frequency of urination and its effects on metabolism...

[CANCER RESEARCH 51. 4371-4377. August 15. 1991]

Frequency of Urination and Its Effects on Metabolism, Pharmacokinetics, BloodHemoglobin Adduci Formation, and Liver and Urinary Bladder DNA AdductLevels in Beagle Dogs Given the Carcinogen 4-Aminobiphenyl1

Fred F. Kadlubar, Kenneth L. Dooley, Candee H. Teitel, Dean W. Roberts, R. Wayne Benson, Mary Ann Butler,John R. Bailey, John F. Young, Paul W. Skipper, and Steven R. TannenbaumThe National Center for Toxicological Research, Jefferson, Arkansas 72079 fF. F. K., K. L. D., C. H. T., D. W. R., R. W. B., M. A. B., J. R. B., J. F. Y.J, and theMassachusetts Institute of Technology, Cambridge, Massachusetts 02178 [P. W. S., S. R. T.]

ABSTRACT

The human urinary bladder carcinogen, 4-aminobiphenyl (ABP), isknown to undergo hepatic metabolism to an A-hydroxy arylamine and itscorresponding /V-glucuronide. It has been proposed that these metabolitesare both transported through the blood via renal filtration to the urinarybladder lumen where acidic pH can facilitate the hydrolysis of the \-glucuronide and enhance the conversion of A"-hydroxy-4-aminobiphenyl

(N-OH-ABP) to a reactive electrophile that will form covalent adductswith urothelial DNA. Blood ABP-hemoglobin adducts, which have been

used to monitor human exposure to ABP, are believed to be formed byreactions within the erythrocyte involving N-OH-ABP that has entered

the circulation from the liver or from reabsorption across the urothelium.To test these hypotheses directly, experimental data were obtained fromfemale beagles given [3H)ABP (p.o., i.V., or intraurethrally), |3H|N-OH-ABP (i.v. or intraurethrally), or |3H|N-OH-ABP W-glucuronide (i.V.).

Analyses included determinations of total ABP in whole blood andplasma, ABP-hemoglobin adducts in blood erythrocytes, ABP and VOH-ABP levels (free and A'-glucuronide) in urine, urine pH, frequency

of urination (controlled by urethral catheter), rates of reabsorption ofABP and N-OH-ABP across the urothelium, and apparent volumes ofdistribution in the blood/tissue compartment. The major ABP-DNAadduct, AHguan-8-yl)-4-aminobiphenyl, was also measured in urothelial

and liver DNA using a sensitive immunochemical method. An analog/digital hybrid computer was then utilized to construct a multicompart-

mental pharmacokinetic model for ABP and its metabolites that separates: (a) absorption; (b) hepatic metabolism and distribution in bloodand tissues; (c) ABP-hemoglobin adduct formation; (d) hydrolysis and

reabsorption in the urinary bladder lumen; and (e) excretion. Using thismodel, cumulative exposure of the urothelium to free N-OH-ABP wassimulated from the experimental data and used to predict ABP-DNA

adduct formation in the urothelium.The results indicated that exposure to N-OH-ABP and subsequent

ABP-DNA adduct formation are directly dependent on voiding frequency

and to a lesser extent on urine pH. This was primarily due to the findingthat, after p.o. dosing of ABP to dogs, the major portion of the total N-OH-ABP entering the bladder lumen was free N-OH-ABP (0.7% of thedose), with much lower amounts as the acid-labile A'-glucuronide (0.3%

of the dose). The fraction of free/conjugated N-OH-ABP then decreased

with greater voiding intervals, as a consequence of the rapid reabsorptionof free N-OH-ABP across the urothelium. Accordingly, the model correctly predicted a 17-fold decrease in the level of urothelial DNA adducts

as average voiding frequency was increased from 3 times a day to acontinuous voiding for the first 12 h after dosing. This was also consistentwith data from intraurethral instillation of N-OH-ABP, which showed a>60-fold higher DNA adduct level than similarly administered ABP. Incontrast, blood ABP-hemoglobin adduct levels, which accounted for 8-

10% of the p.o. dose, were independent of voiding interval; and theamounts of free N-OH-ABP available for reabsorption in the bladderlumen were insufficient to account for the high ABP-hemoglobin adduct

levels observed.Thus, these data indicate that hemoglobin-ABP adduct formation is

Received 1/2/91; accepted 5/30/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by NIH Grants ES-00-597 and ES-02-109 to P. W. S. and

S. R. T.

likely to be a direct consequence of the hepatic /V-oxidation of ABP toN-OH-ABP and its entry into the circulation; and the measurement of

hemoglobin adduct levels can be regarded as valid index of both exposureand metabolic activation of this carcinogen in humans. In addition, thesestudies provide strong evidence for the role of unconjugated urinary N-OH-ABP in carcinogen-DNA adduct formation in the carcinogen-target

tissue and suggest that the frequency of urination should be a criticaldeterminant in human urinary bladder carcinogenesis.

INTRODUCTION

ABP2 is strongly carcinogenic for the urinary bladder of both

dogs and humans (1), and evidence for two separate metabolicactivation pathways has been presented. The first involves ahepatic cytochrome P-450IA2-catalyzed /V-oxidation (2, 3) toyield N-OH-ABP, which can be subsequently conjugated byhepatic glucuronyltransferases to form an /V-hydroxy arylamine/V-glucuronide (4). Both N-OH-ABP and its /V-glucuronide mayenter the circulation, undergo renal filtration, and thus enterthe lumen of the urinary bladder for subsequent excretion. Inthe blood, a major proportion of the free N-OH-ABP appearsto be further oxidized within the erythrocyte to 4-nitrosobi-phenyl which then forms a covalent addition product with asingle cysteine on hemoglobin, accounting for up to 10% of theABP dose administered (5, 6). In the urinary bladder lumen,infrequent voiding and an acidic urine would be expected topromote the hydrolysis of the A'-glucuronide and the release ofmore N-OH-ABP, followed by its reabsorption and covalentbinding to urothelial DNA (7). N-OH-ABP and other /V-hydroxy arylamines have long been shown to be direct-actingmutagens, to react spontaneously with nucleic acids and proteins under slightly acidic conditions, and to induce tumorformation at sites of application (reviewed in Refs. 8 and 9).Accordingly, in a previous study (10), urine pH and voidinginterval were proposed as important pharmacokinetic determinants for arylamine-induced urinary bladder carcinogenesis. Inaddition, a three-compartment pharmacokinetic model was developed, based on data from 2-naphthylamine-treated rats, thatwas predictive of individual and species differences in susceptibility to urinary bladder carcinogenesis by 2-naphthylamine.

A second metabolic activation pathway for arylamine bladdercarcinogens has been suggested to involve the peroxidation ofABP directly in the carcinogen-target tissue by the action ofurothelial prostaglandin hydroperoxidase. This peroxidase,which is present at relatively high levels in the bladder epithelium of both dogs (11) and humans (12), mediates the arachi-donic acid-dependent cooxidation of several arylamines toDNA-bound adducts (13). For benzidine, 2-aminofluorene, and2-naphthylamine, specific diimine or iminoquinone metaboliteshave been implicated as reactive intermediates leading to the

2The abbreviations used are: ABP, 4-aminobiphenyl; N-OH-ABP, iV-hydroxy-4-aminobiphenyl; HPLC, high pressure liquid chromatography.

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URINARY FREQUENCY AND ADDUCT FORMATION AFTER ABP

formation of DNA adducts that are unique to the peroxidativepathway (14-16). Studies with ABP have provided similarresults and indicate that the major peroxidase-derived adduci,although not yet identified, is chromatographically distinctfrom the major adducts formed by reaction of N-OH-ABP withDNA (17). The predominant DNA adducts from N-OH-ABPhave been characterized previously as yV-(deoxyguanosin-S-yl)-ABP, ./V-ideoxyguanosin-A^-yO-ABP, and A/-(deoxyadenosin-8-yl)-ABP (8, 18). While the peroxidative pathway appears to beprimarily responsible for urothelial DNA adduct formationfrom benzidine in the dog (14, 19), only about 20% of the DNAadducts formed from 2-naphthylamine (16) and about 10% ofthose formed from ABP (17) in the dog can be attributed toperoxidative activation.

Therefore, the reaction of urinary N-OH-ABP with urothelialDNA remains a probable mechanism to account for the majorDNA adducts formed from ABP in vivo. In this regard, N-(deoxyguanosin-S-yl)-ABP is not only the predominant adductformed from N-OH-ABP in vitro but has also been shown torepresent 65-70% of the total adducts found in the dog bladderepithelium and to be persistent in the target tissue (17, 18). Inorder to test this hypothesis further and to assess the relativeexposure of the bladder epithelium to N-OH-ABP as a functionof voiding interval and urine pH, experimental studies wereundertaken in the dog and a complex pharmacokinetic modelwas developed to determine the effect of these variables onurothelial ABP-DNA adduct formation. In addition, ABP-blood hemoglobin adduct levels, which have been shown to bean index of human exposure to ABP (6), were determinedconcomitantly in order to examine the relative contributions ofinitial hepatic TV-oxidation and subsequent urinary N-OH-ABPreabsorption to total hemoglobin adduct formation.

MATERIALS AND METHODS

Chemicals. [2,2'-'H]ABP (37 mCi/mmol) and unlabeled ABP were

obtained from Chem-Syn Science Laboratories, Lenexa, KS, and fromthe Aldrich Chemical Co., Milwaukee, WI, respectively. Both werepurified further by silica gel (grade 923; Davison Chem. Co., Baltimore,MD) column chromatography using benzene:ethyl acetate (9:1) as theeluent and by subsequent crystallization from n-hexane. Their puritywas judged to be >99% as determined by HPLC (see below). [2,2'-3H]

N-OH-ABP (92 mCi/mmol) and unlabeled N-OH-ABP were freshlyprepared by ammonium bisulfide reduction (20) of [3H]4-nitrobiphenyl(Chem-Syn) and 4-nitrobiphenyl (Aldrich), respectively. Their puritywas judged to be >95% as determined by HPLC. [2,2'-3H]N-OH-ABPA'-glucuronide (107 mCi/mmol) was prepared biosynthetically from[3H]N-OH-ABP (Chem-Syn) and characterized as described previously

(4), except that solutions were extracted with diethyl ether (and degassed) just prior to animal treatment to remove any aglycone formed.

Animal Studies. Female beagles (7-11 kg) were obtained from Marshall Farms USA, Inc., North Rose, NY, and were housed in dogmetabolism cages (Unifab Corp., Kalamazoo, MI) fitted with a flow-through pH meter that simultaneously recorded urine pH and time ofurination (21). Urine samples were obtained either by normal micturition or from an indwelling Foley urethral catheter. When required forimplacement of urinary catheters and multiple blood collections, dogswere sedated with a short-acting anesthetic (10 mg/kg ketamine and0.5 mg/kg diazepam i.v.) at 15 min prior to carcinogen dosing and wereplaced in a restraining harness (Alice King Chatham Medical Arts, LosAngeles, CA). No effects on carcinogen metabolism, pharmacokinetics,or adduct formation were observed as a consequence of sedation.

For p.o. dosing, [3H]ABP (3.7 mCi/mmol) was administered p.o. at

5 mg/kg in a gelatin capsule. For i.v. injections to determine thevolumes of distribution, [3H]ABP (37 mCi/mmol), [3H]N-OH-ABP (92mCi/mmol),and [3HjN-OH-ABP/V-glucuronide(107 mCi/mmol) were

dissolved in 1.6 ml of 60% dimethyl sulfoxide-water and administeredat 0.04, 0.04, and 0.01 mg/kg, respectively. Intraurethral doses wereinstilled by catheter in argon-saturated, phosphate-buffered (pH 6)saline-0.5 IHMEDTA (5 ml/kg) containing [3H]ABP (37 mCi/mmol)or [3H]N-OH-ABP (92 mCi/mmol) at a delivered dose of 0.4 mg/kg.

After 1 h, the bladder lumen was drained with the catheter and washedwith warm saline (1 ml/kg), and recovery of the administered dose wasdetermined by scintillation counting to be nearly 40% of the instilleddose for both ABP and N-OH-ABP. Thus, the actual dose absorbedacross the urothelium was calculated to be 0.25 mg/kg.

Heparinized whole blood, plasma, washed erythrocytes, and urinewere collected and analyzed immediately at the specified intervals fortotal radioactivity by liquid scintillation counting (7). Urine and eryth-rocyte samples were then quickly frozen over dry ice and stored atâ€”20Â°C.ABP-hemoglobin adduct levels in erythrocytes were determined

by gas chromatography/mass spectrometry (6) and analyses of eachurine sample for ABP, N-OH-ABP, and their A'-glucuronides werecarried out by HPLC, diode-array spectrophotometry, and flow-throughscintillation counting (2, 22, 23) as detailed previously.

Upon terminal sacrifice at 24 h, DNA was isolated from the urothelium and liver of dogs treated p.o. with ABP or intraurethrally withABP or N-OH-ABP; and ABP-DNA adducts were estimated by aspecific immunochemical method that measures the major ABP-baseadduct, jV-(guan-8-yl)-ABP (24).

Pharmacokinetic Modeling. A multicompartmental pharmacokineticmodel was constructed from these data and from data on the rates ofhydrolysis of N-OH-ABP A'-glucuronide at pH 5, 6, 7, and 8.3 Theresults obtained from i.v. administration of ABP, N-OH-ABP, and N-OH-ABP A'-glucuronide were used to simplify the modeling procedure

by subdividing this multicompartment into several of its components.PCNONLIN, a pharmacokinetics program for personal computers(Version 3.0; SCI Software, Lexington, KY), was utilized to deriveinitial estimates of the rate constants from a two-term exponentialequation. These estimates were then used in a two-compartment modelon an analog/digital hybrid computer (PACER 500; Electronics Associates, Inc., West Long Branch, NJ) to obtain least square iterative fitsof each i.v. data set. These results were subsequently used to set andhold constant these portions of the multicompartmental model in orderto allow simulation of the remaining components that were applicableto the p.o. and intraurethral routes of administration. After the individual rate constants were determined for the complete model, bladderexposure to free N-OH-ABP in the lumen was simulated as a functionof pH (5.0, 6.0, 7.0, and 8.0) and voiding interval (1, 2, 3, 4, 6, 8, and10 h). These values encompass the range of observations seen in thedogs that were studied.

RESULTS AND DISCUSSION

Effect of Voiding Interval and Urine pH on the Pharmacody-namics of ABP and Its A/-Hydroxy Metabolites in Dogs. In orderto examine the effect of different voiding intervals on theabsorption, distribution, metabolism, and excretion of ABP, N-OH-ABP, and N-OH-ABP A'-glucuronide, dogs were allowed

to exhibit normal voiding patterns or were fitted with a urinarycatheter to allow emptying of the bladder lumen at specifiedintervals. In dogs given [3H]ABP p.o. at 5 mg/kg (Fig. 1),

absorption of ABP occurred rapidly and the percentage of thedose in whole blood and plasma reached a maximum at 2-4 hafter treatment. Plasma levels then decreased rapidly whereasthe percentage of dose in whole blood reached a plateau at 6 h.This occurred concomitantly with the formation of ABP-hemoglobin adducts, which increased steadily from 2 to 12 h aftertreatment until the percentage of dose bound to hemoglobinapproximated the levels of total ABP in whole blood (8-11 %).Urinary excretion of ABP and its metabolites increased steadilyover this time period and was essentially complete at 24 h after

3 V. Woolen and F. Kadlubar, unpublished studies.

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12 16Time (hrs)

20

-60

12 16

Time (hrs)

a.m< 20

60

50

12 16

Time (hrs)

Fig. 1. Metabolic profiles of four dogs with different voiding intervals showing the time course distribution of radioactivity in whole blood, plasma, boundhemoglobin (Hb-ABP), and urine after p.o. administration of 5 mg/kg [3H]ABP. (A) The dog voided normally with urine samples taken at 10, 13 and 24 h. (B) Thedog voided normally with urine samples taken at 1,2, 4, 12, and 24 h. (C) The dog voided at 2-h intervals by urinary catheter, with samples taken for analysis for upto 12 h, followed by a 24-h sampling. (D) The dog voided continuously by catheter for up to 12 h with urine samples taken for analysis every 2 h, followed by a 24-hsampling. Blood samples were taken on a sampling schedule that was independent of urine sample collections.

treatment, ranging from 38 to 59% of the dose. Fecal excretionaccounted for an additional 35-40% of the dose.

In these dogs, the average voiding interval varied widely andranged from 8 h (Fig. IA) to 4.8 h (Fig. IB) to 3.4 h (Fig. 1C)to continuous voiding for the first 12 h after dosing (Fig. 10).Interestingly, voiding interval had no discernible effect on theoverall metabolic profile of ABP or on ABP-hemoglobin adductlevels. These data clearly indicate that hemoglobin adduct formation, which arises from the reaction of hemoglobin with N-OH-ABP in the erythrocyte (25, 26), occurs independently ofmicturition and appears to coincide with initial metabolism ofABP in the liver and the apparent release of free N-OH-ABP

into the circulation.To examine this possibility further, each urine collection was

analyzed by HPLC (cf. " Materials and Methods") to determine

the relative amounts of ABP, N-OH-ABP, their 7V-glucuro-nides, and other major metabolites. Cumulatively, the predominant urinary metabolites of ABP in the dog were the sulfateand glucuronide conjugates of 3-hydroxy-ABP and 4'-hydroxy-

ABP (27), with ABP and N-OH-ABP (and their 7V-glucuro-nides) accounting for only about 2 and 1% of the dose, respectively. In contrast to the widely held hypothesis that the N-glucuronide is the major transport form for yV-hydroxy arylam-ines (4), HPLC analyses of urine collected continuously bycatheter for the first 12 h indicated that 68-89% of the N-OH-ABP entering the bladder lumen was in the unconjugated form.Moreover, the relative proportion of the unconjugated N-OH-ABP vs. its 7V-glucuronide metabolite was inversely proportional

so

â€¢Ã‹70

60

xO

50

_'cO

30

20

Continuous12 hrs

3456Average Voiding Interval (hrs)

Fig. 2. Effect of voiding interval on the proportion of urinary N-OH-ABPexcreted as the unconjugated metabolite. Average voiding interval for each dogwas determined as the number of voided samples obtained by micturition or byurethral catheter as described in Fig. 1 for Dogs A-D.

to the voiding interval (Fig. 2), ranging from 22 to 73% unconjugated N-OH-ABP, as the average daily voiding interval wasdecreased from 8 h to continuous voiding for the first 12 h afterdosing. For each dog, 70-90% of the total amounts of urinaryN-OH-ABP were excreted during the first 10-12 h after dosing.

These data indicate that the majority of N-OH-ABP entering

4373




50

12 16 20Time (hrs)

24

lower amounts of this conjugate indicate that total bladderexposure to N-OH-ABP should be much more dependent onvoiding interval than on urine pH. In these studies, urine pHwas found to vary markedly from sample to sample (pH 5-8).Moreover, it did not correlate with any metabolic parameterand appeared to become more alkaline with the greater degreeof physical activity exhibited by the animal during theexperiment.

For comparison to the p.o. route of administration and forpurposes of pharmacokinetic modeling (see below), metabolicprofiles were also determined for ABP, N-OH-ABP, and N-OH-ABP /V-glucuronide given i.v. at 0.04, 0.04, and 0.01 mg/kg, respectively (Fig. 3). After injection, blood levels of thesecompounds rapidly decreased and their volumes of distributionwere calculated to be 0.26, 0.16, and 0.19 liter/kg, respectively.Although the percentage of dose in urine at 24 h for eachcompound was similar to that obtained after p.o. dosing ofABP, the kinetics and extent of ABP-hemoglobin adduci formation differed appreciably. As might be expected, i.v. injectionof N-OH-ABP resulted in rapid hemoglobin adduci formation;

ABP given i.v. formed adducts similarly to the p.o. dose, whileN-OH-ABP jV-glucuronide showed the slowest rate and extentof ABP-hemoglobin adduci formation, as well as a more rapidexcretion inlo urine.

In order to approximate the rate of absorption of urinaryABP and N-OH-ABP across the urothelium after p.o. dosingwith ABP, dogs were given both compounds by intraurelhralcalheler at 0.40 mg/kg. After 1 h, the bladder luminal contenlswere removed and the absorbed dose was determined lo be 0.25mg/kg (cf. "Materials and Methods"), which corresponded to

about 60% of Ihe administered dose. This rale of reabsorplion

Fig. 3. Metabolic profiles of dogs showing the time course distribution ofradioactivity in whole blood, plasma, bound hemoglobin (Hb-ABP), and urineafter i.v. injection of ABP (A), N-OH-ABP (B). or N-OH-ABP N-glucuronide(C).

Ihe bladder lumen is unconjugaled and ihus immedialely available for reabsorption and for covalent binding lo urolhelialDNA. On Ihe olher hand, the tolal amounl of N-OH-ABP thatcan be reabsorbed (Â»1%of the dose) is insufficient to accountfor Ihe high levels of ABP-hemoglobin adducts that are formed(~ 10% of the dose). Therefore, il would appear lhal aboul 11% oof Ihe dose of ABP indeed enters the circulation from the liver(and perhaps other tissues) as free N-OH-ABP, of which Ihemajority (~10% of the dose) is absorbed inlo Ihe erylhrocyteand becomes bound to hemoglobin, with only about 0.7 and0.3% of the dose being filtered by the kidney into the bladderlumen as free N-OH-ABP and its /V-glucuronide, respectively.Although the A'-glucuronide may hydrolyze at acidic pH in thebladder lumen and release more N-OH-ABP, Ihe relalively

12 16Time (hrs)

Fig. 4. Metabolic profiles of dogs showing the time course distribution ofradioactivity in whole blood, plasma, bound hemoglobin (Hb-ABP), and urineafter intraurethral instillation of ABP (A) and N-OH-ABP (B).

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URINARY FREQUENCY AND ADDUCI FORMATION AFTER ABP

ABP

ABSORPTION

BLOOD(+ TISSUES)

Hb-ABP

(10%)

ABP N-OH-ABP N-OH-ABP

BLADDER'Ã®

^^ i^'ABP-M\LUMEN

ABPURINE

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\^

N-uiucuromaeIÃ•T~ABP-M

N-OH-ABP Â£-N-OH-ABPÂ«f

>N-Glucuronide4*'N-OH-ABPABP-MN-OH-ABPN.Glucuronide(-50%)

(0.7%) ,n QO/I

Feces(-35%)

Fig. 5. Multicompartmental pharmacokinetic model for the distribution ofABP and its metabolites in the dog. A'umftprs in parentheses, percentage of doseexcreted in the urine or bound to hemoglobin (Hb) after a 5-mg/kg p.o. dose ofABP. The values for N-OH-ABP and N-OH-ABP /V-glucuronide were selectedfrom a dog with the shortest average voiding interval (12-h continuous). ABP-Mis used to designate all metabolites of ABP other than N-OH-ABP or its N-glucuronide.

was comparable to that reported previously (7) for reabsorptionof N-OH-ABP across the rat urinary bladder. The metabolicprofiles (Fig. 4) of these dogs indicated a rapid absorption ofthe intraurethral dose, followed by a steady decrease in wholeblood and plasma levels, with increasing levels of ABP-hemo-

globin adducts and cumulative excretion into the urine. Asobserved with i.v. dosing, intraurethral instillation of N-OH-ABP resulted in an appreciably higher rate and extent of hemoglobin adduct formation.

For each route of administration of ABP, the covalent binding of circulating N-OH-ABP to hemoglobin was about 10-foldhigher than the amount of N-OH-ABP entering the urinarybladder lumen. Thus, ABP-hemoglobin adduct formation canalso be regarded as a major detoxification pathway.

Pharmacokinetic Model to Predict Exposure of the UrinaryBladder to N-OH-ABP as a Function of Voiding Interval andUrine pH. From these data, a multicompartmental pharmacokinetic model was designed to assess the role of the two physiological variables, micturition and urine pH, in determiningthe exposure of the urothelium to N-OH-ABP, an ultimatecarcinogenic metabolite of ABP. This model (Fig. 5) allows for:(a) the absorption of a p.o. dose of ABP into blood and bodytissues; (b) the conversion of ABP to N-OH-ABP, N-OH-ABP/V-glucuronide, and other metabolites, primarily in the liver; (c)the movement of ABP and its metabolite through the circulation, the reaction of N-OH-ABP with hemoglobin, and thedeposition of these metabolites in the urinary bladder lumen;(d) the acidic hydrolysis of urinary N-OH-ABP /V-glucuronideto N-OH-ABP and the reabsorption of N-OH-ABP and ABPacross the urothelium; and (e) the removal of ABP and itsmetabolites from these compartments by excretion into urineand feces. Rate constants for these processes are summarizedin Table 1.

The accumulation of N-OH-ABP in the bladder lumen andconsequently its contact with the urothelium was simulatedwith this model on an analog/digital hybrid computer using pHvalues from 5 to 8 and voiding intervals from 1 to 10 h. Atypical simulation is presented in Fig. 6 for a urine pH of 6.0and a voiding interval of 4 h. As shown in the simulation, thelevels of total N-OH-ABP in the lumen gradually increased

prior to each urination. The amounts in excreted urine alsoincreased in a stepwise manner, while the total exposure of theurothelium to N-OH-ABP (the integral of N-OH-ABP levelsin the lumen) increased as a curvilinear function for the first 8h. These simulations were then conducted for each of the pHvalues and voiding intervals indicated above and values for theintegral of N-OH-ABP were tabulated.

These relative exposure values were subsequently plotted asa function of voiding interval for each pH simulated (Fig. 7).The results show that exposure to the carcinogenic N-OH-ABPmetabolite is directly proportional to the length of time theurine resides in the bladder lumen. Decreased urine pH didincrease the amounts of N-OH-ABP formed from its /V-glucuronide, but the increase in exposure was only about 10% andclearly not as profound as that derived from unconjugated N-OH-ABP as a consequence of prolonged urine retention.

Table 1 Rate constants associated with the multicompartmental pharmacokineticmodel shown in Fig. 5"

ProcessRate constant(min)-'

AbsorptionABP. p.o. 0.060ABP. intraurethral 0.060N-OH-ABP, intraurethral 0.020

Distribution in blood/tissuesABP -. ABP-M* 0.030ABP â€”N-OH-ABP 0.0039N-OH-ABP -. ABP-M 0.020N-OH-ABP ^Hb-ABP 0.012N-OH-ABP â€”N-OH-ABP A'-glucuronide 0.0067N-OH-ABP A'-glucuronide â€”N-OH-ABP 0.0025

Excretion from blood/tissues to bladder lumenABP 0.00071ABP-M 0.011N-OH-ABP 0.0033N-OH-ABP A'-glucuronide 0.00067

Fecal excretionABP-M 0.0087

Â°Elimination from the urinary bladder lumen into the urine was set faster than

any other rate constant and was chosen to completely empty the bladder at thetime of voiding. Rate constants for the hydrolysis of N-OH-ABP A'-glucuronideto N-OH-ABP were pH dependent in the bladder lumen and were set in accordance with the urine pH measured at each micturition.

* ABP-M, ABP converted to other metabolites.

Time (hrs)Fig. 6. Computer-simulated levels of: (A) total ABP in whole blood (x 2); (B)

total free N-OH-ABP in the bladder lumen (x 200); (C) free N-OH-ABP excretedin urine (x 30); and (D) the integral of N-OH-ABP concentration or cumulativeexposure in the bladder lumen (x 400). The simulation was done for a urine pHof 6.0 and a voiding interval of 4 h. Note that Cunes B and C are stepwisefunctions that always decrease and increase, respectively.

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Effect of Voiding Interval on the Formation of ABP-DNAAdducts in the Liver and Urothelium of Dogs. The predictivevalue of this model (cf. Fig. 7) was tested by estimation of theABP-DNA adducts formed in the bladders of ABP-treated dogs(5 mg/kg), whose average daily voiding intervals was varied (cf."Materials and Methods") from 8 h to continuous voiding for

the first 12 h after dosing (Fig. 1). As shown in Fig. 8, whenvoiding interval was decreased, the level of jV-(guan-8-yl)-ABPadducts in the urothelium increased over a 17-fold range; whilelevels in the liver varied less than 3-fold. The potent reactivityof urinary N-OH-ABP toward bladder epithelial DNA wasfurther demonstrated by direct instillation of N-OH-ABP (0.25mg/kg) into the dog urinary bladder, which gave levels of

468VOIDING INTERVAL (MRS)

10

Fig. 7. Computer-simulated estimations of the effect of voiding interval andurine pH on cumulative exposure of the bladder epithelium to urinary N-OH-ABP. Relative exposure is calculated by assigning a zero value to the integral ofthe free N-OH-ABP concentration in the bladder lumen (i.e., cumulative exposure) when the voiding interval is zero. The points on the curve are relativeexposure values obtained from computer simulations of cumulative exposure forvoiding intervals of I-10 h and urine pH values of 5. 6, 7, and 8. Urine pH washeld constant for each simulation of exposure to N-OH-ABP; each point represents cumulative exposure values (as shown by Cum D in Fig. 6) obtained inseparate computer simulations.

0.8

0.6

0.4

0.2

BLADDER| Continuous:!-'3.4 Hfl'.

UVER/34.6 HH' 8 HIÂ»

ABP orN-OH-ABPIntraurethral,0.25mg/kgB

[-1-B

LAI1I1IÂ¡DCRLIVER!.â€¢Â»1210.80.60.40.20â€¢

A hi' .W.OH. ABP

Fig. 8. ABP-DNA adduci levels in the urothelium and liver (x Vj) of six dogs:as a function of voiding interval after p.o. administration of ABP ( 11:or afterintraurethral instillation of ABP or N-OH-ABP (B). For each determination.DNA samples were hydrolyzed and analyzed in triplicate by a competitive avidin-biotin-amplified enzyme-linked immunoassay (24). The percentage coefficient ofvariation was<10%.

urothelial yV-(guan-8-yl)-ABP adducts that were comparable tothat found in dogs given 20-fold higher p.o. doses of ABP. Incontrast, intraurethral administration of ABP (0.25 mg/kg) didnot give detectable binding levels.

Conclusions. These studies provide strong evidence that accumulation of N-OH-ABP in the urinary bladder is primarilyresponsible for ABP-DNA adduci formation in the carcinogen-target tissue. Moreover, the construction of a multicompart-mental pharmacokinetic model that accurately simulates bladder exposure to N-OH-ABP as a function of voiding intervaland urine pH provides an excellent predictive tool for assessment of individual differences in susceptibility to arylamine-induced urinary bladder carcinogenesis. Thus, it would be usefulto collect data on both urine pH and voiding intervals inepidemiological studies in humans, where jV-(guan-8-yl)-ABPhas now been shown to be present as major adduci in theurolhelium of cigarelle smokers (28).

Moniloring of humans for blood Hb-ABP levels should alsoprovide a useful estimation of both exposure and metabolicaclivation. The metabolic activation pathways for ABP in humans and dogs appear to be very similar (23) and previoussludies in humans have shown that Hb-ABP adduci levels area reliable measure of exposure to ABP in cigaretle smoke (24,29). The pharmacokinelic studies reported herein clearly indicate that Hb-ABP adducts are formed from circulating levelsof N-OH-ABP in blood; and receÃ±Ãsludies in vivo in rals usingcylochrome P-450IA2 inhibitors (30) provide direct evidencethat Hb-ABP adduct formalion is derived from the hepatic 7V-oxidalion of ABP lo N-OH-ABP. Finally, Ihe use of Hb-ABPadducts as a predictive tool in humans is further supported byour recent observation lhal smoking-relaled DNA adducls present in exfoliated urolhelium of differenl individuals are signif-icanlly correlaled wilh measured blood levels of Hb-ABP.4

ACKNOWLEDGMENTS

We express our sincere appreciation to Professor Gerard Mulder ofthe University of Leiden for his helpful advice and stimulating discussions throughout the course of this study.

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1991;51:4371-4377. Cancer Res Fred F. Kadlubar, Kenneth L. Dooley, Candee H. Teitel, et al. Given the Carcinogen 4-AminobiphenylLiver and Urinary Bladder DNA Adduct Levels in Beagle DogsPharmacokinetics, Blood Hemoglobin Adduct Formation, and Frequency of Urination and Its Effects on Metabolism,

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