N-Acetylcysteine and Sodium 2-Mercaptoethane Sulfonate As Sources of Urinary Thiol Groups in the Rat1

[CANCER RESEARCH 44, 3797-3800, September 1984]

N-Acetylcysteine and Sodium 2-Mercaptoethane Sulfonate as Sources of Urinary Thiol Groups in the Rat1

Kari Ormstad2 and Yasuo Ohno

Department of Forensic Medicine, Karolinska Inst/tutet. S-104 01 Stockholm. Sweden

ABSTRACT Cells of various organs were isolated according to techniques routinely used in our laboratory; hepatocytes as described by MoldÃ©uset al. (13), Increasing the urinary output of free thiol groups protects kidney cells according to the data of Jones ef al. (10), intestinal epithelial against cyclophosphamide-induced bladder toxicity. In the pres cells according to the data of Dawson and Bridges (8), myocardial cells ent study, intact rats, isolated perfused kidneys, and freshly according to the data of Rajs et al. (19), and pulmonary cells according isolated cells from various rat organs are used to compare the to the data of Dawson et al. (9). In vitro perfusion of isolated kidneys efficacy of A/-acetylcysteine and sodium 2-mercaptoethane sul- was performed as described by Ormstad ef al. (15), and urine was collected from ureteral catheters. fonate (mesna) as sources of urinary thiols. For the in vivo experiments, rats were anesthetized with diethyl ether. In intact rats given a single i.v. dose of mesna, urinary thiol Via a small abdominal incision, a silicone catheter was inserted in the output is approximately 10-fold higher than in rats given an urinary bladder and secured with a ligature, and the urethra was closed equimolar dose of /V-acetylcysteine. This is partly due to the fact immediately below the neck of the bladder. In the left femoral vein, 16 that A/-acetylcysteine is rapidly absorbed by various types of i/mol Of W-acetylcysteme (16 mw; 1 ml) or 8 ^mol of dimesna (8 ITIM; 1 cells, whereas mesna is transported selectively to the kidney, ml) were injected; similarly treated and operated rats which had been and partly to different renal handling of the two compounds. The given i.v. injections of 1 ml of 0.9% NaCI served as controls. To prevent results suggest that mesna is a more favorable drug than A/- hypoperfusion and pollakisuria during the experiment, 10 ml of 0.9% acetylcysteine for increasing urinary thiol excretion. NaCI were injected s.c. at the nape of the neck of all rats. The rats were placed in stainless steel restriction cages, and urine was sampled in 10- min batches, immediately mixed with an equal volume of 6.5% trichlo- INTRODUCTION roacetic acid, and stored on ice for up to 2 hr, whereupon the content of free thiol groups was determined according to the spectrophotometric Oxazaphosphorine compounds (cyclophosphamide, ifosfam- method of Saville (20). ide, and trofosfamide) are frequently used as cytostatics in All in vitro incubations were performed in a medium consisting of a cancer chemotherapy and as immunosuppressants in organ modified Krebs-Henseieit bicarbonate buffer, pH 7.4, supplemented with transplant recipients and in various states of immunological 25 mw 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid in rotating derangement. Among other side effects, a frequently arising round-bottomed, siliconized glass flasks at 37Â°under continuous gassing problem during this treatment is hemorrhagic cystitis, which has with carbogen (95% O2 plus 5% CO2). Samples were removed at preset been attributed to the reactive aldehyde, acrolein (CH;>=CHâ€” time points and, in the case of cell incubations designed to study thiol CHO), a product of Oxazaphosphorine metabolism (6,7). Several oxidation, the cells were sedimented by centrifugation (700 rpm for 1 min) before the supernatant was further processed. Cellular uptake of studies have indicated that compounds which are excreted in a mesna, dimesna, and W-acetylcysteine was determined by incubation of form featuring free thiol groups may counteract the urotoxic isolated cells with the compounds in question, separation of the cells effect of oxazaphosphorines (4, 5, 11), and among these A/- acetylcysteine and mesna3 seem to be most effective. In a recent from the medium by rapid centrifugation in a microfuge, and washing once with fresh medium. Free thiols were determined after deproteini- paper, Berrigan ef al. (2) reported that the latter compounds, if zation by addition of an equal volume of 6.5% trichloroacetic acid given in appropriate doses, are equally effective in protecting according to the spectrophotometric method of Saville (20). against cyclophosphamide-induced urothelial damage and Mesna and dimesna were generously supplied by Asta-Werke AG, depression of hepatic drug metabolism. Degussa Pharma Gruppe, Bielefeld, German Federal Republic. All other Since oxazaphosphorine-derived acrolein, which exerts its chemicals were of at least reagent grade and were purchased from local toxicity on the lining of the urinary passages, is likely to be commercial sources. produced after glomerular filtration (21), the potency of a uropro- tective drug is dependent on its capacity to yield free thiol groups RESULTS in the urine. The aim of the present study was to compare A/- In contrast to previous findings with mesna (17), the sponta acetylcysteine and mesna as sources of urinary thiols in the rat. neous rate of oxidation of A/-acetylcysteine was very slow in the absence of biological material (Chart 1). Neither did addition of MATERIALS AND METHODS citrated plasma to the incubation affect the oxidation rate. Incu bation with isolated cells from rat intestine or kidney did, how Male Sprague-Dawley rats weighing 200 to 300 g were kept in ever, lead to oxidation of A/-acetylcysteine to the corresponding stainless steel cages with free access to tap water and pelleted rat food. disulfide; about twice as rapidly in the presence of renal cells as with intestinal cells. Incubations were also performed with iso 'This work was funded by the Swedish Board for Technical Development and lated hepatocytes and pulmonary cells, but with these cells the by the Swedish Medical Research Council, Grant 03X 2471. "To whom requests for reprints should be addressed, at Department of Forensic oxidation rate did not differ from that observed in the absence Mediane, Karolinska Institute!. Box 60400, S-104 01 Stockholm, Sweden. 3The abbreviations used are: mesna, sodium 2-mercaptoethane sulfonate; di- of cells in the incubation (data not shown). Based upon these mesna, sodium 2-mercaptoethane sulfonate disulfÃ¬de;SH, sulthydryl results and upon those of Bonanomi and Gazzaniga (3), we Received September 28,1983; accepted June 4,1984. chose to perform the following comparative experiments with

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cysteine. After 100 min, approximately 27% of the dimesna injected was excreted in thiol form, whereas only approximately 3% of the A/-acetylcysteine had reached the urine as free thiol. According to previous experience, dimesna does not penetrate into most living tissues, and in a series of experiments, we compared uptake rates for radiolabeled mesna, dimesna, and A/- acetylcysteine into freshly isolated cells from various rat organs (Table 1). Among the cell types tested, only renal and intestinal epithelial cells absorbed mesna or dimesna to any substantial degree, whereas N-acetylcysteine was readily absorbed by all cell types. These data suggest that the discrepancy between dimesna and N-acetylcysteine as source of urinary thiol observed in the in vivo experiments may at least partly be due to the fact 20 40 60 that N-acetylcysteine is retained in various tissues and enters time (min) various metabolic pathways, thus being prevented from ever Chart 1. Oxidation of N-acetylcysteine to the corresponding disulfide during reaching the kidneys. incubation in vitro at 37Â° under a carbogen (95% OÂ¡,5% CO2) atmosphere. Incubation media contained Krebs-Henseleit bicarbonate buffer, pH 7.4 (O), human However, different renal handling of the 2 compounds may citrated plasma (A), intestinal epithelial cells (10* cells/ml) in Krebs-Henseleit buffer also contribute to the observations presented in Charts 2 and 3. (â€¢),andrenal epithelial cells (10* cells/ml) in Krebs-Henseleit buffer (â€¢).Bars,mean To eliminate the influence of other cells and tissues, a series of Â±S.D. of 3 separate incubations. experiments was performed where equimolar (in SH units) con centrations of dimesna (200 nÂ»)and N-acetylcysteine (400 Â¿Â¿M) were added to the perfusate circulating through isolated rat kidneys in vitro. Urine was collected in 5-min batches from ureteral catheters, and thiol excretion was monitored (Chart 4). Like in the in vivo situation, dimesna was the most effective thiol source, but the difference was less pronounced. Furthermore, even urine from kidneys perfused with a medium without any

20 60 t i me (min) _ 2- Chart 2. Urinary concentration of thiol groups in rats given i.v. injections of 8 E /Â¿molofdimesna (O), 16 nmol of N-acetylcysteine (D). or 15.4 ^mol of NaCI (A). mesna in disulfide form (dimesna) and N-acetylcysteine in thiol Ã• form. Urines from mammals normally contain a certain amount of 20 50 100 free thiol groups, mainly originating from cysteine moieties in time ( min ) peptides and other low-molecular-weight compounds. Upon in Chart 3. Urinary excretion of free thiol groups in intact rats given i.v. injections jection of NaCI immediately prior to urine collection, a low and of 8 /Â¿molofdimesna (O), 16 umol of N-acetylcysteine (HI), or 15.4 Â«molof NaCI (A). A. accumulated diuresis; B, accumulated amount of free thiol groups in urine. seemingly constant urinary concentration of thiol groups was One experiment typical of 3. found (Chart 2). In rats given injections of A/-acetylcysteine, the urinary thiol concentration was roughly doubled, whereas urine Table 1 from rats given injections of dimesna contained a thiol concen Uptake of mesna, dimesna, and N-acetylcysteine into freshly isolated cells from tration which initially was more than 10-fold higher than under various rat organs Cells (10* cells/ml) were incubated with the sulfur compounds at a 100 Â¡atfinal control conditions. Although the urinary thiol concentration de concentration. Data are from 3 experiments calculated from the first 5 min of clined rapidly, after 100 min it was still 4-fold higher than in rats incubation. given injections of N-acetylcysteine and 8-fold higher than in cells/min)Cell Uptake rate (pmol/IO* those given injections of NaCI. The urinary volume was quite similar irrespective of which typeHepatocvtes cysteine315 compound had been injected into the rats (Chart 3). After 20 to f Â±40 30 min of relative pollakisuna. diuresis was stabilized at about Renal epithelial 218 Â±25 180 Â±30 205 Â±30 50 /tl/min, which probably is reflecting an excretion of free water Intestinal epithelial 235 Â±19 161 Â±28 306Â±43 Pulmonary 6Â± 3 ND 331 Â±31 excess. Thus, the total amount of free thiol excreted during the Â±18*MyocardialMesna5Â± NDDimesnaND6NDN-acetyl 132 experiment was approximately 10-fold higher in rats given injec Mean Â±S.D. tions of dimesna than in those given injections of N-acetyl " ND, not detectable.

3798 CANCER RESEARCH VOL. 44

in dimesna-treated rats. This could be due to different renal handling of the 2 com pounds or to different whole-body disposition, i.e., that a smaller fraction of a dose of W-acetylcysteine may actually reach the kidneys than is the case with dimesna. The data obtained from _ 2 the uptake experiments with various types of isolated cells suggest that this is at least part of the explanation; whereas dimesna only is absorbed by intestinal and renal cells, W-acetyl cysteine readily penetrates into all cell types tested. Thus, it must be expected that also in vivo W-acetylcysteine is actively extracted from the circulating blood to be incorporated in various cells and organs. In addition, the kidney seems to modify thiol excretion from 20 40 60 the 2 sources studied. During perfusion of isolated kidneys, all time ( mm ) extrarenai influences are eliminated, and also under these con Chart 4. Accumulated urinary excretion of free thiol groups in isolated rat ditions the urinary output of dimesna-derived thiols was about kidneys perfused with a medium supplemented with 200 ^M dimesna (O) or 400 twice that of W-acetylcysteine-derived. This suggests a difference Â¡MW-acetylcysteine (O) or without addition of thiol source (A). Time point zero, addition of thiol source after approximately 20-min preperfusion to stabilize the in renal handling of the 2 compounds. Previous studies have kidney preparation. One experiment typical of 4. indicated that dimesna is freely filtered and actively reabsorbed; dimesna undergoes an enzyme-catalyzed reduction in the cyto extra thiol source contained a low concentration of â€”SHgroups, plasm of the tubular epithelial cells and is subsequently returned possibly representing a nonphysiological loss of material from to the tubular lumen in thiol form, whereas any surplus of the nephronal lining during perfusion. The excretion of dimesna- absorbed disulfide is returned to the perivascular capillaries (17, derived thiol groups amounted to more than twice that of W- 18). W-Acetylcysteine, however, may undergo a net reabsorption acetylcysteine-derived thiols and occurred at a more rapid rate, after filtration and partly enter the metabolic system of the kidney particularly during the initial phase after addition of thiol source. itself or be returned to the circulating plasma. Our results are These observations suggest differences in the renal handling of well in line with pharmacokinetic data obtained previously4 (17), dimesna and A/-acetylcysteine. that in the rat as well as in humans only approximately 15% of an i.v.-injected dose of W-acetylcysteine was excreted in urine, DISCUSSION and mainly in disulfide form, whereas after injection of mesna or It has previously been shown (5, 17) that, irrespective of the dimesna, 40 to 70% appeared in urine, almost exclusively in thiol form administered and the route of administration, mesna will form. exist in a chemically stable and pharmacologically inert disulfide Thus, the present study indicates that mesna or dimesna may form in peripheral blood. Most living cells, with the exception of be a more favorable choice than W-acetylcysteine for increasing intestinal and renal epithelium, do not absorb low molecular thiol output in urine. weight disulfides (cf. RÃ©f.17), and therefore dimesna is likely to be quantitatively excreted in the kidney. W-Acetylcysteine, how REFERENCES ever, remains in reduced form in plasma (3) (Chart 1), and 1. Ashkar, S., Binktey, F., and Jones, D. P. Resolution of a renal sulfhydryl therefore is accessible for protein binding as well as for chemical (glutathione) oxidase from >-glutamyltransferase. FEBS Lett., 124: 166-168, reactions of the free thiol groups with other plasma components 1981. while circulating. During passage of the renal peritubular capillary 2. Berrigan, M. J., Mannello, A. J., Pavelic, Z., Williams, C. J., Struck, R. F., and Gurtco, H. J. Protective role of thiols in cyclophosphamide-induced urotoxicity beds, A/-acetylcysteine is exposed to the thiol-oxidizing enzyme and depression of hepatic drug metabolism. Cancer Res., 42: 3688-3695, which is located in the basolateral part of the plasma membrane 1982. of renal epithelium (12,14,15,16) and for which A/-acetylcysteine 3. 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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1984 American Association for Cancer Research. N-Acetylcysteine and Sodium 2-Mercaptoethane Sulfonate as Sources of Urinary Thiol Groups in the Rat

Kari Ormstad and Yasuo Ohno

Cancer Res 1984;44:3797-3800.

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