[CANCER RESEARCH 47, 2028-2031, April 15, 1987]

yV-Glucuronidation of Carcinogenic Aromatic Amines Catalyzed by Rat

Hepatic Microsomal Preparations and Purified Rat Liver Uridine5 '-Diphosphate-Glucuronosyltransferases1

Mitchell D. Green and Thomas R. Tephly2

Department of Pharmacology, University of Iowa, Iowa City, Iowa 52242

ABSTRACT

The .\-|>lururonidut ¡onof three carcinogenic aromatic amines (4-ami-nobiphenyL, a-naphthylamine, and 0-naphthylamine) was investigated in

hepatic microsomal preparations from two rat strains. In preparationsfrom Wistar rats, individual variability was observed for the glucuroni-

dation of the arylamines. This variability correlated with high and lowlevels of 3a-hydroxysteroid UDP-glucuronosyltransferase (UDPGT) in

hepatic microsomal preparations from Wistar rats. This individual variability was not observed in Sprague-Dawley rat hepatic microsomalpreparations because hepatic 3a-hydroxysteroid UDPGT levels do not

vary in this strain of rats.Five highly purified rat liver UDPGTs were investigated for their

ability to catalyze the conjugation of the aromatic amines. Of the purifiedenzymes investigated, only 3a-hydroxysteroid UDPGT catalyzed theglucuronidation of 4-aminobiphenyl. a-Naphthylamine and 0-naph-thylamine conjugations were catalyzed by 3a-hydroxysteroid, 17/8-hy-droxysteroid, and 3-methylcholanthrene-inducible p-nitrophenol

UDPGTs. The three aromatic amines did not serve as substrates forpurified digitoxigenin monodigitoxoside or phenobarbital-inducible mor

phine UDPGTs.The results show that /V-glucuronide formation can be catalyzed by

UDPGT isofonns which also catalyze the formation of 0-glucuronides.In addition, variable levels of 3a-hydroxysteroid UDPGT in Wistar rat

liver may have toxicological significance for substrates of this isoenzyme.

INTRODUCTION

Many aromatic amines, such as /3-naphthylamine and 4aminobiphenyl, are known to induce tumors, most notably inthe urinary bladder (1,2). It has been shown that the metabolicactivation of arylamines can be an important factor in determining their carcinogenic potential (2). 7V-Hydroxylation ofarylamines has been demonstrated to be involved in producingtoxicity from these compounds (3,4). Another major metabolicpathway for aromatic amines, which may compete with N-hydroxylation and thereby influence carcinogenic potential, isJV-glucuronidation (5, 6). Glucuronidation of xenobiotics isgenerally considered to be a significant step in their detoxicationand elimination from the body. However, arylamine W-glucu-ronides are labile and may be easily hydrolyzed to the parentamine under weakly acidic conditions which can exist in theurinary bladder (7). Therefore, accumulation of the W-glucuro-nide in the bladder may result, ultimately, in exposure of thisorgan to the relatively lipophilic and potentially carcinogenicparent arylamine.

Conjugation of endogenous and exogenous chemicals withglucuronic acid is a process catalyzed by UDPGTs3 (EC

2.4.1.17). A number of UDPGTs have been purified to homo-

Received10/6/86; revised 1/20/87; accepted 1/23/87.The costs of publication of this article were defrayedin part by the payment

of page charges. This article must therefore be hereby marked advertisementinaccordancewith 18 U.S.C. Section 1734solelyto indicatethis fact.

1This work was supported by NIH Grant GM26221.2To whom requests for reprints should be addressed.5The abbreviations used are: UDPGT, UDP-glucuronosyltransferase; HA,

high activity; LA, low activity; A',,, apparent competitive inhibition constant

determined from slope versus inhibitor concentration of secondary plots derivedfrom primary double reciprocal plots.

geneity from rat hepatic microsomes, and their substrate specificities towards important endogenous substrates and xenobiotics have been determined. Several purified UDPGTs havestringent substrate specificities towards physiological substratessuch as 17j3-hydroxysteroids (8), 3a-hydroxysteroids and bileacids (9), estrone (10, 11), and bilirubin (12, 13). In contrast,investigations of the reactivity of some xenobiotics such as p-nitrophenol, a-naphthol, or 4-methylumbelliferone with purified UDPGTs have demonstrated overlapping substrate specificities (8, 11). Other xenobiotics, such as morphine (14) anddigitoxigenin monodigitoxoside (15), are also conjugated byspecific UDPGT isozymes. It is not known whether the N-glucuronidation of carcinogenic aromatic amines is catalyzedby a single form or more than one form of hepatic UDPGT.Isofonns III and IV of Roy Chowdhury et al. (11) have beenshown to catalyze the /V-glucuronidation of aniline.

Preliminary studies on the JV-glucuronidation of aromaticamines catalyzed by 17/3-hydroxysteroid and 3a-hydroxysteroidUDPGTs showed that a-naphthylamine conjugation could becatalyzed by both isozymes (16). The purpose of the presentstudy was to extend that work and to compare 5 highly purifiedUDPGTs for their ability to mediate the Y-glucuronidation ofthree arylamines: a-naphthylamine; ß-naphthylamine; and 4-aminobiphenyl. The UDPGTs were characterized as to theirreactivity with the aromatic amine substrates.

MATERIALS AND METHODS

Chemicals. [4-14C]Testosterone (50 mCi/mmol) and [l,2-3H]andros-

terone (40 to 50 Ci/mmol) were purchased from New England Nuclear(Boston, MA). p-Nitro [i/-14C]phenol (79 mCi/mmol) was obtainedfrom Amersham (Arlington Heights, IL). a-Naphthylamine, /3-naph-thylamine, 4-aminobiphenyl, UDP-glucuronic acid (ammonium salt),testosterone, androsterone, /7-nitrophenol, and L-a-phosphatidylcholine(egg yolk, type XI-E) were obtained from Sigma Chemical Co. (St.Louis, MO). Emulgen 911 was a gift from Kao Atlas (Tokyo, Japan).

Tissues and Enzyme Preparations. Male Wistar (Charles River, Wilmington, MA) or Sprague-Dawley (Biolabs, St. Paul, MN) rats (180 to220 g) were used in these studies. The animals were housed over wiremesh in stainless-steel cages and were allowed food (Purina rodentchow; Purina, St. Louis, MO) and water ad libitum. The light-darkcycle was 6:00 a.m. to 6:00 p.m. All rats were deprived of food overnightbefore preparation of hepatic microsomes.

For preparation of hepatic microsomes, the rats were killed bycerebral concussion and decapitation, and their livers were perfused insitu with ice-cold 1.15% K( I. Livers were then removed and homogenized in 4 volumes of 1.15% KC1. Microsomes were then isolated bydifferential centrifugation and stored at -70°C with a 1.15% KC1

overlay (17).3o-Hydroxysteroid, 17/3-hydroxysteroid, and a 3-methylcholan

threne-inducible UDPGT were purified to homogeneity from hepaticmicrosomal preparations by chromatofocusing and affinity chromatog-raphy (8, 9). Morphine UDPGT was purified to homogeneity from thelivers of phenobarbital-treated rats as reported by Puig and Tephly (14).Digitoxigenin monodigitoxoside UDPGT activity was separated andpartially purified as described by von Meyerinck et al. (15).

Enzyme Assays and Kinetic Analysis. For the determination of mi-

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AROMATIC AMINE /V-GLUCURONIDATION

crosomal UDPGT activities, rat liver microsomes were suspended in25 mM monoethanolamine buffer, pH 9.4, containing 20% glycerol and0.1 mM dithiothreitol. The suspensions were then solubilized by theaddition of 0.5 mg of Emulgen 911/mg of microsomal protein. Aliquotsof the preparations were used in the enzyme assays. Testosterone andandrosterone UDPGT assays were performed as described previously(8, 17). Steroid UDPGT activities were determined by incubatingsolubilized microsomes (20 to 50 ¿ig)in reaction mixtures containingradiolabeled steroid, 50 mM Tris-HCl buffer, pH 8.0, 10 mM MgCl2,100 /ig/ml phosphatidylcholine, and 5 mM UDP-glucuronic acid. Theconcentrations of testosterone and androsterone were 50 and 56 uM,respectively. a-Naphthylamine, 0-naphthylamine, and 4-aminobiphenyljV-glucuronidation assays were conducted using the spectrofluorometricmethod of Lilienblum and Bock (18). The reaction mixtures containedsolublized microsomes (20 to 50 //g), 100 mM Tris-HCl buffer, pH 7.4,10 mM MgCh, 100 fig/ml phosphatidylcholine, 5 mM UDP-glucuronicacid, and 1 mM arylamine. The final volume of all reaction mixtureswas 1.0 ml. The enzymatic reaction was initiated by the addition ofUDP-glucuronic acid, and the assays were conducted at 37°Cfor 10

min. All reactions were run under conditions of linearity with respectto time and protein concentration. Protein was determined by themethod of Bradford (19) using the Bio-Rad protein assay kit.

For quantitation of the arylamine glucuronides, the reaction mixturewas extracted using chloroform to remove the unreacted amine substrate. An aliquot (1.0 ml) of 1.0 M HC1 was then added, and the Aglucuronides were allowed to hydrolyze overnight. The acidic reactionmixture was then neutralized using 2 M Tris (pH 9.0). The amount ofglucuronide formed was quantitated using fluorescence concentrationstandard curves of the aromatic amines as standards. Fluorescencewavelengths of 325-nm excitation and 447-nm emission (a-naphthyl-amine), 299-nm excitation and 388-nm emission (4-aminobiphenyl),and 335-nm excitation and 413-nm emission(/3-naphthylamine) wereused for quantitation.

The apparent A',,, for the aromatic amines was determined from

double reciprocal plots of initial velocity versus substrate concentration(20). Inhibition studies were conducted using variable concentrationsof substrate (/>-nitmplu-nol, testosterone, or androsterone) at a fixedconcentration of UDP-glucuronic acid (5 mM) in the presence orabsence of arylamine. A„for the various aromatic amines was determined as described (20).

RESULTS

Aromatic Amine Glucuronidation by Rat Liver Microsomes.Glucuronidation rates for a-naphthylamine, /3-naphthylamine,and 4-aminobiphenyl were determined using Sprague-Dawleyrat hepatic microsomal preparations (Table 1). The microsomalrate for glucuronidation of a-naphthylamine is high, beingcomparable to that observed for p-nitrophenol. The rate ofconjugation for /i naphtlnlamine is approximately one-tenththat of a-naphthylamine. The microsomal glucuronidation ratefor 4-aminobiphenyl in these preparations was one-twentieththat for a-naphthylamine.

Table 1 Glucuronidation rates of arylamines in solubilized Hepatic microsomalpreparations from Sprague-Dawley and Wistar rats

Solubilization of microsomes and assay conditions are described in "Materialsand Methods." Enzymatic rates are expressed as nmol substrate conjugated/min/

mg protein.

UDPGT activity

p-NitrophenolTestosteroneAndrosteronea-Naphthylaminetf-Naphthylamine4-AminobiphenylSprague-Dawley"89.4

±3.7*

5.5 ±0.312.5 ±1.260.3 ±0.5

6.6 ±0.53.4 ±0.5HAWistar71.5

±2.65.9 ±0.2

10.8 ±0.565.2 ±4.311.9 ±2.03.4 ±0.2LA

Wistar68.1

±4.85.7±0.2

0.8 ±0.140.3 ±1.5

5.8 ±2.50.8 ±0.1

" Some of these data are taken from Green et al. (16) and are included for

comparative purposes.* Mean ±SE for at least 4 animals.

Unlike Sprague-Dawley rats, Wistar rats possess individualvariability with respect to androsterone glucuronidation becauseof genetic differences in levels of 3«-hydroxysteroid UDPGT(21). Therefore, Wistar rat liver microsomes, prepared fromindividual rats, can be used to predict the reactivity of varioussubstrates with this UDPGT isoform. The rate of conjugationof androsterone in HA Wistar rat hepatic microsomal preparations is approximately 10 times higher than that of LApreparations (Table 1). As has been demonstrated previously(22), no differences were observed in the glucuronidation ratesfor testosterone or p-nitrophenol. In contrast, differences inglucuronidation rates were observed for the arylamine substrates in HA and LA Wistar rat hepatic microsomal preparations. The glucuronidation activity towards a-naphthylaminein LA hepatic microsomal preparations was approximately 60%of that seen in HA preparations. The rates of glucuronidationof j8-naphthylamine (—50%)and 4-aminobiphenyl (—75%)werealso lower in hepatic microsomal preparations from LA Wistarrats. These results strongly suggest that the three aromaticamines may serve as substrates for 3o-hydroxysteroid UDPGTin rat liver.

The apparent Km and Vm„for androsterone (a substrate for3a-hydroxysteroid UDPGT) and 4-aminobiphenyl were determined in activated hepatic microsomal preparations from HAand LA Wistar rats. As we reported earlier (22) the apparentA",,,for androsterone in HA microsomes (5 MM)was approxi

mately 20-fold lower than in LA microsomes (120 UM). Incontrast, the apparent A,,, for 4-aminobiphenyl in HA microsomes (1.6 mM) is 8-fold higher than that in LA microsomes(0.2 mM). Interestingly, the maximal velocity for 4-aminobiphenyl iV-glucuronidation in hepatic microsomal preparationsfrom HA Wistar rats (8.5 nmol/min/mg protein) is also 8-foldhigher than that from LA rats (1.0 nmol/min/mg protein).Thus the ratio of Km to Vmaxfor 4-aminobiphenyl is constantin LA and HA Wistar rat liver microsomal preparations. Attempts to determine the kinetic parameters for a-naphthyla-mine and p'-naphiIn lamine- in hepatic microsomal preparations

resulted in nonlinear double reciprocal plots. We have foundthis to be the case when a substrate is conjugated by more thanone UDPGT in a complex preparation such as hepatic microsomes.

^-Glucuronidation of Arylamines by Purified Rat Liver UDP-Glucuronosyltransferases. a-Naphthylamine and /3-naphthyla-mine were tested as substrates for five purified UDPGTs andfound to be substrates for glucuronide conjugation catalyzed byp-nitrophenol UDPGT (3-methylcholanthrene inducible), 17/3-hydroxysteroid UDPGT, and 3a-hydroxysteroid UDPGT (Table 2). Morphine (phénobarbital inducible) and digitoxigeninmonodigitoxoside UDPGTs appear to be incapable of catalyzing the conjugation of arylamines with glucuronic acid. Animportant finding was that glucuronidation of 4-aminobiphenylwas selectively catalyzed by the purified 3a-hydroxysteroidUDPGT.

Even though three separate UDPGTs catalyze the conjugation of a-naphthylamine and onaphi In lamine, the kinetic parameters towards these substrates are different for the differentisozymes (Table 3). The lowest apparent Km values for a-naphthylamine and /3-naphthylamine were observed using the/Miifrophenol UDPGT (3-methylcholanthrene inducible) iso-form. In contrast, the highest V^ for these substrates wasobserved with 17/3-hydroxysteroid UDPGT, suggesting that thismay be the major constitutive isoform responsible for theglucuronidation of these arylamines.

All three aromatic amines were competitive inhibitors of2029

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AROMATIC AMINE W-GLUCURONIDATION

Table 2 N-Glucuronidation of aromatic amines by purified UDPGTs

Specific activities are expressed as nmol substrate conjugated/min/mg protein for the purified enzyme.

UDPGTisoenzymeAr)

laminea-Naphthylamined-Naphlhylaminc

4-Aminobiphenylp-Nitrophenol

(3-methylcholanthreneinducible)380

±27°

26 ±30170-Hydroxy-

sleroid1750

±7279 ±15

03o-Hydroxy-

steroid730

±6089 ±32

140 ±42Morphine

(phénobarbitalinducible)"0

ND0Digitoxigenin

monodigitox-oside*NO*

00

' From Puig and Tephly (14).* From von Meyerinck a al. (15).' Mean ±SD of at least three different preparations of purified enzyme.' ND. not determined.

Table 3 Kinetic characterization ofarylamine glucuronidationby purified UDPGTs

The apparent A'mvalues are expressed in inMand were determined at a constant

(5 IHMi concentration of UDP-glucuronic acid. The maximal velocity is expressedas nmol substrate conjugated/min/mg purified enzyme.

UDPGT isoenzyme

p-Nitrophenol(3-methylcholanthrene 17/3-Hydroxy-

Arylamine inducible)steroida-Naphthylamine

KmV—„V~JKmd-NaphthylamineKmv«,V«JA.4-Aminobiphenyl

Kmv««.v«^*.0.58

+ 0.10°

610 ±6210500.60

±0.1546 ±5.0771.6

±0.53900 ±650

24401.3

±0.2211 ±60

1603a-Hydroxy-

steroid1.9

±0.42050 ±170

11404.2

±0.2350 ±40831.3

±0.3266 ±80

205' Mean •SD of determination using at least three different enzyme prepara

tions.

Table 4 Inhibition of UDPGTs by arylaminesAU the aromatic amines were competitive inhibitors. p-Nitrophenol was the

primary substrate (0.2 to 4.0 m\ii for p-nitrophenol (3-methylcholanthrene inducible) UDPGT. Testosterone (5.0 to 50 «IM)was the primary substrate for 170-hydroxysteroid UDPGT. Androsterone (5.6 to 56 ,<M)was the primary' substratefor 3o-hydroxysteroid UDPGT. The concentration of UDP-glucuronic acid was5.0 niM.

Apparent A .

p-Nitrophenol(3-methylcholanthrene 17/3-Hydroxy-

inducible) steroidInhibitor (UDPGT)(UDPGT)a-Naphthylamine

/j-Naphthylamine4-Aminobiphenyl0.38*

0.420.390.42

0.280.363a-Hydroxy-

steroid(UDPGT)0.07

NI»

0.20" Values represent mM.* NI, not inhibitory at 2 mM.

glucuronidation catalyzed by /»-nitrophenol (3-methylcholanthrene inducible), 170-hydroxysteroid, and 3a-hydroxysteroid

UDPGTs even though they were not substrates for all the givenisozymes. The inhibition constants for the aromatic amines aregiven in Table 4. The KK values for a-naphthylamine and ß-naphthylamine inhibition of p-nitrophenol glucuronidation catalyzed by the 3-methylcholanthrene-inducible UDPGT are similar to their apparent A'mvalue as substrates. In contrast, the A,,

values for inhibition of glucuronidation of testosterone (17.;hydroxysteroid UDPGT isoform) and androsterone (3a-hy-

droxysteroid UDPGT isoform) are much lower than the apparent A'mvalues. /3-NaphthyIamine (at a concentration of 2 IHM)

did not inhibit androsterone glucuronidation activity catalyzedby 3a-hydroxysteroid UDPGT. This observation is probably

due to the relatively poor reactivity of ß-naphthylamine withthis isozyme (Table 3).

DISCUSSION

The present study has shown that A'-glucuronidation of aro

matic amines is catalyzed by purified UDPGTs. Surprisingly,UDPGTs which were thought to be specific for steroid O-conjugation can also catalyze the JV-glucuronidation of aryl-amines. UDPGTs purified from rat liver have been shown toexhibit strict substrate specificity for important endogenoussubstrates such as estrone (IO, 11), bilirubin (12, 13), testosterone (8), androsterone (8), and bile acids (9). However, as shownin this study and others (8, 11), purified UDPGTs can be lessspecific for small phenolic, planar molecules. The exception tothis observation appears to be 4-aminobiphenyl which wasconjugated exclusively by 3a-hydroxysteroid UDPGT.

Human liver microsomal preparations (18) and more recentlypurified UDPGTs isolated from human liver (23) have beenshown to catalyze the conjugation of carcinogenic aromaticamines with glucuronic acid. As with UDPGTs purified fromrat liver, two and possibly three, purified human UDPGTsexhibited less specificity for the conjugation of small planar,aromatic phenols and amines (24). One human isoform (elutingfrom a chromatofocusing column around pH 7.4) catalyzed theglucuronidation of a-naphthylamine, p-nitrophenol, and 4-methylumbelliferone. A second human isoform (eluting from achromatofocusing column around pH 6.2) catalyzed the glucuronidation of the preceding three substrates as well as 4-aminobiphenyl. A possible third isoform was also separated(pH 7.7) which had a similar substrate profile as the pH 6.2isoform.4

The present study has shown that a-naphthylamine and ß-naphthylamine conjugation in rat liver is mediated by a 3-methylcholanthrene-inducible UDPGT, but that 4-aminobiphenyl glucuronidation is not. These data are in agreement withinduction studies of others (18) where it was observed that a-naphthylamine and 0-naphthylamine, but not 4-aminobiphenylglucuronidation were increased by 3-methylcholanthrene pretreatment. Our results using morphine (a phenobarbital-in-ducible isoform) UDPGT indicate that this isoform is notresponsible for the increase in hepatic microsomal a-naphthylamine and ß-naphthylamine glucuronidation activities observed after phénobarbitalpretreatment (18, 25).

Sprague-Dawley rat hepatic microsomal rates for the glucuronidation of. ; naphi h\ lam ¡IK'and 4-aminobiphenyl were one-tenth and one-twentieth, respectively, that for the conjugationof a-naphthylamine. These results are identical to those obtained by Lilienblum and Bock (18) using activated microsomal

4 Y. Irshaid and T. R. Tephly, unpublished observation.

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AROMATIC AMINE ^-GLUCURONIDATION

preparations from untreated male Wistar rat livers. However,in contrast to the work of these investigators, we found differential conjugation rates for these substrates in microsomes fromWistar rats when the rat liver preparations had been separatedbased on their hepatic 3-a-hydroxysteroid UDPGT activity. Inaddition the results of Wang et al. (25) seem to confirm ourdata. These workers reported a highly variable rate (1.3 ±1.3nmol/min/mg protein) for 4-aminobiphenyl glucuronidation inactivated microsomal preparations from female CD rats. Theirresults were probably due to the presence of high and low 3a-hydroxysteroid UDPGT activity in animals of this strain. Theuse of HA and LA 3«-hydroxysteroid UDPGT activity microsomal preparations from Wistar rat liver appears to be aneffective means of screening substrates for their reactivity towards this isoform.

The present study shows that the apparent Km values foraromatic amines for the p-nitrophenol UDPGT are similar totheir apparent Afisvalues as inhibitors of /»-nitrophenol glucuronidation. To date the p-nitrophenol UDPGT has been shownto conjugate only planar phenols, and now planar aromaticamines. In addition, we have recently shown that, when steroidsare used as alternate substrate inhibitors for steroid UDPGTs,the apparent A",,is similar to the apparent A',,,(26). In most

cases the apparent K¡,of an alternate substrate is similar to theapparent Km(27) provided that the reaction mechanism of thealternate substrate is the same as that of the variable substrate.Our data, therefore, suggest that the glucuronidation of structurally similar substrates proceeds by the same enzymatic mechanism. The relationship between the apparent Km and KKdoesnot hold for the arylamines as competitive inhibitors of steroidglucuronidation by the two purified steroid-specific UDPGTs.These results could be due to the dissimilar chemical nature ofthe steroids and arylamines. Our data, therefore, strongly suggest that the enzymatic reaction mechanism of glucuronidationcatalyzed by purified steroid UDPGTs for these two chemicallydiverse classes of substrates is different. Further kinetic studieswill be necessary to fully elucidate the enzymatic mechanismsfor these enzymes.

The present study indicates that caution should be used whenWistar rats, or other strains of rats with variable levels of 3a-hydroxysteroid UDPGT (28), are used in carcinogenesis studiesinvolving aromatic amines. Low hepatic levels of 3«-hydroxy-steroid UDPGT may place a strain on the in vivo ability of theanimals to detoxify 4-aminobiphenyl and /3-naphthylaminethrough glucuronide conjugation. Matsui and Hakozaki (29)have demonstrated that LA Wistar rats also exhibited impairedbiliary excretion of androsterone glucuronide after administration of the steroid. Thus, the deficiency of 3a-hydroxysteroidUDPGT activity which is observed in vitro appears to also holdfor the in vivo situation.

REFERENCES

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6. Poupko, J. M., Hearn, W. L., and Radomski. J. L. ,V-Glucuronidation of/V-hydroxy aromatic amines: a mechanism for their transport and bladder-specific carcinogenicity. Toxicol. Appi. Pharmacol., 50:479-484, 1979.

7. Kadlubar, F. F., Unruh, L. E.. Flammang, T. J., Sparks, D., Miu hum, R.K., and Mulder, G. J. Alteration of urinary levels of the carcinogen, /V-hydroxy-2-naphthylamine, and its jY-glucuronide in the rat by control ofurinary pH, inhibition of metabolic sulfation, and changes in biliary excretion. Chem.-Biol. Interact., 33: 129-147, 1981.

8. Falany, C. N., and Tephly, T. R. Separation, purification, and characterization of three isoenzymes of UDP-glucuronyltransferase from rat liver micro-somes. Arch. Biochem. Biophys., 227: 248-258, 1983.

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10. Falany, C. N., Roy Chowdhury, J., Roy Chowdhury, N., and Tephly, T. R.Steroid 3- and I7-OH UDP-glucuronosyltransferase activities in rat andrabbit liver microsomes. Drug Metab. Dispos., Il: 426-432, 1983.

11. Roy Chowdhury, J., Roy Chowdhury, N., Falany, C. N.. Tephly, T. R., andArias, I. M. Isolation and characterization of multiple forms of rat liverUDP-glucuronate glucuronosyltransferase. Biochem. J., 233:827-837,1986.

12. Burchell, B., and Blanckaert, N. Bilirubin mono- and di-glucuronide formation by purified rat liver microsomal bilirubin UDP-glucuronyltransferase.Biochem. J., 223:461-465, 1984.

13. Roy Chowdhury, N., Arias, I. M., Lederstein, M., and Roy Chowdhury, J.Substrates and products of purified rat liver bilirubin UDP-glucuronosyltransferase. Hepatology, 6: 123-128, 1986.

14. Puig, J. F., and Tephly, T. R. Isolation and purification of rat liver morphineUDP-glucuronosyltransferase. Mol. Pharmacol., 30: 558-565, 1986.

15. von Meyerinck, L., Coffman, B. L., Green, M. D., Kirkpatrick, R. B.,Si Immilli. A., and Tephly, T. R. Separation, purification, and characterization of digitoxigenin mono-digitoxoside UDP-glucuronosyltransferase activity. Drug Metab. Dispos., 13: 700-704, 1985.

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17. Tukey, R. H., Billings. R. E., and Tephly, T. R. Separation of oestrone UDP-glucuronyltransferase and p-nitrophenol UDP-glucuronyltransferase activities. Biochem. J.. 171:659-663, 1978.

18. Lilienblum, W., and Bock. K. W. A'-Glucuronide formation of carcinogenicaromatic amines in rat and human liver microsomes. Biochem. Pharmacol.,55:2041-2046, 1984.

19. Bradford, M. A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein-dye binding. Anal.Biochem., 72: 248-253, 1976.

20. Rudolph, F. B., and Fromm, H. J. Plotting methods for analyzing enzymerate data. In: D. L. Purich (ed.). Contemporary Enzyme Kinetics and Mechanism, pp. 53-73. New York: Academic Press, 1983.

21. Matsui, M., and Nagai, F. Genetic deficiency of androsterone UDP-glucuronosyltransferase activity in Wistar rats is due to the loss of ezyme protein.Biochem. J., 234: 139-144, 1986.

22. Green, M. D., Falany, C. N., Kirkpatrick, R. B., and Tephly, T. R. Straindifferences in purified rat hepatic 3a-hydroxysteroid UDP-glucuronosyltransferase. Biochem. J., 230:403-409, 1985.

23. Irshaid, Y., Nghiem, D. D., and Tephly, T. R. Purification of a human UDP-glucuronosyltransferase. Fed. Proc., 45:933, 1986.

24. Irshaid. Y., and Tephly, T. R. Isolation and purification of two human liverUDP-glucuronosyltransferases. Mol. Pharmacol., 57:27-34. 1987.

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1987;47:2028-2031. Cancer Res   Mitchell D. Green and Thomas R. Tephly 

-Diphosphate-Glucuronosyltransferases′Uridine 5by Rat Hepatic Microsomal Preparations and Purified Rat Liver

-Glucuronidation of Carcinogenic Aromatic Amines CatalyzedN

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