Murine strain differences in metabolism and bladder toxicity of cyclophosphamide

Toxicology. 75 (1992) 257-272 Elsevier Scientific Publishers Ireland Ltd.

257

Murine strain differences in metabolism and bladder toxicity of cyclophosphamide Lucy Fraiser and James P. Kehrer Division of Pharmacology and Toxicology. College of Pharmacy University of Texas at Austin. Austin, Texas 78712-1074 (USA/

(Received May 1st, 1992: accepted July 7th, 1992)

Summary Cyclophosphamide (CP) undergoes metabolic activation, generating phosphoramide mustard and acrolein which are believedto be responsiblefor the cytostatic and toxic effects, respectively.In this study, CP-induced bladder toxicity (hemorrhagic cystitis) was found to be significantlygreater in the ICR than the C57BL/6N (C-57) strain of mice. Strain differences exist in the distribution of CP metabolites to the bladder, as evidenced by consistently higher levels of acrolein equivalents measured in the urine of the sensitive ICR strain. These differences may arise from strain variation in the oxidative metabolism of CP by the mixed-functionoxidase system. However, intrinsic factors within the bladder may also be involved in the resistanceexhibited by C-57 mice. Support for this hypothesis is provided by the significantincrease in hemorrhagic response and permeability of ICR compared to C-57 bladders exposed to equivalent levels ofacrolein by intravesicleinstillation. Basal protein thiol levelswere higher in C-57 than in the ICR strain. However, the effects of acrolein on protein thiol content did not correlate with toxicity suggesting that these groups are not the critical targets for CP-induced bladder injury. Key words." Cyclophosphamide; Hemorrhagic cystitis; Strain difference: Metabolism; Bladder injury: Acrolein

Introduction C y c l o p h o s p h a m i d e (CP), a widely used a n t i t u m o r a n d i m m u n o s u p p r e s s a n t drug, is classified as an alkylating agent although the parent drug exerts only minimal alkylating activity and is not cytostatic in vitro. CP does, however, exert several biological effects following in vivo activation to metabolites capable of alkylation, including target-organ toxicity to lung and bladder [1]. C P is activated following oxidative m e t a b o l i s m by the microsomal cytochrome P-450 mixed-function oxidase system ( M F O ) [2,3] a n d possibly also by cooxidation via the prostaglandin H synthase complex [4]. The initial hydroxylated metabolite, 4-hydroxycyclophosphamide Correspondence to." James P. Kehrer, Div. of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin. TX 78712-1074, USA.

03tKI-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

258 (4-OH-CP), is in equilibrium with its tautomer, aldophosphamide. Aldophosphamide is cleaved by a H-elimination reaction generating stoichiometric amounts of phosphoramide mustard and acrolein which are believed responsible for the cytostatic and toxic effects of cyclophosphamide, respectively [5]. Urotoxic complications are often limiting factors in the therapeutic use of CP [6]. CP-induced urotoxicity ranges from transient irritative voiding symptoms to lifethreatening hemorrhagic cystitis [7,8]. The incidence of cystitis has been reported to be as high as 78% in unprotected individuals given high doses of CP (greater than 200 mg/kg) with 4% mortality from uncontrolled hemorrhage [7,9]. This doselimiting effect is believed to be due to contact between the bladder wall and acrolein present in the urine [10,11]. Previous studies have shown that C57BL/6N (C-57) mice are resistant and ICR mice are sensitive to the lung fibrotic effects of CP [12]. In the current study, a corresponding strain variation in CP-induced bladder toxicity is demonstrated. The reasons for the strain difference in susceptibility to the urotoxic effects of CP are currently unknown. Hoyt and Lazo have suggested that the differential sensitivity of Balb/c and C-57 mice to CP-induced lung injury is the result of differences in repair processes [13,14]. Alternately, the sensitivity of murine strains to CP-induced toxicities could be due to differences in several in vivo processes. For example, strain differences in in situ activation or inactivation, distribution of the active species to target-organs, permeation of the active species into target-organ cells, or the cytotoxic response of target-organ cells may be involved. Currently, none of these mechanisms can be ruled out. We have, therefore, investigated the relationship between CP-induced bladder toxicity and strain differences in: (i) levels of acrolein equivalents measured in urine following hydrolysis; (ii) covalent binding of CPderived radioactivity; (iii) MFO activity; (iv) cytotoxic responses of the bladder to acrolein and (v) protein thiol status of the bladder. Materials and methods

Animals Male ICR and C-57 mice, weighing between 25-40 g and 20-35 g respectively, were supplied by Harlan Sprague Dawley and housed at the Animal Resource Center at this institution. The animals were maintained on a 12/12-light dark cycle and were provided with food and water ad libitum.

Chemicals Cyclophosphamide was obtained from Mead Johnson (Cytoxan®) (Evansville, IN) or Sigma Chemical Co. (St. Louis, MO). [ring-4-14C]cyclophosphamide (98% pure by HPLC) was purchased from New England Nuclear (Boston, MA) and diluted with unlabeled CP. [chlorethyl side chain-3H]CP (99% pure by HPLC) was custom synthesized by Amersham Corporation, (Arlington Heights, IL) and diluted with unlabeled CP. Final specific activities were 0.013-0.014 Ci/mol and 0.045-0.048 Ci/mole for 14C and 3H, respectively. All other chemicals were reagent grade.

259

Preparation of bladder homogenates Mice were euthanized by cervical dislocation. Bladders were removed, expressed, trimmed of extraparenchymal tissues and rinsed with cold 50 mM Tris-HCl containing 0.1 mM EDTA (pH 7.6). The bladders were homogenized for 30 s with a Tekmar Tissuemizer in 3 ml of 50 mM Tris-HCl. The resulting crude homogenate was filtered through four layers of gauze and made to a volume of 4 ml/bladder with the same buffer.

Toxicity studies Cyclophosphamide was administered to animals as a single intraperitoneal (i.p.) injection at doses of 100, 200, or 300 mg/kg and damage was allowed to progress for I - 5 days. The volume of injection was 0.15 ml/10 g body weight. Bladder damage was assessed by measuring the blood content of bladders. Because of its high extinction coefficient and its high concentration in blood, oxyhemoglobin was used as a marker for whole blood in a spectrophotometric assay. The small amount of interfering absorbance and the low residual blood content of bladder homogenates from untreated mice made it possible to quantitate the blood content of bladder homogenates from CP-treated mice by directly measuring the absorbance at 415 nm. A 500-tA aliquot of bladder homogenate was diluted to 1 ml with 0. I M Tris-HCI (pH 7.0) containing 1% (v/v) Triton-X-100 and centrifuged for 10 min at I 1 000 x g. The absorbance at 415 nm of the resulting supernatant was determined. Bloodenriched aliquots of bladder homogenates used to construct calibration curves were treated in an identical manner.

Preparation of subcellular fractions and determination of enzyme activities Mouse hepatic and renal microsomes were prepared by standard differential centrifugation procedures. The cytosolic fractions were saved and the washed microsomal pellets were resuspended (20 mg/ml) in 0.1 M sodium-potassium phosphate buffer (pH 7.4). Protein determinations were carried out by the method of Lowry [15]. Aminopyrine N-demethylase (AD) activity was measured by incubating 1 mg microsomai protein for 10 min with 5 mM aminopyrine and 50 mM NADPH at 37°C in a total volume of 2 ml 0.1 M phosphate buffer (pH 7.4). The reaction was terminated by adding 0.5 ml of 20% (w/v) trichloroacetic acid. After centrifuging at 40 000 x g, 2 ml of the clear supernatant was mixed with 1 ml Nash reagent (6 M ammonium acetate, 60 mM acetylacetone, 0.15 M acetic acid, pH 6.7). Samples were then incubated for 10 min at 60°C and allowed to cool to room temperature. Formaldehyde released was determined by measuring the absorbance of each sample at 412 nm using water as a reference [161. Ethoxyresorufin O-deethylase (EROD) activity was determined fluorimetrically [17]. The reaction mixture contained 1.5 ml of 0.1 M sodium-potassium phosphate buffer (pH 7.4), 1 mg microsomal protein and 10 p,l ethoxyresorufin (50 ttM in 1.25% Tween-80). A 50-/zl aliquot of 50 mM N A D P H was added and the progressive increase in fluorescence as ethoxyresorufin was deethylated to resorufin was recorded for 5 min with excitation and emission wavelengths of 510 and 586 nm, respectively.

260

Determination of acrolein and~or re&ted compounds in the urine The reaction between m-aminophenol and acrolein, substituted acroleins, or compounds that can generate them under acid conditions results in the formation of the fluorescent compound 7-hydroxyquinoline [18]. In the current study, ICR and C-57 mice were treated with 300 mg/kg CP or an equal volume of saline. Mice were housed five to a cage and urine was collected on ice at hourly intervals for 6 h. The ability of urine to form 7-hydroxyquinoline when reacted with m-aminophenol was determined. Briefly, a 50-t~l aliquot of urine was diluted to 2 ml, reacted with 0.5 ml of a reagent containing 250 mg m-aminophenol and 300 mg hydroxylamine, followed by the addition of 0.5 ml 5 M HCI. Blanks were similarly prepared with urine from saline-treated mice. The resulting mixture was heated in a boiling water bath for 10 min, cooled under tap water, centrifuged at 2500 × g for 3 min and fluorescence was determined at excitation and emission wavelengths of 355 and 510 nm. A standard curve was generated using acrolein as substrate. The data are expressed as nanomoles of acrolein equivalents per 100 g body weight. Areas under the curve for excretion of acrolein and related compounds were calculated by the trapezoidal rule using the formula: Areai = [ C(ti) + C(ti+O/2 } × Ati] where C(ti) and C(ti+l) are two consecutive observations and Ati is the time interval [19]. Covalent binding Mice received a 200-mg/kg dose of labeled CP with a specific activity of 0.013-0.014 Ci/mol for lac and 0.045-0.048 Ci/mol for 3H and were euthanized at 0.5, 1, 2, 4, 8 and 12 h. Radioactive labeling of both the ring (lac) and the chlorethyl side chain (3H) allows the metabolism of this compound to both of the postulated covalent binding species (acrolein and phosphoramide mustard, respectively) to be traced [12]. Organs were homogenized in 0.1 M KPO 4 buffer (pH 7.4) and the total radioactivity was determined in a 200-400-t~1 aliquot of solubilized protein (NCS tissue solubilizer). Protein was precipitated with 5 volumes of 10% trichloroacetic acid and the samples were stored overnight at 0°C. Following centrifugation, the protein pellet was assayed for irreversible binding of lac and 3H CP. Briefly, the protein precipitate was extracted once with 5 volumes ethylacetate:methanol (50:50) and then exhaustively with methanol (approximately 6 washes) until no significant radioactivity was seen in the extracts. Extracted precipitates were dissolved in 1 N NaOH at 50°C. A 50-#1 aliquot was removed for protein estimation (Lowry) and the remainder was neutralized with 10% glacial acetic acid and analyzed for radioactivity by liquid scintillation counting. Acrolein instillations Intravesicle instillation of 0.05, 0.5, or 6.5 mM acrolein was carried out by means of a simple needle puncture of the urinary bladder of mice under pentobarbital anesthesia. The acrolein solution was prepared in isotonic phosphate buffer (0.1 M KPO4, 0.9% NaC1, pH 7.4). Controls received an equal volume of isotonic phosphate buffer. Mice were killed 24 h later and bladder damage was determined by estimation of bladder blood content.

261

Bladder permeability The permeability of excised bladders was determined by measuring the ability of water to enter a 50% sucrose solution contained within the bladder. Excised bladders were cannulated and filled with either a 0.1 M phosphate-buffered saline solution or acrolein (0.05, 0.5, or 6.5 mM), prepared in the same buffer. Bladders were immersed in water at 37°C for 1 h, then emptied and refilled (through the cannula) with a 50% (w/v) solution of sucrose. Fine polyethylene tubing of known diameter bore (0.28 mm), was attached to the cannula (0.3 mm × 1.2 cm) and the sucrose containing bladder was immersed in a water bath (distilled water; 22-25°C). The distance that the liquid moved in the capillary tubing was recorded after 30 min. The volume of water entering the bladder was determined by calculating the cylindrical volume (~rr2h) of the tubing, where r was the radius of the tubing and h was the height of the column of liquid [20]. Protein thiols Following determination of bladder permeability, bladders were opened, emptied of their contents, then rinsed in 0.25 M sucrose, 6 mM EDTA (pH 7.4). Protein thiols in whole-bladder tissue were determined in T r i s - E D T A buffer (0.2 M Tris, 20 mM EDTA, pH 8.2) by reaction with 5,5-dithiobis-2-nitrobenzoic acid [21] (0.2 mM final concentration). Data and statistical analysis Data are expressed as the mean ± SE and were analyzed using the unpaired Student's t-test, where appropriate, or a one way analysis of variance. Post hoc analyses were carried out using the Student-Newman-Keuls test. A P value of < 0.05 was considered significant. Results

Bladder damage in mice treated with cyclophosphamide No deaths occurred in any of the mice treated with CP or saline. The sequence of events following a single i.p. injection of CP (200-400 mg/kg) has been well characterized in rat bladder [22,23]. The present study demonstrates a similar pattern for the development of hemorrhagic cystitis in ICR mice. Gross observations included the development of hemorrhagic patches, appearance of nodules and thickening of the bladder wall as early as 24 h. These findings were accompanied by dose- and time-dependent changes in bladder hemoglobin content (Fig. I). All three doses of CP tested in ICR mice (100, 200, and 300 mg/kg) caused statistically significant increases in bladder hemoglobin content on days 1 and 2. The bladder hemoglobin content remained significantly elevated up to 3 days after 200 mg/kg and up to 4 days after 300 mg/kg. Bladder damage was maximal 48 h after 300 mg/kg CP and this dose was used in all subsequent studies, with the exception of in vivo covalent binding. C-57 mice were relatively more resistant than ICR mice to CP-induced hemorrhagic cystitis (Fig. I). The C-57 strain exhibited significant elevations over saline

262

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[ ] ICR 200 mg/kg [ ] ICR 100 mg/kg •

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Day Fig. I. Time course of bladder hemoglobin content in ICR and C-57 mice treated i.p. with a single bolus dose of 100, 200, or 300 mg/kg cyclophosphamide. Control mice received a single injection of saline and had hemoglobin contents of 0.042 4- 0.006/~1 blood/bladder at all days tested. The results represent the mean hemoglobin content of bladder homogenates 4- SE (n = 3-6). *Significantly different from salinetreated controls, tSignificantly less than ICR mice at the same time point (P < 0.05).

controls in bladder hemoglobin on day 3, but the damage was less severe at all times than that seen in ICR mice (21% of peak ICR levels). Differences in bladder wet weight (44 4- 4 and 42 4- 3 mg/bladder for ICR and C-57 strains, respectively) or protein content (8.8 4- 1.6 and 6.7 4- 0.3 mg/bladder for ICR and C-57 strains, respectively) were not significant and could not explain the observed strain differences.

Urinary content of acrolein and related compounds The formation of 7-hydroxyquinoline has been used as a measurement for acrolein in the urine of rats following infusion of CP [24]. Based on the ability of conjugated acroleins as well as compounds that generate acrolein or its conjugates following hydrolysis to form 7-hydroxyquinoline, formation of this fluorescent compound upon reaction of biologic samples with m-aminophenol does not necessarily indicate the presence of free acrolein. However, since acrolein is a decomposition product of CP, measurement of free acrolein, conjugated acrolein, or compounds that can generate them (i.e. aldophosphamide) in the urine can be used as an indicator of CP metabolism in vivo and may reflect reactive CP species. The concentration of acrolein equivalents in the urine was significantly lower in the C-57 mice 2 and 3 h after CP (Fig. 2). Although, the cumulative levels measured in the urine were not significantly different at 6 h between the two strains, the rate of release of acrolein and/or related compounds into the urine was significantly slower in the resistant C-57 strain, particularly at early time points. Area under the curve calculations revealed that total exposure of C-57 mice to acrolein and/or related compounds via the urine was significantly (27 4- 9%) less than ICR.

263

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Time (hours) Fig. 2. Urinary acrolein concentrations. Mice were administered a 300-mg/kg dose of cyclophosphamide and were placed in metabolism cages (5 mice/cage). Urine was collected at hourly intervals and assayed the same day for free acrolein. Urine volumes voided over the 6-h collection period were 3.6 ± 0.2 and 3.3 ± 0.3 ml for ICR and C57 mice, respectively. Data are expressed as the mean ± SE (n = 3-4). AUC, Area Under the Curve (expressed as nmol x h/100 g body wt.). *Significantly higher than the corresponding determination in C-57 mice (P < 0.05).

The stability of acrolein equivalents in urine over an 18-h time period was determined in animals treated with CP. The concentration of acrolein equivalents did not change significantly when urine was kept at room temperature for 2 h. (Table I). A significant loss of acrolein equivalents was noted after 18 h. However, when authentic acrolein was added to urine collected from untreated mice, approximately 40% was lost within 30 min.

TABLE 1 STABILITY O F A C R O L E I N IN U R I N E Data are expressed as means ± SE (n = 3) or single observations. Values in parentheses are the percentage of time 0. Urine was collected for 3 h from untreated ICR mice or mice administered a single 300 mg/kg dose of CP. Assay conditions were as described in methods, n.d., not determined. Time (h)

Acrolein added to control urine (nmol/50 ~.1)

Acrolein in urine following CP administration (nmol acrolein equivalents/50 ,o,I)

0 0.5 1 2 18

150 92 62 41 n.d.

71 + 12 n.d. n.d. 66 ± 9 (93%) 36 (50%)*

+ I ± 2 (61%)* (41%)* (27%)*

*Significantly different from zero-time.

264 Organ distribution and covalent binding of radiolabeled cyclophosphamide The time courses for distribution of radiolabeled CP, following a dose of 200 mg/kg, to kidney and bladder are shown in Fig. 3A and Fig. 4A. CP-derived radioactivity was rapidly distributed to liver [12], kidney and bladder in both the ICR and C-57 strains. Peak levels were attained within 30-60 min, followed by a rapid and continuous fall over the next 12 h. Area under the curve calculations revealed that 45 ± 14% more 3H and 49 ± 18% m o r e 14C was distributed to the kidneys of ICR than to C-57 mice. However, these differences were not statistically significant. Covalent binding of radioactivity to kidney protein reached a maximum by 30 min and remained relatively constant over the next 12 h (Fig. 3B). The only difference in covalent binding between strains was seen at 8 h, with C-57 mice showing significantly higher binding of laC-labeled material. Similar findings were obtained with liver tissue [121. The resistant C-57 strain showed higher levels of both 3H and lac in the bladder

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T i m e (hours) Fig. 3. A, Total distribution of 3H- and 14C-labeled cyclophosphamide to kidney. B, Covalent binding of 3H- and 14C-labeled cyclophosphamide to kidney protein. Mice received 200 mg/kg cyclophosphamide containing both 3H- and 14C-labeled material and were killed at the indicated times. Data are expressed as the mean (n = 3-4 ) ± SE. *Significantly different from the corresponding determination in ICR mice (P < 0.05).

265

than the ICR strain at all time points examined (Fig. 4A). Area under the curve calculations indicated that 240 + 53% more 3H and 321 + 50% more ~4C was distributed to the bladders ofC-57 mice relative to ICR mice. The differences in total distribution of 14C-labeled material was statistically significant. Likewise, significantly more radioactivity was bound to the bladders of the C-57 strain (Fig. 4B). Area under the curve calculations revealed that the C-57 strain bound 293 ± 58% more 3H and 526 ± 216% more 14C than the 1CR strain.

Mixed-function oxidase activity No strain differences were observed in renal AD activity, nor was hepatic EROD activity different between strains. However, hepatic microsomes isolated from ICR mice exhibited significantly higher AD activity than those from C-57 mice (Table II). Lineweaver Burke kinetics indicated that CP competitively inhibited Ndemethylation of aminopyrine with a Ki of 2.1 mM.

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266 TABLE II STRAIN DIFFERENCE IN MONOOXYGENASE ACTIVITIES Values represent mean 4- SE (n = 3). Assay conditions were as described in methods, n.d., not determined. Aminopyrine N-demethylase (nmol formaldehyde/min/mg protein)

Ethoxyresorufin O-deethylase (nmol resorufin/min/mg)

ICR C-57

Liver

Kidney

Liver

Kidney

4.8 4- 0.2 3.4 4- 0.3*

1.1 4- 0.1 1.1 4- 0.2

0.17 ± 0.02 0.15 ± 0.02

n.d. n.d.

*Significantly different from ICR.

Intravesicle instillation of acrolein The instillation o f acrolein directly into the u r i n a r y b l a d d e r s o f I C R a n d C-57 mice p r o d u c e d d a m a g e similar to that o b s e r v e d with systemic a d m i n i s t r a t i o n o f CP, except at high doses where h e m o r r h a g e was m o r e diffuse. T h e severity o f h e m o r rhage was c o n c e n t r a t i o n - d e p e n d e n t in b o t h strains (Table III). However, the h e m o r rhagic response was less p r o n o u n c e d in the C-57 mice at all c o n c e n t r a t i o n s tested. W i t h instillation o f the highest acrolein c o n c e n t r a t i o n (6 m M ) , the m a g n i t u d e o f the strain difference in b l a d d e r h e m o g l o b i n c o n t e n t was a p p r o x i m a t e l y 3-fold.

Strain differences in bladder permeability and protein thiols A c r o l e i n - i n d u c e d increases in b l a d d e r p e r m e a b i l i t y were dose- a n d strain-related (Fig. 5). Increases in p e r m e a b i l i t y were statistically significant in the I C R strain at acrolein c o n c e n t r a t i o n s as low as 0.05 m M (1 l - f o l d increase), while this concentration failed to p r o d u c e significant increases in the p e r m e a b i l i t y o f the b l a d d e r s from

TABLE III STRAIN DIFFERENCE IN HEMORRHAGIC RESPONSE FOLLOWING INTRAVESICLE INSTILLATION OF ACROLEIN Values are expressed as means 4- SE (n = 3-4) of bladder hemoglobin content (#1 blood/bladder), n.d., not determined. Acrolein (raM)

ICR

C-57

1.5 3.0 4.5 6.0

1.4 ± 0.10t 2.4 ± 0.25t 3.5 4- 0.70t 10.0 4- 1.85t

n.d. 1.9 ± 0.14t 2.6 4- 0.35t 3.5 4- 1.10t*

*Significantly different from ICR. *Significantly different from phosphate-buffered saline control.

267

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A c r o l e i n (mM) Fig. 5. Strain difference in bladder permeability following intravesicle instillation of acrolein. Results arc expressed as multiples of saline control values. Saline control values were 0.73 + 0.2 and 1.4 :~ 0.3 nl of water for ICR and C-57 mice. respectively. Data represent means + SE of 3-4 determinations. *Significantly greater than saline control, tSignificantly less than the corresponding determination in ICR mice (P < 0.05).

the C-57 strain. Acrolein concentrations of 6.5 mM were required to produce statistically significant increases in bladder permeability in the C-57 strain. The permeability increases of bladders from ICR mice exposed to 6.5 mM acrolein were significantly greater (77-fold versus 41-fold) than that seen in C-57 mice at the same acrolein concentration. The basal protein thiol levels of the resistant C-57 strain, as well as thiol levels following 0.05 mM acrolein, were significantly elevated over that seen in the ICR strain (Table IV). As expected, a significant loss in protein thiols was observed when 6.5 mM acrolein was instilled into the bladders of both strains. However, when blad-

TABLE IV STRAIN D I F F E R E N C E IN PROTEIN INSTILLATION OF ACROLEIN

THIOL

STATUS

FOLLOWING

INTRAVESICLE

Values represent means ± SE (n = 3-4 mice). Protein thiols are expressed as nmol GSH equivalents/rag bladder wet wt. Assay conditions were as described in methods. Acrolein (mM)

ICR

0 0.05 0.50 6.5

4.8 6.5 8.3 2.5

± ± ± +

(?-57

0.3 0.6 1.3 0.23t

*Significantly different from ICR. tSignificantly different from saline control.

6.3 8.5 10.8 3.5

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0.3* 0.3t* 0.6t OAt

268 ders were exposed to 0.05 or 0.5 mM acrolein, bladder protein thiols appeared to increase, although these elevations were statistically significant in the C-57 strain only. Discussion

CP is an oxazaphosphorine alkylating agent that is widely used to treat a variety of malignant and non-malignant diseases. Chemotherapy with CP has been associated with considerable target-organ toxicity to the bladder [25-27]. In this study, murine strain differences were demonstrated to exist in the susceptibility to this injury. Although C-57 mice treated with 300 mg/kg CP exhibited a significant increase in bladder hemoglobin levels over saline controls, by comparison to ICR mice, they were almost completely resistant to the toxic effects of CP. This finding is in agreement with previous studies indicating that ICR and Balb/c mice are sensitive while C-57 mice are resistant to the pulmonary fibrotic effects of CP [12,13]. Multiple factors probably contribute to the observed strain differences in susceptibility to CP-induced toxicities. The resistance of C-57 mice to systemically administered CP could occur on a pharmacokinetic basis through (i) decreased metabolism of CP, (ii) rapid elimination of CP (or metabolites) from the blood, (iii) decreased distribution of CP or the active species to the bladder, (iv) exclusion of the toxic species from critical targets within the bladder, or (v) detoxification prior to entry into or within the bladder. Pharmacokinetic differences clearly influence the expression of CP-induced bladder injury. In ICR and C-57 mice treated with a 200mg/kg dose of radiolabeled CP, area under the curve for total radioactivity was significantly lower in the blood and systemic clearance significantly higher in the resistant C-57 strain [12]. Severe damage to the urinary bladder has only been observed following treatment with CP analogs that are metabolized to acrolein [11]. Acrolein is, therefore, suspected as the CP-metabolite responsible for hemorrhagic cystitis [27]. Several lines of evidence support the contention that CP-induced injury to the urinary bladder results from contact between the urothelium and acrolein present in the urine rather than a selective cytotoxic effect of bloodborne substances. Particularly, Sladek et al. [28] demonstrated a linear correlation between urinary acrolein concentrations and the incidence of hemorrhagic cystitis. Although the overall pattern of excretion was similar in both strains, consistently higher levels of acrolein equivalents were measured in the urine of the sensitive ICR strain. This appeared to result, at least in part, from a more rapid release of acrolein and/or related compounds into the urine when compared to C-57 mice. In the current study, a non-specific assay in which m-aminophenol reacts with c~3unsaturated compounds or substances which can be converted into such compounds in acid solution to produce the fluorescent compound 7-hydroxyquinoline, was used as a determinant of CP metabolism. Consequently, this assay is positive not only for acrolein, but conjugated acrolein or any compound that can generate acrolein following hydrolysis. Interestingly, when urine collected from mice treated with CP was kept at room temperature for 2 h, the amount of acrolein equivalents that could be measured did not change significantly. However, when acrolein was added to

269

urine collected from untreated or CP-treated mice, approximately 40% was lost within 30 min. These results suggest that acrolein is excreted into the urine in a stabilized form rather than as free acrolein, thereby providing a mechanism by which this reactive species may be transported from its site formation in the liver to its site of action in the bladder. Acrolein added to urine may react with nucleophiles in the urine which differ from nucleophiles in the liver. The adducts thus formed may differ in stability under the highly acidic conditions of the assay used for determination of acrolein equivalents. The release of electrophilic metabolites from labile GSH or cysteine adducts represents an attractive mechanism for target-organ toxicity in tissues which have only weak drug-metabolizing activity. It has been suggested that this mechanism may explain the selective bladder carcinogenicity of isothiocyanate [29]. It is possible that a similar mechanism is involved in the target-organ toxicity of CP. There were higher levels of total radioactivity in the kidneys of ICR mice, although the strain difference in this parameter did not reach statistical significance. Surprisingly, consistently higher levels of total and covalently bound radioactivity were seen in the bladders from the resistant C-57 strain of mice. These results demonstrate that organ distribution and covalent binding of radiolabeled CP do not correlate with CP-induced bladder toxicity. Possible explanations for these results include: (i) non-toxic CP metabolites (alcophosphamide; 4-ketocyclophosphamide; carboxycyclophosphamide) may also possess the 3H and 14C label and covalently bind to tissue macromolecules [30]; (ii) only a portion of the covalent binding target sites are related to toxicity; or (iii) distribution of radiolabeled material to the bladder may largely reflect bloodborne radioactivity. The toxic species may, therefore, be excluded from critical target sites which are believed to be concentrated at the lumenal border of the bladder (more easily accessed by toxins in the urine) [20]. These results indicate that kinetically distinct compartments for CP and its metabolites may exist in the bladder and these compartments differ in their accessibility to critical target sites within the bladder. Bladder damage induced by intravesicle administration of acrolein was dosedependent in both strains, but was greater in the sensitive ICR strain at all concentrations tested. There was a dramatic increase in the bladder hemoglobin levels in ICR mice when the concentration of acrolein was increased from 4.5 mM to 6.0 mM, suggesting the existence of a threshold level in this strain. No such increase was observed in the resistant C-57 strain, although it is possible that such a threshold may exist at higher acrolein concentrations. These results suggest an intrinsic murine strain difference in the cytotoxic response of the bladder to acrolein present in the urine. Since acrolein arises from oxidation, the results from the present study suggest that the murine strain difference in susceptibility to CP-induced bladder damage may, in part, be due to differences in CP metabolism. ICR mice exhibited approximately 30% higher hepatic AD activity than C-57 mice. Kinetic studies revealed that the same hepatic microsomal mixed-function oxidase system metabolizes both aminopyrine and CP. This supports the hypothesis that differences in AD activity are partially responsible for the strain variation in toxicity. The initial response of the bladder to toxic CP metabolites appears to be fragmen-

270 tation of the lumenal membrane [31 ]. Because of its extreme reactivity toward thiols, many of the biologic effects of acrolein have been attributed to interaction with cellular thiols [32]. Cytochemical staining of the bladder for thiols and disulfides indicates that the urothelium contains an abundance of cysteine-rich proteins at the lumenal margin. The urothelium represents a barrier to the passage of salts and water between urine and blood and this barrier function is destroyed by sodium thioglycollate (a known keratin solvent), which is presumed to act by breaking disulfide bridges and physically disrupting the lumenal membrane. Based on this finding, it has been suggested that the integrity of the bladder as a selective permeability barrier may depend on thiol content [20]. Acrolein increased bladder permeability in both ICR and C-57 mice in a doserelated manner. Strain differences were seen, with bladders from C-57 mice exhibiting relatively more resistance to acrolein. Total protein thiol levels were greater in the bladders from C-57 mice than ICR mice. At high doses of acrolein (6.5 mM), the increases in bladder permeability correlated with protein thiol loss in both strains. Surprisingly, at lower doses of acrolein (0.05 and 0.5 mM), increases over saline controls in bladder protein thiol status were noted, although it only reached statistical significance in the C-57 strain. Bladders from ICR and C-57 mice sustained different degrees of injury upon exposure to the same concentration of acrolein suggesting that the murine strain difference in response to acrolein in the urine is not due simply to differences in the repair process. Furthermore, the data indicate that acrolein-induced increases in bladder permeability do not correlate with loss of protein thiols. However, changes in the protein thiols measured in bladder tissue do occur and may be the result of conformational changes in bladder proteins due to alkylation by acrolein. Alternatively, these results may be suggestive of an ability of acrolein to break disulfide bridges, although no evidence to support this hypothesis could be found in the literature. The results from this study suggest that the murine strain variation in the susceptibility to CP-induced hemorrhagic cystitis is simultaneously modulated by several in vivo processes. Strain differences exist in distribution of CP metabolites to the bladder, as evidenced by the consistently higher levels of acrolein equivalents measured in the urine of the sensitive ICR strain. These differences may arise from strain variation in oxidative metabolism of CP by the MFO system or perhaps by a recently suggested novel bioactivation mechanism that involves sulfoxidation in the kidney of an acrolein S-conjugate initially formed in the liver [33 ]. Intrinsic factors within the bladder may also be responsible for a portion of the resistance to hemorrhagic cystitis seen in the C-57 strain. Support for this hypothesis is provided by the increased hemorrhagic response and permeability of ICR bladders exposed to acrolein by direct instillation. While basal protein thiol levels in the bladder were different between the two strains, loss of these nucleophiles did not correlate with acrolein-induced injury. These results indicate that either protein thiols do not represent critical target sites within the bladder or measurements of total tissue thiols are unable to detect changes in the fraction critical for injury.

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Acknowledgements This work was supported by NIH grants HL35689 and HL48035. JPK is the Gustavus and Louise Pfeiffer Professor of Toxicology. References I 2 3 4 5 6 7 8 9 10 11 12 13

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