Developmental alteration of hepatic UDP-glucuronosyltransferase and sulphotransferase towards androsterone and 4-nitrophenol in Wistar rats.

Postnatal development of hepatic UDP-glucuronosyltransferase and sulphotransferase activities towards androsterone and 4-nitrophenol as well as cytochrome P-450 contents was studied in male and female Wistar rats. The rats with high and low UDP-glucuronosyltransferase activity towards androsterone were classified by the genotype of the parent animals. UDP-glucuronosyltransferase activity towards androsterone began rapidly to enhance after 30 days of age in the high-activity group, whereas the transferase activity remained low throughout in the low-activity group. Such a striking difference was not observed in UDP-glucuronosyltransferase activity towards 4-nitrophenol, sulphotransferase activity towards androsterone and 4-nitrophenol, and cytochrome P-450 contents. Sex-based difference in the sulphotransferase activity was marked after 30 days of age. Sulphotransferase activity towards androsterone was much higher in adult females than in adult males, whereas higher sulphation activity towards 4-nitrophenol was found in adult males. The results also indicate that the low level of the UDP-glucuronosyltransferase activity did not lead to compensatory stimulation of the sulphotransferase activity.     

Strain differences in purified rat hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase.

Qualitative and quantitative differences of purified hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase were investigated in Wistar and Sprague-Dawley rats. Individual differences in the glucuronidation rate of androsterone and chenodeoxycholic acid were observed in hepatic microsomal fractions from Wistar but not Sprague-Dawley rats. No individual variation was observed in the glucuronidation of testosterone, p-nitrophenol or oestrone. The 3 alpha-hydroxysteroid UDP-glucuronosyltransferases from livers of Wistar and Sprague-Dawley rats were isolated and highly purified by using Chromatofocusing and affinity chromatography. The amount of 3 alpha-hydroxysteroid UDP-glucuronosyltransferase in the liver of Wistar rats exhibiting low rates for androsterone glucuronidation is about 10% or less than that found in hepatic microsomal fractions obtained from Wistar rats having high rates for androsterone glucuronidation. The apparent Km for androsterone with purified 3 alpha-hydroxysteroid UDP-glucuronosyltransferase from Wistar rats with high glucuronidation activity (6 microM) was not different from that observed for the enzyme purified from Sprague-Dawley animals, whereas that for the enzyme purified from Wistar rats with low glucuronidation activity was substantially higher (120 microM). Despite the differences in apparent Km values for androsterone, the apparent Km for UDP-glucuronic acid (0.3 mM) was not different in the different populations of rats.     

Biosynthesis of hydroxyl-linked glucuronides of short-chain bile acids by rat liver 3-hydroxysteroid UDP-glucuronosyltransferase

Microsomal preparations from livers of Sprague-Dawley rats catalyze the glucuronidation of 3 alpha-hydroxy-5 beta-H (3 alpha, 5 beta) short-chain bile acids (C20-C23), predominantly at the hydroxyl group, while the glucuronidation of 3 beta, 5 beta short-chain bile acids occurs exclusively at the carboxyl group. A similar pattern of conjugation was also observed in Wistar rats having normal levels of 3-hydroxysteroid UDP-glucuronosyltransferase. Significant reductions of formation rates for hydroxyl-linked, but not carboxyl-linked, short-chain bile acid glucuronides were observed in hepatic microsomes from Wistar rats with low 3-hydroxysteroid UDP-glucuronosyltransferase activity. 3-Hydroxysteroid UDP-glucuronosyltransferase, purified to homogeneity from Sprague-Dawley liver microsomes, catalyzed the 3-O-glucuronidation of 3 alpha, 5 beta C20-23 bile acids, as well as of lithocholic and isolithocholic acids (C24). The apparent Michaelis constants (KM) for short-chain bile acids were similar to the value obtained for androsterone. 3 alpha, 5 beta-C20 and 3 beta, 5 beta-C20 competitively inhibited glucuronidation of androsterone by the purified 3-hydroxysteroid UDP-glucuronosyltransferase. Purified 17 beta-hydroxysteroid and p-nitrophenol UDP-glucuronosyltransferases did not catalyze the glucuronidation of bile acids. In addition, none of the purified transferases catalyzed the formation of carboxyl-linked bile acid glucuronides. The results show that 3-hydroxysteroid UDP-glucuronosyltransferase, an enzyme specific for 3-hydroxyl groups of androgenic steroids and some conventional bile acids, also catalyzes the glucuronidation of 3 alpha-hydroxyl (but not carboxyl) groups of 3 alpha, 5 beta short-chain bile acids.     

Expression of four phenobarbital-inducible cytochrome P-450s in liver, kidney, and lung of rats

Specific antibodies were prepared against cytochromes P450 PB-1, PB-2, PB-4, and PB-5 purified from hepatic microsomes of male rats treated with phenobarbital. With these antibodies, the levels of these four cytochrome P450s in hepatic, renal, and pulmonary microsomes of male rats that were untreated, treated with phenobarbital, or treated with 3-methylcholanthrene were examined. P450 PB-1 and PB-2 were present in moderate amounts in hepatic microsomes of untreated male rats and were induced 2- to 3-fold with phenobarbital. Also, the expression of these forms was suppressed by 3-methylcholanthrene. These forms were not detected in the renal or pulmonary microsomes of untreated rats or rats treated with phenobarbital or 3-methylcholanthrene. P450 PB-4 and PB-5 were found in the hepatic microsomes of untreated male rats at a low level but were induced with phenobarbital more than 50-fold. P450 PB-4 and PB-5 were not detected in renal microsomes; only P450 PB-4 or a closely related form was present in the pulmonary microsomes of untreated male rats, and its level was not changed by phenobarbital treatment. The constitutive presence of P450 PB-4 in pulmonary microsomes was confirmed by the investigation of testosterone metabolism. Purified P450 PB-4 had high testosterone 16 alpha- and 16 beta-hydroxylation activity in a reconstituted system. The testosterone 16 beta-hydroxylation activity of hepatic microsomes was induced with phenobarbital, and more than 90% of the testosterone 16 beta-hydroxylation activity of hepatic microsomes from rats treated with phenobarbital was inhibited by anti-P450 PB-4 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of phenobarbital and 3-methylcholanthrene on the hepatic cytochrome P-450 metabolism of various alkoxyresorufin ethers in the cotton rat (Sigmodon hispidus)

1. Microsomes isolated from phenobarbital and 3-methylcholanthrene induced cotton rats (Sigmodon hispidus) were tested for o-dealkylase activity with methoxy-, ethoxy-, pentoxy- and benzoxyresorufin ethers. 2. The activity of 3-methylcholanthrene induced microsomes was greater than controls. 3. Activity of phenobarbital induced microsomes was not different from controls. 4. There was a distinct difference between male and female animals. 5. The results obtained from cotton rats are markedly different from results obtained from Sprague-Dawley (S-D) rats.     

Comparison of phenobarbital-and carcinogen-induced aldehyde dehydrogenases in the rat

There is a genetically determined variation in the inducibility of a high-K_m cytoplasmic aldehyde dehydrogenase activity in the rat liver by treatment with phenobarbital. In the present experiments this activity increased after phenobarbital administration in the phenobarbital-responsive rats also in the intestinal postmitochondrial supernatant fraction. Phenobarbital-nonresponsive rats did not exhibit such an increase after drug treatment. Intraperitoneal administration of 2,3,7,8,-tetrachlorodibenzo-pdioxin, strongly enchanced the cytoplasmic enzyme activity in the liver of both responsive and nonresponsive rats. This effect was also seen in the serum but not in the intestinal or hte kidney. Intragastric administration of 3-methylcholanthrene, 3,4,-benzpyrene or chrysene induced the activity in liver and intestine but not in serum or kidney. The activity in liver was also induced by long-term feeding with 2-acetamido-fluorene. The activities induced by tetrachlorodibenzodioxin or the carcinogens had similar behaviour in isoelectric focusing in gel slabs and in gel chromatography, suggesting a possible common identity of these induced enzymes. The activity induced by these agents could be clearly differentiated both from the activity induced by phenobarbital and from the normal cytoplasmic activities.     

The molecular basis of the inherited deficiency of androsterone UDP-glucuronyltransferase in Wistar rats

A major UDP-glucuronyltransferase isoenzyme in rat liver (51 kDa), corresponding to androsterone glucuronidating activity, has been identified by immunoblot analysis. This isoenzyme is absent from Wistar rats exhibiting the low androsterone (LA) UDP-glucuronyltransferase activity exhibiting the low androsterone (LA) UDP-glucuronyltransferase activity phenotype. Northern blot analysis of total RNA from normal and androsterone glucuronidation deficient Wistar rats demonstrated that the mRNA encoding this protein was not synthesised. Differences in restriction fragment length observed on Southern blotting of genomic DNA from LA Wistar rats indicate that this inherited deficiency is the result of a deletion in the androsterone UDP-glucuronyltransferase gene.     

Large deletion of androsterone UDP-glucuronosyltransferase gene in the inherited deficient strain of Wistar rats

LA Wistar rats have a deficiency of androsterone UDP-glucuronosyltransferase (UDPGT) and are present in Wistar rat colonies around the world. In order to clarify the molecular mechanism of the deficiency, androsterone UDPGT cDNA clone, pGT2 was isolated from rat liver cDNA library and was digested with restriction enzymes to afford three probes for Northern and Southern blot analyses in HA (normal), heterozygous LA and LA Wistar rats. In Northern blot analysis, androsterone UDPGT mRNA was totally absent in LA Wistar rat liver. Southern blot analysis suggested a large deletion of androsterone UDPGT gene in the rats. Genomic DNA amplifications with synthetic primers which have nucleotide sequences corresponding to the 5′-region of androsterone UDPGT cDNA, suggested that androsterone UDPGT gene has exon 1 with a length of some 700 bp and that this exon is deleted in LA Wistar rats. Based on these lines of evidence, it is concluded that the large portion of androsterone UDPGT gene is deleted in LA Wistar rats, which results in the absence of androsterone UDPGT mRNA and consequently the corresponding enzyme protein.     

Large deletion of androsterone UDP-glucuronosyltransferase gene in the inherited deficient strain of Wistar rats.

LA Wistar rats have a deficiency of androsterone UDP-glucuronosyltransferase (UDPGT) and are present in Wistar rat colonies around the world. In order to clarify the molecular mechanism of the deficiency, androsterone UDPGT cDNA clone, pGT2 was isolated from rat liver cDNA library and was digested with restriction enzymes to afford three probes for Northern and Southern blot analyses in HA (normal), heterozygous LA and LA Wistar rats. In Northern blot analysis, androsterone UDPGT mRNA was totally absent in LA Wistar rat liver. Southern blot analysis suggested a large deletion of androsterone UDPGT gene in the rats. Genomic DNA amplifications with synthetic primers which have nucleotide sequences corresponding to the 5'-region of androsterone UDPGT cDNA, suggested that androsterone UDPGT gene has exon 1 with a length of some 700 bp and that this exon is deleted in LA Wistar rats. Based on these lines of evidence, it is concluded that the large portion of androsterone UDPGT gene is deleted in LA Wistar rats, which results in the absence of androsterone UDPGT mRNA and consequently the corresponding enzyme protein.     

Enzymatic induction with phenobarbital and 3-methylcholanthrene in rats exposed to normobaric oxygen

Induction by phenobarbital and 3-methylcholanthrene of enzymes metabolizing xenobiotics in rat exposed 55 hrs to hyperoxia, is maintained. The level of microsomal pulmonary and hepatic cytochrome P-450 has even increased. In rat protected against a hyperoxia 6 days, stimulation of UDP-glucuronosyltransferase does not decrease and malondialdehydes level does not change. These results assume the probable role of enzymatic induction in the tolerance to hyperoxia induced in rast by treatment with phenobarbital and 3- methylcholanthrene.     

Effect of flumecinol (Zixoryn) on the cytochrome P450 and cytochrome P448 dependent hepatic microsomal monooxygenase activities in male rats.

The effect of three-day oral administration of 50 mg/kg bw. and 100 mg/kg bw. flumecinol (Zixoryn, Gedeon Richter Chemical Works Ltd., Budapest, Hungary) and intraperitoneal administration of 50 mg/kg bw. phenobarbital as well as the single intraperitoneal administration of 20 mg/kg bw. 3-methylcholanthrene on various cytochrome P450 and P448 dependent hepatic microsomal enzyme activities was studied in male albino Wistar rats. 50 mg/kg bw. flumecinol had no significant effect. 100 mg/kg bw. flumecinol had an inducing effect comparable to the one of phenobarbital. The activity of the cytochrome P448 dependent 7-ethoxyresorufin O-deethylase was enhanced by all three substances, but flumecinol's effect was by far behind that of 3-methylcholanthrene, so the carcinogenic promoter effect of flumecinol can be questioned.     

Induction of P-450 isoenzyme activities in Syrian golden hamster liver compared to rat liver as probed by the rate of 7-alkoxyresorufin-O-dealkylation

The activities of 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-deethylase (PROD), 7-ethoxycoumarin-O-deethylase (ECOD) and aromatic hydrocarbon hydroxylase (AHH) were measured in hepatic microsomes from male and female Wistar rats and Syrian golden hamsters in order to probe the basal activity and the inducibility by phenobarbital (PB) and 3-methylcholanthrene (MC) of different P-450 isoenzymes. The basal activities of EROD and ECOD, but not PROD and AHH, were higher in male hamsters than in male rats. No sex-related difference in enzyme activities was observed with hamsters, whereas male rats had a higher ECOD and AHH activity than female rats. Induction by PB led to a 450-fold and 250-fold increase in PROD activity in male and female rat liver microsomes, respectively, while MC had a more pronounced inductive effect on EROD activity in this species. In hamsters, EROD activity was induced by MC but not by PB. Unexpectedly PROD activity in male and female hamster liver microsomes was only moderately induced by PB, the extent being lower than on induction by MC. Therefore, the activity of PROD, which is useful as a specific enzymatic assay for P-450 IIB in the rat liver, cannot be used to probe PB-like inducers in the hamster liver.     

Phenolsulphonphthalein conjugation and biliary excretion in the rat: influence of phenobarbital and 3-methylcholanthrene

The effect of phenobarbital and 3-methylcholanthrene pretreatment on the biliary excretion of phenolsulphonphthalein (PSP) was investigated in male Wistar rats. The dye was injected at a single dose of 200 mumol/kg body wt. About 20% of the compound was excreted as a glucuronide in the controls, the liver UDP-glucuronyltransferase activity toward PSP being 0.064 +/- 0.005 nmol.min-1.mg protein-1. Treatment for two weeks with phenobarbital (354 mumol.kg body wt-1.day-1) caused a transient increase in conjugated and unconjugated PSP excretion, but glucuronyltransferase activity was not modified. 3-Methylcholanthrene pretreatment for 4 days (75 mumol.kg body wt-1.day-1) also enhanced biliary excretion of the dye, but the increase corresponded only to the glucuronide and glucuronyltransferase activity was significantly enhanced by 20%. Our data indicate that not only the rate of biotransformation but also other factors could be responsible for increased PSP biliary excretion following administration of microsomal enzyme inducers.     

Demonstration of functional heterogeneity of hepatic uridine diphosphate glucuronosyltransferase activities after administration of 3-methylcholanthrene and phenobarbital to rats.

After the administration of 3-methylcholanthrene to adult male rats, activities of hepatic UDP-glucuronosyltransferase towards six from a group of 12 substrates were stimulated by 250-350%. Activities towards the remaining six substrates were unaffected. Conversely, after phenobarbital administration, activities formerly stimulated by 3-methylcholanthrene remained unchanged, and the other six activities were stimulated by 160-280%. The relationship of these two groups of transferase activities to other evidence suggesting the same heterogeneity of the enzyme is discussed.     

Adaptive responses of rat liver to the gestagen and anti-androgen cyproterone acetate and other inducers. I. Induction of drug-metabolizing enzymes

Treatment of female Wistar rats with cyproterone acetate (CPA) was shown to cause pronounced increases of hepatic microsomal monooxygenase activity towards the following substrates: ethylmorphine (EM), aminopyrine (AP), benzphetamin (BPA) and benzo[a]pyrene (BP). Minor increases were seen using p-nitroanisole (pNA) and aniline (AN). Monooxygenase activity reached maximal levels within 24 h. The effects were dose-dependent, the threshold dose being about 4 mg/kg, and were reversible within 6 days. The results of comparative studies with several ‘classical’ microsomal enzyme inducers, i.e. pregnenolone-(16α)-carbonitrile (PCN), phenobarbital (PB), α-hexachlorocyclohexane (α-HCH) and 3-methylcholanthrene (3-MC) suggest that CPA belongs to the PCN-type and α-HCH to the phenobarbital type of inducers. In male rats CPA induced only moderate increases of monooxygenase activities which can be explained by decreased testosterone secretion due to anti-gonadotropic effect of CPA.     

Induction of drug metabolizing enzymes in human liver cell line Hep G2

Human cytochrome P-450, UDP-glucuronosyltransferase and sulphotransferase activities have been measured in the cell line Hep G2 following treatment of cells with 3-methylcholanthrene or phenobarbital. 3-Methylcholanthrene treatment caused a 20-30-fold increase in the O-deethylation of 7-ethoxycoumarin. The glucuronidation and sulphation of the product 7-hydroxycoumarin were increased 36 and 7 fold, respectively. In comparison, phenobarbital treatment did not increase these activities significantly. However, phenobarbital-inducible proteins were identified on "Western blots' using antibodies to a rat liver phenobarbital inducible P-450 form. The molecular masses of the proteins did not coincide with those expected for cytochromes P-450. However, characteristic of P-450 forms, the synthesis of these proteins was suppressed by 3-methylcholanthrene treatment. The Hep G2 cell line represents a potentially useful model for studying the regulation of human P-450 genes.     

Effect of microsomal enzyme inducing agents on hepatic biotransformation in cotton rats (Sigmodon hispidus): comparison to that in Sprague-Dawley rats.

1. Activities of several biotransformation enzymes were determined in male and female Sigmodon hispidus. Benzphetamine N-demethylase and glutathione S-transferases toward 1-chloro-2,4-dinitrobenzene and sulfobromophthalein were higher in male Sigmodon hispidus than the female animals. 2. The study also determined the effect of microsomal enzyme inducing agents on hepatic biotransformation in male Sigmodon hispidus. 3. Cytochrome P-450 concentration was similar in cotton and Sprague-Dawley rats, and was increased after phenobarbital, pregnenolone-16 alpha-carbonitrile, or 3-methylcholanthrene treatment. 4. Benzphetamine N-demethylase was 4-fold higher in Sigmodon hispidus and was induced by 75-100% after phenobarbital. 5. UDP-Glucuronosyltransferase toward estrone, 1-naphthol, diethylstilbestrol and testosterone was 2- to 4-fold higher in cotton rats and was not altered by treatment with the inducing agents. 6. Conjugation of 1-chloro-2,4-dinitrobenzene, ethacrynic acid and sulfobromophthalein with glutathione was similar in both rodent species and was not inducible. 7. Sulfation of 2-naphthol was 15-30% of that in Sprague-Dawley rats and was not increased by inducer administration.     

Phenobarbital inducible UDP-glucuronosyltransferase is responsible for glucuronidation of 3'-azido-3'-deoxythymidine: characterization of the enzyme in human and rat liver microsomes

Glucuronidation by liver microsomes of 3'-azido-3'-deoxythymidine (AZT) was characterized in human and in various animal species. The glucuronide isolated by HPLC, was identified by mass spectrometry (fast atom bombardment, desorption in chemical ionization), and beta-glucuronidase hydrolysis. AZT glucuronidation reaction in liver microsomes of human and monkey proceeded similarly with an apparent Vmax of 0.98 nmol/min/mg protein and apparent Km of 13 mM. Oleoyl lysophosphatidylcholine activated more than twofold the formation of the glucuronide. Human kidney microsomes could also biosynthesize AZT glucuronide, although to a lower extent (six times less than the corresponding liver). Probenecid, which is administered to AIDS patients, decreased hepatic AZT glucuronidation in vitro (I50 = 1.5 mM), whereas paracetamol did not exert any effect at concentrations up to 21.5 mM. Morphine also inhibited the reaction (I50 = 2.7 mM). AZT glucuronidation presented the highest rate in human and in monkey (0.50 nmol/min/mg protein); pig and rat glucuronidated the drug two and three times less, respectively. In Gunn rat, the specific activity in liver microsomes was similar (0.18 nmol/min/mg protein) to that of the congenic normal strain; this suggests that an isozyme other than bilirubin UDP-glucuronosyltransferase catalyzed the reaction. In rats, AZT glucuronidation was stimulated fourfold by phenobarbital; 3-methylcholanthrene or clofibrate failed to increase this activity. This result was consistent with the bulkiness of the AZT molecule (thickness 6.7 A), which is a critical structural factor for glucuronidation of the drug by phenobarbital-induced isozymes. Altogether, the results strongly indicate that UDP-glucuronosyltransferase (phenobarbital inducible forms) is responsible for AZT glucuronidation.     

Studies on the genetic linkage of bilirubin and androsterone UDP-glucuronyltransferases by cross-breeding of two mutant rat strains.

Gunn rats, which have defects in bilirubin and 4-nitrophenol UDP-glucuronyltransferases (GT), were crossed with LA Wistar rats with a defect in androsterone GT. The F1 hybrids showed normal GT activities towards androsterone, bilirubin and 4-nitrophenol, demonstrating that Gunn and LA ('low activity') Wistar rats inherit a homozygous dominant trait for androsterone GT and bilirubin GT respectively. The F2 progeny showed four different combinations of bilirubin and androsterone GT activities: defects in both GT activities, a single defect in bilirubin GT activity, a single defect in androsterone GT activity and two normal GT activities. They were segregated in the approximate ratio of 1:3:3:9, which is compatible with Mendel's Principle of Independent Assortment. These results provide evidence that androsterone GT and bilirubin GT are located on different chromosomes. In the F2 generation, defective bilirubin and 4-nitrophenol GT activities were not segregated, indicating that these two mutant genes are closely linked on the same chromosome.