[CANCER RESEARCH 44, 3797-3800, September 1984]
N-Acetylcysteine and Sodium 2-Mercaptoethane
Urinary Thiol Groups in the Rat1
Kari Ormstad2 and Yasuo Ohno
Department of Forensic Medicine, Karolinska Inst/tutet. S-104 01 Stockholm. Sweden
ABSTRACT
Increasing the urinary output of free thiol groups protects
against cyclophosphamide-induced bladder toxicity. In the pres
ent study, intact rats, isolated perfused kidneys, and freshly
isolated cells from various rat organs are used to compare the
efficacy of A/-acetylcysteine and sodium 2-mercaptoethane sul-
fonate (mesna) as sources of urinary thiols.
In intact rats given a single i.v. dose of mesna, urinary thiol
output is approximately 10-fold higher than in rats given an
equimolar dose of /V-acetylcysteine. This is partly due to the fact
that A/-acetylcysteine is rapidly absorbed by various types of
cells, whereas mesna is transported selectively to the kidney,
and partly to different renal handling of the two compounds. The
results suggest that mesna is a more favorable drug than A/-
acetylcysteine for increasing urinary thiol excretion.
INTRODUCTION
Oxazaphosphorine compounds (cyclophosphamide, ifosfam-
ide, and trofosfamide) are frequently used as cytostatics in
cancer chemotherapy and as immunosuppressants in organ
transplant recipients and in various states of immunological
derangement. Among other side effects, a frequently arising
problem during this treatment is hemorrhagic cystitis, which has
been attributed to the reactive aldehyde, acrolein (CH;>=CH—
CHO), a product of Oxazaphosphorine metabolism (6,7). Several
studies have indicated that compounds which are excreted in a
form featuring free thiol groups may counteract the urotoxic
effect of oxazaphosphorines (4, 5, 11), and among these A/-
acetylcysteine and mesna3 seem to be most effective. In a recent
paper, Berrigan ef al. (2) reported that the latter compounds, if
given in appropriate doses, are equally effective in protecting
against cyclophosphamide-induced urothelial damage and
depression of hepatic drug metabolism.
Since oxazaphosphorine-derived acrolein, which exerts its
toxicity on the lining of the urinary passages, is likely to be
produced after glomerular filtration (21), the potency of a uropro-
tective drug is dependent on its capacity to yield free thiol groups
in the urine. The aim of the present study was to compare A/-
acetylcysteine and mesna as sources of urinary thiols in the rat.
MATERIALS AND METHODS
Male Sprague-Dawley rats weighing 200 to 300 g were kept in
stainless steel cages with free access to tap water and pelleted rat food.
'This work was funded by the Swedish Board for Technical Development and
by the Swedish Medical Research Council, Grant 03X 2471.
"To whom requests for reprints should be addressed, at Department of Forensic
Mediane, Karolinska Institute!. Box 60400, S-104 01 Stockholm, Sweden.
3The abbreviations used are: mesna, sodium 2-mercaptoethane sulfonate; di-
mesna, sodium 2-mercaptoethane sulfonate disulfìde;SH, sulthydryl
Received September 28,1983; accepted June 4,1984.
SEPTEMBER 1984
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Sulfonate as Sources of
Cells of various organs were isolated according to techniques routinely
used in our laboratory; hepatocytes as described by Moldéuset al. (13),
kidney cells according to the data of Jones ef al. (10), intestinal epithelial
cells according to the data of Dawson and Bridges (8), myocardial cells
according to the data of Rajs et al. (19), and pulmonary cells according
to the data of Dawson et al. (9). In vitro perfusion of isolated kidneys
was performed as described by Ormstad ef al. (15), and urine was
collected from ureteral catheters.
For the in vivo experiments, rats were anesthetized with diethyl ether.
Via a small abdominal incision, a silicone catheter was inserted in the
urinary bladder and secured with a ligature, and the urethra was closed
immediately below the neck of the bladder. In the left femoral vein, 16
i/mol Of W-acetylcysteme (16 mw; 1 ml) or 8 ^mol of dimesna (8 ITIM; 1
ml) were injected; similarly treated and operated rats which had been
given i.v. injections of 1 ml of 0.9% NaCI served as controls. To prevent
hypoperfusion and pollakisuria during the experiment, 10 ml of 0.9%
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-
roacetic acid, and stored on ice for up to 2 hr, whereupon the content
of free thiol groups was determined according to the spectrophotometric
method of Saville (20).
All in vitro incubations were performed in a medium consisting of a
modified Krebs-Henseieit bicarbonate buffer, pH 7.4, supplemented with
25 mw 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid in rotating
round-bottomed, siliconized glass flasks at 37°under continuous gassing
with carbogen (95% O2 plus 5% CO2). Samples were removed at preset
time points and, in the case of cell incubations designed to study thiol
oxidation, the cells were sedimented by centrifugation (700 rpm for 1
min) before the supernatant was further processed. Cellular uptake of
mesna, dimesna, and W-acetylcysteine was determined by incubation of
isolated cells with the compounds in question, separation of the cells
from the medium by rapid centrifugation in a microfuge, and washing
once with fresh medium. Free thiols were determined after deproteini-
zation by addition of an equal volume of 6.5% trichloroacetic acid
according to the spectrophotometric method of Saville (20).
Mesna and dimesna were generously supplied by Asta-Werke AG,
Degussa Pharma Gruppe, Bielefeld, German Federal Republic. All other
chemicals were of at least reagent grade and were purchased from local
commercial sources.
RESULTS
In contrast to previous findings with mesna (17), the sponta
neous rate of oxidation of A/-acetylcysteine was very slow in the
absence of biological material (Chart 1). Neither did addition of
citrated plasma to the incubation affect the oxidation rate. Incu
bation with isolated cells from rat intestine or kidney did, how
ever, lead to oxidation of A/-acetylcysteine to the corresponding
disulfide; about twice as rapidly in the presence of renal cells as
with intestinal cells. Incubations were also performed with iso
lated hepatocytes and pulmonary cells, but with these cells the
oxidation rate did not differ from that observed in the absence
of cells in the incubation (data not shown). Based upon these
results and upon those of Bonanomi and Gazzaniga (3), we
chose to perform the following comparative experiments with
3797
K. Ormstad and Y. Ohno

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 37° under a carbogen (95% O¡,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.
(•),and renal epithelial cells (10* cells/ml) in Krebs-Henseleit buffer (•).
Bars, mean
To eliminate the influence of other cells and tissues, a series of
±S.D. of 3 separate incubations.
experiments was performed where equimolar (in SH units) con
centrations of dimesna (200 n»)and N-acetylcysteine (400 ¿¿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

/¿molof dimesna (O), 16 nmol of N-acetylcysteine (D). or 15.4 ^mol of NaCI (A).

mesna in disulfide form (dimesna) and N-acetylcysteine in thiol Õ

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
of 8 /¿molof dimesna (O), 16 umol of N-acetylcysteine (HI), or 15.4 «molof NaCI
jection of NaCI immediately prior to urine collection, a low and
(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 ¡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 ±40
30 min of relative pollakisuna. diuresis was stabilized at about Renal epithelial 218 ±25 180 ±30 205 ±30
50 /tl/min, which probably is reflecting an excretion of free water Intestinal epithelial 235 ±19 161 ±28 306±43
Pulmonary 6± 3 ND 331 ±31
excess. Thus, the total amount of free thiol excreted during the ±18*
MyocardialMesna5± NDDimesnaND6 NDN-acetyl 132
experiment was approximately 10-fold higher in rats given injec Mean ±S.D.
tions of dimesna than in those given injections of N-acetyl " ND, not detectable.

3798 CANCER RESEARCH VOL. 44

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_ 2
20 40 60
time ( mm )
Chart 4. Accumulated urinary excretion of free thiol groups in isolated rat
kidneys perfused with a medium supplemented with 200 ^M dimesna (O) or 400
¡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
kidney preparation. One experiment typical of 4.
extra thiol source contained a low concentration of —SHgroups,
possibly representing a nonphysiological loss of material from
the nephronal lining during perfusion. The excretion of dimesna-
derived thiol groups amounted to more than twice that of W-
acetylcysteine-derived thiols and occurred at a more rapid rate,
particularly during the initial phase after addition of thiol source.
These observations suggest differences in the renal handling of
dimesna and A/-acetylcysteine.
DISCUSSION
It has previously been shown (5, 17) that, irrespective of the
form administered and the route of administration, mesna will
exist in a chemically stable and pharmacologically inert disulfide
form in peripheral blood. Most living cells, with the exception of
intestinal and renal epithelium, do not absorb low molecular
weight disulfides (cf. Réf.17), and therefore dimesna is likely to
be quantitatively excreted in the kidney. W-Acetylcysteine, how
ever, remains in reduced form in plasma (3) (Chart 1), and
therefore is accessible for protein binding as well as for chemical
reactions of the free thiol groups with other plasma components
while circulating. During passage of the renal peritubular capillary
beds, A/-acetylcysteine is exposed to the thiol-oxidizing enzyme
which is located in the basolateral part of the plasma membrane
of renal epithelium (12,14,15,16) and for which A/-acetylcysteine
is a substrate (1) (Chart 1). However, under in vivo conditions,
W-acetylcysteine will be filtered in the renal glomeruli before
reaching the site of thiol oxidase in the proximal convoluted
tubules, and it therefore seems likely that it will be excreted in
thiol form, which is also indicated by pharmacokinetic studies
(cf. Réf.3).
The concentration of thiol in the urine from rats preloaded with
fluid and given injections of a bolus dose of dimesna initially was
more than 10-fold higher than in urine from similarly treated rats
given injections of an equimolar amount (SH units) of W-acetyl
cysteine (Chart 2). However, the thiol excretion rapidly declined
and after 100 min was approximately 4-fold higher than in rats
given injections of W-acetylcysteine and approximately 8-fold
higher than in control animals. No systematic difference in di
uresis could be noted among the 3 groups of animals, and the
accumulated thiol excretion thus was proportional with the uri
nary concentrations of SH groups; approximately 10-fold higher
SEPTEMBER 1984
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Acetylcysteine and Mesna as Urinary Thiol Sources
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
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
the 2 sources studied. During perfusion of isolated kidneys, all
extrarenai influences are eliminated, and also under these con
ditions the urinary output of dimesna-derived thiols was about
twice that of W-acetylcysteine-derived. This suggests a difference
in renal handling of the 2 compounds. Previous studies have
indicated that dimesna is freely filtered and actively reabsorbed;
dimesna undergoes an enzyme-catalyzed reduction in the cyto
plasm of the tubular epithelial cells and is subsequently returned
to the tubular lumen in thiol form, whereas any surplus of
absorbed disulfide is returned to the perivascular capillaries (17,
18). W-Acetylcysteine, however, may undergo a net reabsorption
after filtration and partly enter the metabolic system of the kidney
itself or be returned to the circulating plasma. Our results are
well in line with pharmacokinetic data obtained previously4 (17),
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,
and mainly in disulfide form, whereas after injection of mesna or
dimesna, 40 to 70% appeared in urine, almost exclusively in thiol
form.
Thus, the present study indicates that mesna or dimesna may
be a more favorable choice than W-acetylcysteine for increasing
thiol output in urine.
REFERENCES
1. Ashkar, S., Binktey, F., and Jones, D. P. Resolution of a renal sulfhydryl
(glutathione) oxidase from >-glutamyltransferase. FEBS Lett., 124: 166-168,
1981.
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
and depression of hepatic drug metabolism. Cancer Res., 42: 3688-3695,
1982.
3. Bonanomi, L., and Gazzaniga, A. lexicological, pharmacokinetic and metabolic
studies on acetylcysteine. Eur. J. Respir. Dis., 6Ã-(Suppl.).-111: 45-51,1980.
4. Botta, J. A., Nelson, L. W., and Weikel, J. H. Acetylcysteine in the prevention
of cyclophosphamide-induced cystitis in rats. J. Nati. Cancer Inst., 5?; 1051-
1057,1973.
5. Brock, N.. Pohl. J., and Stekar, J. Studies on the urotoxicity of oxazaphos-
phorine cytostatics and its prevention. II. Comparative study on the uroprotec-
tive efficacy of thiols and other sulfur compounds. Eur. J. Cancer, 17: 1155-
1163.1981.
6. Brock, N., Stekar, J., Pohl, J., Niemeyer, U., and Scheffler, F. Acrotein, the
causative factor of urotoxic side-effects of cydophosphamide, ifosfamide,
trofosfamide and sulfosfamide. Drug Res., 29: 659-661,1979.
7. Cox, P. J. Cydophosphamide cystitis—identification of acrolein as the causa
tive agent. Biochem. Pharmacol., 28: 2045-2049.1979.
8. Dawson, J. R., and Bridges, J. R. Xenobiotic metabolism by isolated intestinal
epithelial cells from guinea pigs. Biochem. Pharmacol., 28: 3299-3305,1979.
9. Dawson, J. R., Norbeck, K.. and Moldeus. P. The isolatioin of rat lung cells for
the purpose of studying drug metabolism. Biochem. Pharmacol., 37: 3549-
3553.1982.
10. Jones, D. P., Sundby. G.-B. Ormstad, K., and Orrenius, S. Use of isolated
kidney cells for study of drug metabolism. Biochem. Pharmacol., 28:929-935,
«N.Brock, K. Ormstad, S, Orrenius, T. Lâstbom, N. Uehara, J. Pohl, and J.
Stekar, unpublished results.
3799
K. Ormstad and Y. Ohno
1979.
11. Kedar, A., Simpson, C. L., Williams, P., Moore, R.. Tritsch, G.. and Murphy,
G. P. The prevention of cydophosphamide-mduced bladder swelling in the rat
by the i.v. administration of sodium-2-mercaptoethane sulfonate. Res. Com
mun. Chem. Pathol. Pharmacol., 29: 339-348,1980.
12. Lash, L. H . and Jones, 0. P. Localization of the membrane-associated thiol
oxidase of rat kidney to the basal-lateral plasma membrane. Biochem. J . 203:
371-376,1982.
13. Moldeus. P., Hogberg. J., and Orrenius, S. Isolation and use of liver cells.
Methods Enzymol., 5Õ:60-71,1978.
14. Ormstad, K., Lastbom, T., and Orrenius, S. Characteristics of renal glutathione
oxidase activity. FEBS Lett., 730. 239-243,1981.
15. Ormstad, K., Lâstbom, T., and Orrenius, S. Evidence for different localization
of glutathione oxidase and •>
-giutamyItransferase activities during extracellular
glutathione metabolism in isolated perfused rat kidney. Biochem. Biophys.
Acta, 700: 148-153,1982.
16. Ormstad, K., Moldeus, P., and Orrenius, S. Partial characterization of a
3800
Downloaded from cancerres.aacrjournals.org on July 16, 2019. © 1984 American Association for Cancer Research.
glutathione oxidase present in rat kidney plasma membrane fraction. Biochem.
Biophys. Res. Commun., 89: 497-503,1979.
17. Ormstad. K., Orrenius, S., Lâstbom, T., Uehara, N., Poni, J., Stekar. J., and
Brock, N. Pharmacokinetics and metabolism of sodium-2-mercaptoethanesul-
fonate in the rat. Cancer Res., 43:333-338,1983.
18. Ormstad, K., and Uehara, N. Renal transpon and disposition of Na-2-mercap-
toethane sulfonate disulfide (dimesna) in the rat. FEBS Lett., 750: 354-357,
1982.
19. Rajs, J., Sundberg, M., Sundby, G.-B., Dane«,N., Tomling, G., Biberfeld, P.,
and Jakobsson, S. W. A rapid method for the isolation of viable cardiac
myocytes from adult rat. Exp. Cell Res., Õ75:183-189,1978.
20. Saville, B. A scheme for the colorimetrie determination of microgram amounts
of thiois Analyst, 83: 670-672,1958.
21. Wildenauer, 0. B., and Oehlmann C. E. Interaction of cyclophosphamide
metabolites with membrane proteins: an in vitro study with rabbit liver micro-
somes and human red blood cells. Effect of thtols. Biochem. Pharmacol., 3f:
3535-3541,1982.
CANCER RESEARCH VOL. 44
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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