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.

AH receptor-controlled transcriptional regulation and function of rat and human UDP-glucuronosyltransferase isoforms.

Transcriptional regulation and function of rat and human PAH-inducible UDP-glucuronosyltransferase (UGT) isoforms have been studied. 1. At least two PAH-inducible UGT isoforms are expressed in a variety of tissues, the rat isoforms UGT1A6 and UGT1A7, and the human isoforms UGT1A6 and UGT1A9. 2. For the rat and human UGT1A6 isoforms two modes of tissue- and cell-specific regulation were found, PAH-inducible and constitutive expression. 3. Transient transfection studies, using human UGT1A6/CAT fusion constructs and colon carcinoma Caco-2 cells, revealed that PAH induction of human UGT1A6 is mediated by the Ah receptor. 4. Cell-expressed UGT isoforms were used to study their function in PAH metabolism. Rat UGT1A7 and human UGT1A9 appear to be more efficient than the corresponding UGT1A6 isoforms in catalyzing glucuronide formation of PAH phenols and diphenols. Several isoforms may act together in the formation of benzo(a)pyrene-3.6-diol diglucuronide, the major glucuronide found in rat bile. The results suggest complex modes of transcriptional regulation of PAH-inducible UGTs. They also suggest a major role of these UGT isoforms in detoxication of PAHs.

Density-dependent growth of normal and nodular hepatocytes.

To elucidate factors responsible for altered proliferation of preneoplastic hepatocytes in rat hepatocarcinogenesis in vivo, EGF-stimulated DNA synthesis of normal and nodular hepatocytes in primary culture was studied. In addition, the influence of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated to clarify whether this potent tumor promoter differentially affects normal and nodular hepatocyte cultures. Unexpectedly it was found that in nodular hepatocytes spontaneous and EGF-stimulated DNA synthesis was enhanced with increasing cell density while DNA synthesis was inhibited in dense cultures of normal hepatocytes. Mitogenic responses were detected both by [3H]thymidine incorporation into DNA and by 5-bromo-2'-deoxyuridine labeling indices. TCDD (1 nM) acted as a mitoinhibitor both in normal and in nodular hepatocytes. The results suggest marked differences in growth behavior of nodular versus normal hepatocyte cultures probably due to paracrine stimulation by growth factors and altered cell-cell interaction.

Release of mutagenic metabolites of benzo[a]pyrene from the perfused rat liver after inhibition of glucuronidation and sulfation by salicylamide.

The role of glucuronide and sulfate conjugation in presystemic inactivation of benzo[a]pyrene (BP) metabolites was investigated with rat livers perfused with BP (12 mumol). Comparisons were made between metabolite profiles and mutagenicity of medium from perfusions with and without salicylamide, a selective inhibitor of glucuronide and sulfate conjugation. After 4 h perfusion in the presence of salicylamide, certain BP metabolites (diols, quinones, phenols, and metabolites more polar than BP-9,10-diol) were significantly increased at the expense of quinones and phenols in the glucuronide fraction. Mutagenicity of medium (detected by the Ames test, using tester strains TA98 and TA100) was low in perfusion without salicylamide. Mutagenicity detected with tester strain TA98 was significantly increased in perfusions with salicylamide. Involvement of glucuronidation in BP inactivation was also observed at the subcellular level; when cofactors of glucuronidation were added to liver homogenates along with the NADPH regenerating system in the Ames test, BP mutagenicity was markedly decreased. Both the activation of BP to mutagenic metabolites and the inactivation of BP metabolites by glucuronidation was much more pronounced with liver homogenates from 3-methylcholanthrene-treated rats than with those from phenobarbital-treated animals or untreated controls. The results suggest an important role for glucuronidation and sulfation in the inactivation and elimination of polycyclic aromatic hydrocarbons.

Activating and inactivating reactions controlling 2-naphthylamine mutagenicity.

Factors controlling 2-naphthylamine mutagenicity were studied using the Ames test. 1) Both rat liver microsomes and cytosolic proteins were required for generation of mutagenic metabolites. 2) 1-Hydroxy-2-naphthylamine, the major metabolite of 2-naphthylamine, was not mutagenic but cytotoxic to bacteria. 3) Ascorbic acid, reduced glutathione and conjugation reactions, such as glucuronidation, were strongly inhibiting 2-naphthylamine mutagenicity. 4) When isolated hepatocytes were used as the activating system mutagenic metabolites could not be detected. However cytotoxicity was detectable at doses of 2-naphthylamine greater than 0.2 mumol/10(6) cells. The results suggest that the formation of genotoxic metabolites of 2-naphthylamine is largely prevented in the intact, non-dividing rat hepatocyte.

Protection against toxic redox cycles between benzo(a)pyrene-3,6-quinone and its quinol by 3-methylcholanthrene-inducible formation of the quinol mono- and diglucuronide.

Cytotoxic effects of quinones are thought to be mediated by redox cycles between quinones and quinols whereby reactive oxygen species are generated. The role of glucuronidation in preventing these toxic redox cycles was investigated by using benzo(a)pyrene-3,6-quinone and isolated rat hepatocytes or Reuber hepatoma cells (H4IIE). Inhibition of quinol glucuronidation by salicylamide enhanced quinone-dependent oxygen uptake and cytotoxicity. Conjugation of benzo(a)pyrene-3,6-quinol was shown to proceed via the 6-monoglucuronide to the diglucuronide. Diglucuronide formation was low in hepatocytes from untreated controls and phenobarbital-treated rats. However, it was highly stimulated (26-fold) in hepatocytes from 3-methylcholanthrene-treated rats and was also high in Reuber hepatoma cells. Kinetic analysis with liver microsomes indicated that 3-methylcholanthrene-stimulated glucuronidation was due to an increased Vmax of UDP-glucuronosyltransferase which was enhanced 10- and 40-fold or mono- and diglucuronide formation, respectively. These findings suggest that the investigation of quinol glucuronidation (in particular the formation of benzo(a)pyrene-3,6-quinol diglucuronide) is a most useful probe for the 3-methylcholanthrene-inducible isoenzyme(s) of UDP-glucuronosyltransferase. Moreover, this isoenzyme may be particularly suited to protect against toxic redox cycles between benzo(a)pyrene quinones and quinols.

Glucuronidation of carcinogenic arylamines and their N-hydroxy derivatives by rat and human phenol UDP-glucuronosyltransferase of the UGT1 gene complex.

Since carcinogenic arylamines are sequentially oxidized and conjugated with glucuronic acid, differences in glucuronidation may critically determine the toxic potential of these compounds. Therefore, N-glucuronidation of 1- and 2-naphthylamine (1-NA and 2-NA),4-aminobiphenyl(4-ABP) and their N-hydroxy derivatives was investigated using rat and human liver microsomes and V79 cell-expressed phenol UDP-glucuronosyltransferases (UGT) of the UGT1 gene complex. Cell-expressed UGTs included rat and human UGT1.6, which are known to conjugate planar phenols, and human UGT1.7, conjugating both planar and bulky phenol. (i) N-Glucuronidation of 1- and 2-NA and of N-hydroxy-2-NA was inducible by 3-methylcholanthrene in rat liver microsomes whereas N-glucuronidation of the bulky arylamines 4-ABP and N-hydroxy-4-ABP was not. In support of these findings mutagenicity of N-hydroxy-2-NA in the Ames test was markedly reduced upon addition of UDP-glucuronic acid using liver homogenates from 3-methylcholanthrene-treated rats. (ii) With cell-expressed rat UGT1.6, non-carcinogenic 1-NA was conjugated with the highest rate and with higher affinity than 2-NA. UGT1.6 showed poor activity towards N-hydroxy-4-ABP and 4-ABP. (iii) Substrate specificity of human UGT1.6 also appeared to be limited to planar 1-NA, 2-NA and its N-hydroxy derivative, whereas UGT1.7 showed broader substrate specificity, including the bulky arylamine 4-ABP and its N-hydroxy derivative. The results suggest marked differences in substrate specificity of different UGT isozymes for arylamines and their N-hyroxy derivatives.

Induction of drug-metabolizing enzymes by xenobiotics.

1. Isozymes of the cytochromes P-450, UDP-glucuronosyl transferases (UDPGT) and glutathione S-transferases appear to be differentially inducible by prototype inducers, such as 3-methylcholanthrene (MC), phenobarbital, pregnenolone 16 alpha-carbonitrile and clofibrate. 2. Mechanisms of induction include both transcriptional and post-transcriptional control. MC-type inducers (representing a large number of planar polycyclic aromatics, beta-naphthoflavone and polyhalogenated aromatics) bind with high affinity to the Ah receptor which controls gene expression similar to steroid hormone receptors. The Ah receptor controls the expression of several drug metabolizing enzymes. For example, both cytochrome P450 IA1 and UDPGT-1 appear to be co-induced by inducers with widely differing potencies, such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin, 1,2,3,7,8-pentachloro-dibenzo-p-dioxin and benz(a)anthracene. Much less is known about the mechanism of action of other inducer prototypes. 3. Induction and co-induction of drug-metabolizing enzymes are generally considered as adaptive responses leading to more efficient elimination and detoxication of xenobiotics such as benzo(a)pyrene. For example, when the mutagenicity of benzo(a)pyrene and benzo(a)pyrene-3,6-quinone was studied in the Ames test, glucuronidation or glutathione conjugation (concomitant with cytochrome P-450-dependent reactions) markedly decreased their mutagenicity. The protective effect was more pronounced with the homogenate S9 fraction of MC-treated rats. However, at 'non-physiological' levels of exposure enzyme induction may lead to increased toxic risk.

Growth modulation of hepatocytes and rat liver epithelial cells (WB-F344) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Modulation of DNA synthesis by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied in primary cultures of hepatocytes and in rat liver epithelial cells (WB-F344) to develop models for studies on the interactions between the activated Ah receptor and cellular growth control. In hepatocytes TCDD either positively or negatively modulated EGF-stimulated DNA synthesis. In the presence of ethinylestradiol 10(-12) M TCDD moderately increased EGF-stimulated DNA synthesis (approximately 30%). In contrast, 10(-9) M TCDD in the absence of ethinylestradiol decreased DNA synthesis (approximately 30%). Analysis of variance revealed that the TCDD effects were highly significant. The response of 'early genes' of the jun/fos family and the corresponding proteins was also studied under these two conditions. In agreement with the DNA synthesis data, the level of c-Jun was increased or decreased in nuclear extracts. Furthermore, DNA binding of Jun/Fos proteins, including c-Jun and Fra-1, was decreased under conditions of mitoinhibition, while the level of Fra-1 in nuclear extracts was increased. In WB-F344 cells TCDD treatment for 44 h increased DNA synthesis 2- to 3-fold in comparison with controls, based on measuring [3H]thymidine incorporation into DNA or on determining the nuclear labeling index with bromodeoxyuridine. This effect is probably due to inhibition of high density growth arrest by TCDD. The proposed cellular models may be useful to elucidate the interactions between the activated Ah receptor and signaling pathways of growth homeostasis.

Induction of human UDP glucuronosyltransferases (UGT1A6, UGT1A9, and UGT2B7) by t-butylhydroquinone and 2,3,7,8-tetrachlorodibenzo-p-dioxin in Caco-2 cells.

Human colon carcinoma Caco-2 cells were used to study the induction of UDP glucuronosyltransferase (UGT) isoforms UGT1A6, UGT1A9, and UGT2B7 by aryl hydrocarbon receptor agonists and by antioxidant-type inducers with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and t-butylhydroquinone (TBHQ), respectively. Early- (PF11) and late-passage clones (TC7) of Caco-2 cells, which show low and high constitutive UGT1A6 expression, respectively, were selected. The following results were obtained: 1) In Caco-2 cells UGT activity (4-methylumbelliferone as substrate) was significantly enhanced by 10 nM TCDD or 40 to 80 microM TBHQ and 2) duplex reverse-transcription-polymerase chain reaction analysis showed for the first time that the expression of human UGT1A6, UGT1A9, and UGT2B7 was enhanced by 40 to 80 microM TBHQ; both UGT1A6 and UGT1A9 were induced by 10 nM TCDD, whereas UGT2B7 was not induced by TCDD. The results suggest that at least two human UGTs (UGT1A6 and UGT1A9) are inducible by aryl hydrocarbon receptor agonists and even more isoforms (UGT1A6, UGT1A9, and UGT2B7) are inducible by antioxidant-type inducers in Caco-2 cells.

Functions and transcriptional regulation of PAH-inducible human UDP-glucuronosyltransferases.

Functions and regulation of selected human UDP-glucuronosyltransferases (UGT1A1, UGT1A4, UGT1A6, UGT1A9, UGT2B7, UGT2B15) are summarized. Evidence for at least two PAH-inducible UGTs (UGT1A6 and UGT1A9) is presented, which, however, are also constitutively expressed in a tissue- and cell-specific manner. These isoforms have recently been characterized to conjugate planar and bulky phenols, respectively. Using a selective RT-PCR method, UGT1A6 expression was detected in a variety of tissues (liver, kidney, lung, intestine, and pharyngeal mucosa). PAH-inducible UGTs may cooperate in the metabolism of phenolic metabolites of benzo(a)pyrene. Studies with stably expressed isoforms suggest that UGT1A9 is responsible for the formation of benzo(a)pyrene-3.6-diphenol diglucuronide, the major biliary metabolite of benzo(a)pyrene.