers at equal rates. In line with high constitutive expression of UGT1A1 in extrahepatic tissues, a high R/S ratio of naproxen glucuronidation was found in rat testes, intestine, lung and kidney. The results demonstrate that (R)-naproxen represents a stereoselective substrate of rat UGT1A1, but not of the human orthologous UGT1A1.

Differential induction of human liver UDP-glucuronosyltransferase activities by phenobarbital-type inducers.

(1) UDP-glucuronosyltransferase (UDP-GT) activities and their inducibility were investigated in human liver microsomes from a "liver bank". (2) UDP-GT activities were differentially induced in liver microsomes from patients treated with the phenobarbital-type inducers phenytoin or pentobarbital. UDP-GT activity towards bilirubin was induced 3-fold. Enzyme activities towards paracetamol, benzo(a)pyrene-3,6-quinol, 4-methylumbelliferone and 1-naphthol were moderately induced and to similar extents (2-fold). In contrast, morphine and 4-hydroxybiphenyl glucuronidation were not significantly affected. Cytochrome P-450 dependent 7-ethoxycoumarin O-deethylase was increased 5-fold. (3) A human hepatoma cell line (Hep G2) was studied to obtain information on the inducibility of human UDP-GT activities by 3-methylcholanthrene-type inducers. UDP-GT activities towards benzo(a)pyrene-3,6-quinol and 1-naphthol were moderately but significantly induced by 3-methylcholanthrene-treatment of the cells (2-fold), whereas 7-ethoxyresorufin O-deethylase and 7-ethoxycoumarin O-deethylase were increased over 100- and 10-fold, respectively. (4) The results suggest the existence of differentially inducible UDP-GT isoenzymes in human liver. The findings may be useful as a guide to characterize human liver UDP-GT isoenzymes.

Tissue-specific constitutive and inducible expression of rat phenol UDP-glucuronosyltransferase.

To investigate constitutive and inducible expression of rat phenol UDP-glucuronosyltransferase (UGT1A1) in liver and extrahepatic tissues, a selective cDNA probe for its unique exon 1 was utilized. 6-Hydroxychrysene was used as a functional probe of UGT1A1 activity. Constitutive expression of UGT1A1 was low in liver, but high in kidney, testis, epididymis and ovary. After treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 micrograms/kg for 7 days) the UGT1A1 mRNA level was markedly increased in liver (ca. 10-fold), and only moderately enhanced (up to 2-fold) in extrahepatic tissues where constitutive enzyme expression was high. UGT activity toward 6-hydroxychrysene was strongly inducible in liver (ca. 9-fold) and only moderately inducible in extrahepatic tissues (up to 2-fold). The results suggest complex tissue-specific regulation of UGT1A1 including positive and negative transcriptional factors and marked inducibility by TCDD in liver.

In situ hybridization studies of UDP-glucuronosyltransferase UGT1A6 expression in rat testis and brain.

UDP-glucuronosyltransferases (UGTs), in addition to their role in overall pharmacokinetics, play important roles in local protection of cells against toxins and in the control of endogenous receptor ligands. UGT1A6, which conjugates planar phenols, appears to be expressed in many organs, but information on cell-specific expression in these organs is controversial or absent. Therefore, a non-isotopic in situ hybridization method was developed and applied to localize UGT1A6 expression in rat testis and brain. It was found that UGT1A6 is expressed in Sertoli cells and spermatogonia of rat testis and in brain neurons, in particular in hippocampal pyramidal cells and Purkinje cells of the cerebellum.

Functional heterogeneity of UDP-glucuronosyltransferase activities in C57BL/6 and DBA/2 mice.

Functional heterogeneity of liver microsomal UDP-glucuronosyltransferase activities towards 1-naphthol, 4-methylumbelliferone or 3-hydroxybenzo(a)pyrene (UDP-GT1 activities) and morphine or 4-hydroxybiphenyl (UDP-GT2 activities) was studied in two inbred strains of mice which are genetically responsive (C57BL/6) or non-responsive (DBA/2) to 3-methylcholanthrene-induction of drug metabolizing enzymes. 3-Methylcholanthrene preferentially induced UDP-GT1 activities in C57BL/6 mice. Phenobarbital, however, at low doses (50 mg/kg), selectively induced UDP-GT2 activities. Higher doses of phenobarbital (80 mg/kg) induced both UDP-GT1 and UDP-GT2 activities. In DBA/2 mice 3-methylcholanthrene-induction of UDP-glucuronosyltransferase activities was not detectable whereas enzyme induction by phenobarbital appeared to be unimpaired. UDP-GT1 activities were ubiquitously detectable in mouse tissues whereas appreciable UDP-GT2 activities were only found in liver and small intestinal mucosa. UDP-GT1 (1-naphthol as substrate) was not inhibited by morphine suggesting different active sites for the conjugation of these substrates. The results suggest the presence of at least two functionally different forms of UDP-glucuronosyltransferase in mice. In conjunction with the results of Owens (J. biol. Chem. 252, 2827 (1977)) it is evident that one of these enzyme forms is regulated by the Ah locus.

Differential induction of rat liver microsomal UDP-glucuronosyltransferase activites by various inducing agents.

The selectivity of various inducers of UDP-glucuronosyltransferase was investigated in rat liver microsomes and compared with their effect on monooxygenase reactions. (1) Similar to 3-methyl-cholanthrene beta-naphthoflavone selectively stimulated the glucuronidation of 1-naphthol and 4-methylumbelliferone (GT1 substrates). (2) In contrast, DDT preferentially enhanced the glucuronidation of morphine, 4-hydroxybiphenyl (GT2 substrates) and bilirubin, similar to phenobarbital. (3) Colfibric acid and bezafibrate selectively enhanced bilirubin glucuronidation without affecting GT1 and GT2 reactions. (4) Similar to ethoxyquin and Aroclor 1254, trans-stilbene oxide enhanced both GT1 and GT2 activities but not bilirubin glucuronidation. (5) In contrast to 3-methylcholanthrene-type inducers which induce both cytochrome P-450MC and GT1, probably through a common receptor protein, ethoxyquin and trans-stilbene oxide markedly induced GT1 reactions without affecting benzo[a]pyrene monooxygenase.

Properties of a 3-methylcholanthrene-inducible phenol UDP-glucuronosyltransferase from rat liver.

Functional and molecular probes are described which are useful to identify a 3-methylcholanthrene-inducible phenol UDP-glucuronosyltransferase (GTMC) from rat liver. Two different procedures for isolation of GTMC were compared, method 1 utilizing DEAE-Sepharose chromatography or method 2, chromatofocusing. Method 2 appeared to be superior in separating different isoenzymes. Subsequently the enzyme was purified by affinity chromatography on UDP-hexanolamine Sepharose. With both methods a protein was purified with a subunit Mr of 55,000, catalyzing glucuronidation of a variety of planar phenols and, in particular, of benzo(a)pyrene-3,6-quinol to its mono- and diglucuronide. Antibodies to GTMC recognized a polypeptide with a subunit Mr of 55,000 as the major 3-methylcholanthrene-inducible isoenzyme in rat liver microsomes. The described functional and molecular probes may help to differentiate GTMC from similar isoenzymes conjugating planar phenols and to elucidate its regulation and biological function.

Induction of UDP-glucuronyltransferase and arylhydrocarbon hydroxylase activity in mouse skin and in normal and transformed skin cells in culture.

Methods have been developed which allow quantitative determination of UDP-glucuronyltransferase (UDPGT) and arylhydrocarbon hydroxylase (AHH) activities in unfractionated mouse skin. These methods were used for comparative studies of basal and induced enzyme activities in whole skin and cultured skin cells. After topical application of Aroclor 1254 to the skin UDPGT activities towards 1-naphthol, 3-hydroxybenzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol were increased 3-fold and AHH activity was increased 15-fold. Topical application of the inducer also led to a marked increase of these enzyme activities in liver. UDPGT activity towards 1-naphthol was comparable in whole skin and in cultured keratinocytes and fibroblasts. In contrast, AHH activity was higher in cultured keratinocytes than in skin. In transformed epithelial cell lines the pattern of drug metabolizing enzymes was altered: UDPGT activity was increased 4- to 6-fold whereas AHH activity was decreased. However, AHH activity was still inducible by benz[a]anthracene or 7,12-dimethylbenz[a]anthracene in cultured cells. The altered pattern of AHH and UDPGT in transformed epithelial cell lines is consistent with toxin-resistance of initiated cells, similar to the toxin-resistance phenotype characterized in liver after initiation of hepatocarcinogenesis.

Glucuronidation and isomerization of all-trans- and 13-cis-retinoic acid by liver microsomes of phenobarbital- or 3-methylcholanthrene-treated rats.

Glucuronidation and isomerization of all-trans-retinoic acid (tr-RA) and 13-cis-retinoic acid (13-cis-RA) were investigated in an in vitro system using liver microsomes of differently pretreated rats. In agreement with their thermodynamic stability, more retinoic acid was isomerized from the 13-cis form to the all-trans form than vice versa. Also some 9-cis-retinoic acid (9-cis-RA) could be found. Isomerization was reduced, but in contrast to glucuronidation was still important if boiled microsomes were used. This supports the view that isomerization can proceed as a non-enzymatic process. 3-Methylcholanthrene (MC) pretreatment of the rats increased the microsomal glucuronidation of 13-cis-RA and tr-RA and the formation of 13-cis-retinoyl-beta-glucuronide was enhanced up to 7-fold by MC-induced rat microsomes. The rates of glucuronidation by uninduced and phenobarbital-induced rat microsomes differed only slightly. In addition to glucuronides of the applied retinoic acid isomers (13-cis-RA and tr-RA), 9-cis-RA and its glucuronide were found. Induction of retinoid glucuronidation by pretreatment with MC indicates that this metabolic reaction is catalysed by a MC-inducible UGT isozyme. After two recently described pathways (conversions of retinol to retinal and of retinyl methyl ether to retinol) this is a third step of retinoid metabolism, induced by pretreatment with MC. With human microsomes no more than traces of glucuronides were detected; also, incubations with human microsomes resulted in a lower degree of isomerization than with rat microsomal fractions.