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Frequency of Urination and Its Effects on Metabolism, Pharmacokinetics, Blood Hemoglobin Adduct Formation, and Liver and Urinary Bladder DNA Adduct Levels in Beagle Dogs Given the Carcinogen 4-Aminobiphenyl¹

The National Center for Toxicological Research, Jefferson, Arkansas 72079 [F. F. K., K. L. D., C. H. T., D. W. R., R. W. B., M. A. B., J. R. B., J. F. Y.], and the Massachusetts Institute of Technology, Cambridge, Massachusetts 02178 [P. W. S., S. R. T.]

The human urinary bladder carcinogen, 4-aminobiphenyl (ABP), is known to undergo hepatic metabolism to an N-hydroxy arylamine and its corresponding N-glucuronide. It has been proposed that these metabolites are both transported through the blood via renal filtration to the urinary bladder lumen where acidic pH can facilitate the hydrolysis of the N-glucuronide and enhance the conversion of N-hydroxy-4-aminobiphenyl (N-OH-ABP) to a reactive electrophile that will form covalent adducts with urothelial DNA. Blood ABP-hemoglobin adducts, which have been used to monitor human exposure to ABP, are believed to be formed by reactions 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 from female beagles given [³H]ABP (p.o., i.v., or intraurethrally), [³H]N-OH-ABP (i.v. or intraurethrally), or [³H]N-OH-ABP N-glucuronide (i.v.). Analyses included determinations of total ABP in whole blood and plasma, ABP-hemoglobin adducts in blood erythrocytes, ABP and N-OH-ABP levels (free and N-glucuronide) in urine, urine pH, frequency of urination (controlled by urethral catheter), rates of reabsorption of ABP and N-OH-ABP across the urothelium, and apparent volumes of distribution in the blood/tissue compartment. The major ABP-DNA adduct, N-(guan-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 multicompartmental pharmacokinetic model for ABP and its metabolites that separates: (a) absorption; (b) hepatic metabolism and distribution in blood and tissues; (c) ABP-hemoglobin adduct formation; (d) hydrolysis and reabsorption in the urinary bladder lumen; and (e) excretion. Using this model, cumulative exposure of the urothelium to free N-OH-ABP was simulated 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 finding that, 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 the dose), with much lower amounts as the acid-labile N-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 reabsorption of 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 a continuous voiding for the first 12 h after dosing. This was also consistent with data from intraurethral instillation of N-OH-ABP, which showed a >60-fold higher DNA adduct level than similarly administered ABP. In contrast, blood ABP-hemoglobin adduct levels, which accounted for 8–10% of the p.o. dose, were independent of voiding interval; and the amounts of free N-OH-ABP available for reabsorption in the bladder lumen were insufficient to account for the high ABP-hemoglobin adduct levels observed.

Thus, these data indicate that hemoglobin-ABP adduct formation is likely to be a direct consequence of the hepatic N-oxidation of ABP to N-OH-ABP and its entry into the circulation; and the measurement of hemoglobin adduct levels can be regarded as valid index of both exposure and metabolic activation of this carcinogen in humans. In addition, these studies 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 critical determinant in human urinary bladder carcinogenesis.

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

Received 1/ 2/91. Accepted 5/30/91.

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