[CANCER RESEARCH 40. 3950-3955.
0008-5472/80/0040-OOOOS02.00
November 1980]
Uptake and Decomposition of Chlorozotocin in L5178Y Lymphoblasts
in Vitro'
Hing-Yat Peter Lam, Michael M. Talgoy, and Gerald J. Goldenberg
Manitoba
Institute of Cell Biology ¡H-Y. P. L.. M. M. 7. G. J. GJ. and the Department
of Medicine.
University of Manitoba ¡H-Y. P. L., G. J. GJ, Winnipeg,
Manitoba R3E OV9, Canada
ABSTRACT
Uptake and metabolism of 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose
(Chlorozotocin) by L5178Y lymphoblasts in vitro was investigated, using both glucose- and chloroethyl-labeled Chlorozotocin. A time course of uptake of total
radioactivity revealed a greater cell/medium distribution ratio
of activity in cells treated with chloroethyl-labeled Chlorozotocin
compared to cells treated with the glucose-labeled compound.
Thin-layer Chromatographie analysis showed that uptake of
intact Chlorozotocin was identical in cells treated with either
glucose- or chloroethyl-labeled drug and that the cell/medium
distribution ratio never exceeded unity. Accumulation of 14Cchlorozotocin was not inhibited by an excess of unlabeled
Chlorozotocin, the structural analogs glucose and glucosamine,
or several metabolic inhibitors or by sodium ion depletion.
These observations, together with the relatively low tempera
ture quotient for the uptake process, suggested that Chloro
zotocin uptake occurs by passive diffusion.
In cells treated with glucose-labeled Chlorozotocin, a bicyclic
urethan derivative and polar metabolites soluble in trichloroacetic acid were formed. In cells exposed to chloroethyl-la
beled drug, nonpolar as well as polar metabolites were noted.
Formation of metabolites from the glucose moiety was impeded
by the presence of an excess of unlabeled Chlorozotocin, the
structural analogs glucose and glucosamine, the glucose trans
port inhibitors phlorizin and phloretin, the metabolic inhibitor
m-chlorophenyl carbonyl cyanide hydrazone and by sodium
depletion. Appearance of metabolites arising from the chloroethyl moiety was also blocked by the presence of m-chloro
phenyl carbonyl cyanide hydrazone and by sodium ion deple
tion. These results suggested that metabolism of Chlorozotocin
in L51 78Y lymphoblasts appears to be enzyme mediated.
INTRODUCTION
The glucose-containing nitrosourea, 2-[3-(2-chloroethyl)-3—
nitrosoureido]-D-glucopyranose
(Chlorozotocin), has antitumor
activity against murine L1210 leukemia comparable to that of
BCNU2 but without significant myelotoxicity (1). It has been
suggested that the glucose moiety in Chlorozotocin and other
nitrosoureas may play an important role in this selective sparing
of normal bone marrow cells (6, 15, 16).
We have described the mechanism of uptake of several
alkylating agents including the nitrosoureas BCNU and 1-(2' This work was supported
by a grant from the National Cancer Institute of
Canada.
2 The abbreviations used are: BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea;
Dulbecco's PBS. Dulbecco's phosphate buffered saline; TLC, thin-layer chromatography; TCA. trichloroacetic acid; CCCP. m-chlorophenyl
hydrazone.
Received March 21. 1980; accepted July 30. 1980.
3950
carbonyl cyanide
chloroethyl)-3-cyclohexyl-1
-nitrosourea in normal and neoplastic cells. Uptake of BCNU and 1-(2-chloroethyl)-3-cyclohexyl1-nitrosourea by murine leukemia L5178Y cells was by passive
diffusion (2); however, glucose transport in several mammalian
cells is carrier mediated (3, 7, 13, 17). An investigation of the
mechanism of transport of Chlorozotocin in L51 78Y cells was
undertaken to determine if the uptake process is by simple
diffusion or is carrier mediated and if the preferential cytotoxicity of the drug for leukemia cells might be explained by the
transport mechanism.
MATERIALS AND METHODS
[g/ucose-1-14C]Chlorozotocin
(specific
mmol), [2-ch/oroef/7y/-L/-"'C]chlorozotocin
activity, 19.6 mCi/
(specific activity,
15.8 mCi/mmol), and unlabeled Chlorozotocin were kindly
supplied by Dr. Robert R. Engle, Drug Development Branch,
Division of Cancer Treatment, National Cancer Institute, Bethesda, Md.
L51 78Y cells were maintained in culture in Fischer's medium
supplemented with 10% horse serum. Studies of drug uptake
were performed on cell suspensions at a concentration of 4
x 106 cells/ml in Dulbecco's PBS at pH 7.4, as described
previously (8, 9). Radioactive Chlorozotocin was dissolved in
80% ethanol and diluted appropriately so that the final concen
tration of ethanol never exceeded 1%.
TLC was used to identify intact Chlorozotocin. Cells treated
with labeled drug were chilled to 4°, centrifuged through a
layer of 0.25 M sucrose to remove extracellular radioactivity,
and lysed in distilled water at 4°for 15 min. To determine the
amount of free intracellular Chlorozotocin, TLC of cell lysate
and medium was performed in a solvent system consisting of
chloroform/methanol
(2/1); in this system, glucose and glu
cosamine remain at the origin. Either silica gel 60-precoated
plastic sheets (Merck, Darmstadt, West Germany) or silica gel
G-precoated glass plates (Analtech, Inc., Newark, Del.) were
used. The plastic sheets were cut in 0.5-cm strips or the silica
gel was scraped off the glass plates, and radioactivity was
determined by liquid scintillation spectrometry.
The distribution of radioactivity between TCA-soluble and
-insoluble fractions was determined for cells treated with glu
cose- and chloroethyl-labeled Chlorozotocin. An aliquot of cell
lysate was analyzed by TLC to determine the amount of radio
activity located at the origin, a zone that would contain macromolecules and polar metabolites. Ice-cold TCA was added to
another aliquot of cell lysate to give a final concentration of
10% and was maintained on ice for 1 hr. The TCA solution was
filtered through a Millipore membrane (HA type; pore size, 0.45
/im); the filter was washed with ice-cold 10% TCA, and radio
activity was determined. The filtrate and washings were com
bined and analyzed by TLC; the total recovery of radioactivity
in the filtrate was greater than 90%.
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Uptake and Decomposition
Cell volume was measured using a Model ZBi Coulter
Counter (Coulter Electronics, Inc., Hialeah, Fla.), calibrated
with giant ragweed pollen (mean cell diameter, 19.5 /¿m)and
paper mulberry spores (mean cell diameter, 12.5 /¿m).
RESULTS
Time Course of Uptake of Total Radioactivity by Cells
Treated with Ring- or Chloroethyl-labeled Chlorozotocin. A
time course of uptake of radioactivity by L51 78Y cells treated
with either [g/t/cose-1-1*C]chlorozotocin
or [2-chloroethyl-U14C]chlorozotocin is shown in Chart 1. For both compounds,
uptake of radioactivity approximated linearity for 60 min and
was temperature dependent. At 37°,the uptake of radioactivity
by cells treated with chloroethyl-labeled
chlcrozotocin was
higher than that of cells treated with glucose-labeled Chloro
zotocin, with gradients of total radioactivity of 1.8 and 0.4,
respectively, at 60 min; uptake of radioactivity at 4° was
markedly reduced to gradients of 0.06 and 0.03, respectively.
The difference in uptake of total radioactivity by cells treated
with ring- and chloroethyl-labeled Chlorozotocin suggested dif
ferential accumulation of the glucose and chloroethyl moieties.
Decomposition of Chlorozotocin. Since Chlorozotocin is
labile in aqueous solution, we studied the decomposition of
Chlorozotocin in Dulbecco's PBS, the same medium used in all
the transport studies. Either glucose- or chloroethyl-labeled
Chlorozotocin was incubated in Dulbecco's PBS at pH 7.4 at
37°. The decomposition of the parent compound, which mi
magnetic resonance spectrum identical to the product derived
from streptozotocin. Therefore, we conclude that the Chloro
zotocin decomposition product is also a bicyclic urethan deriv
ative (Chart 2).
A different decomposition product, which migrated with an
RF of 0.80 but was not characterized further, arose from the
chloroethyl moiety. Decomposition of either ring- or chloro
ethyl-labeled Chlorozotocin also yielded polar decomposition
products which remained at the origin on TLC. Decomposition
of Chlorozotocin was very slow at 4°;only 10% decomposition
was detected after 70 min.
Time Course of Accumulation of Intact Drug in L5178Y
Cells. To determine the uptake mechanism of intact drug, cell
contents and medium were analyzed by TLC after L51 78Y cells
were treated with ring- and chloroethyl-labeled Chlorozotocin
(Chart 3). In cells treated with glucose-labeled drug, the parent
compound migrated with an RF of 0.62, and the decomposition
product, bicyclic urethan, migrated with an RF of 0.38 (Chart
3, A and ß).High levels of radioactivity accumulated in the
origin fraction of the cell lysate relative to that observed in the
medium. Radioactivity at the origin was considered to represent
polar metabolite(s) of Chlorozotocin in the cell, although binding
of Chlorozotocin or its glucose moiety to macromolecules could
not be excluded.
TLC analysis of cell lysate and medium following incubation
with chloroethyl-labeled
Chlorozotocin showed that the intact
_ °.
/
grated with an RF of 0.62 on TLC plates, appeared to follow
pseudo-first order kinetics with a half-life of 33 min with either
the glucose- or chloroethyl-labeled compound.
Decomposition of glucose-labeled Chlorozotocin resulted in
the appearance of a radioactive peak with an RF value of 0.38.
This apparent decomposition product contained the glucose
but not the chloroethyl moiety, since a radioactive fraction of
similar RF was not found among the decomposition products
arising from the chloroethyl-labeled
preparation. Decomposi
tion of streptozotocin in alkaline solution was also studied and
yielded a product, which had been identified previously as a
bicyclic urethan derivative (10). The decomposition product of
Chlorozotocin had Chromatographie properties and a proton
of Chlorozotocin
N°M >°H
H°N -
(
DECOMPOSITION
US
pH 7.t
IN
NO
HO
-
1
HN-C-N-CH2CH2CI
O
CHLOROZOTOCIN
BICYCLIC URETHANE
DERIVATIVE
Chart 2. Formation of the bicyclic urethan decomposition
rozotocin.
i
1—
product from Chlo
~â„¢lorigin
10.621hn1
A•origin-RfRf
1
Ctsolvent
8060£
40U
< 20
0o<<
Rf 0.62
'""l-1
0.38ii-T-^-,—
*..
j-i^_rTDPL
r-TL,B-L^-rl
60OÔ
40
6?200—
iLr7"1
30
40
00
TIME (min)
0.2
0.4
0.6
0.8
,1
1.0 O
r-TL
0.2
0.4
0.6
0.8
;806
1.0
Rf
Chart 1. Time course of the uptake of radioactivity by L5178Y cells treated
with 0.1 RIM [g/ucose-1-14C]chlorozotocin
in vitro at 37°(O) and at 4°(•)or by
cells treated with 0.1 HIM [2-cWoroerhy/-U-14C]-chlorozotocin
at 37°O and at
4°(•).Uptake is expressed as the cell/medium distribution ratio of radioactivity
Chart 3. Thin layer chromatograms of extracts of cell contents (A) and tissue
culture medium (6) after incubation of L5178Y cells for 20 min at 37°in medium
containing 0.1 HIM [glucose-1-"C}Chlorozotocin and of cell contents (C) and
culture medium (D) after treatment of cells with 0.1 mw [2-chloroethyl-U-'"C]-
as described in the text and previously (8, 9). Points, average of two determina
tions.
chlorozotocin. TLC was performed on silica gel 60 plates in a solvent system
consisting of chloroform/methanol
(2/1) as described in the text.
NOVEMBER
1980
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3951
H-Y. P. Lam et al.
drug migrated with an RF of 0.62, identical to that noted with
the glucose-labeled counterpart (Chart 3, C and D). Radioac
tivity was observed at the solvent front in the cell lysate but not
in the medium; such a radioactive peak was not observed when
decomposition of chloroethyl-labeled chlorozotocin was stud
ied in cell-free medium. These findings suggested that nonpolar
derivatives are generated from the chloroethyl moiety as a
result of cellular metabolism. The accumulation of radioactivity
at the origin in the cell lysate once again suggested the for
mation of polar metabolite(s) or binding of the intact drug or its
chloroethyl moiety to macromolecules.
Uptake of intact chlorozotocin by cells treated with either
ring- or chloroethyl-labeled
chlorozotocin appeared to follow
an identical time course as shown in Chart 4; the cell/medium
distribution ratio of intact drug was below 0.15 at 60 min.
Evidence for the Formation of Small Polar Metabolites of
Chlorozotocin in L5178Y Cells. As with other nitrosoureas,
chemical decomposition of chlorozotocin may yield active in
termediates such as isocyanate and carbonium ions capable
of reacting with cellular components. To determine the amount
of chlorozotocin associated with macromolecules in the cell,
the distribution of radioactivity in the TCA-soluble and -insolu
ble fractions was measured.
In cells treated with glucose-labeled chlorozotocin for 10
min, the percentage of total radioactivity that was TCA-insoluble was 4.4 ± 0.3% (S.E.). TLC analysis of the cell lysate
revealed that 56.2 ±0.9% of the total radioactivity remained
at the origin. Assuming that the macromolecules adhere firmly
to the silica gel and constitute part of the origin fraction, then
approximately 8% of origin counts (4.4/56.2 x 100%) can be
attributed to metabolites or chlorozotocin irreversibly bound to
TCA-insoluble macromolecules, and 92% of the origin counts
may represent TCA-soluble polar metabolites or parent drug
reversibly bound to macromolecules. When cells were treated
with chloroethyl-labeled
chlorozotocin, 22.5 ± 0.2% of the
total radioactivity was found to be TCA insoluble, and TLC
showed that 74.2 ±3.0% of the radioactivity remained at the
origin. Therefore, about 30% of the activity at the origin was
TCA-insoluble, and 70% was due to the presence of small
polar metabolites or reversibly bound drug.
The amount of intact drug recovered by TLC analysts of the
cell lysate was virtually identical to that observed on TLC of the
TCA-soluble extract in cells treated with either glucose- or
chloroethyl-labeled drug. This finding suggests that the radio
activity at the origin is due to small polar metabolites rather
.16
Õ.IJ
10
20
30
40
TIME (min)
50
Chart 4. Time course of uptake of intact drug by L5178Y cells treated with
[g/ucose-1-MC]-chlorozotocin
(O) or [2-cWoroettiy/-U-"'C]-chlorozotocin
(US).
Uptake is expressed as the cell/medium distribution ratio of intact drug as
determined by TLC and described in the text. Points, the average of 2 determi
nations.
3952
than reversibly bound drug, since reversibly bound drug would
be expected to dissociate during TCA extraction leading to a
greater recovery of free intact drug.
Temperature Dependence of Chlorozotocin Uptake. To
determine the effect of temperature on accumulation of intact
drug, we measured uptake of glucose-labeled chlorozotocin at
37°and 27°.TLC analysis of the following radioactive fractions
gave a temperature quotient, 0,0, of 1.7 for accumulation of
intact drug, 2.0 for appearance of origin counts, and 2.2 for
accumulation of the bicyclic urethan derivative.
Effect of Chlorozotocin and Sugar Analogs on the Uptake
of Chlorozotocin. The uptake of chlorozotocin by L5178Y cells
was studied in the presence of an excess of unlabeled drug,
glucose, or glucosamine (Table 1). All 3 analogs had no effect
on drug uptake in cells exposed to chloroethyl-labeled chlo
rozotocin; however, uptake of total radioactivity was inhibited
in cells treated with glucose-labeled chlorozotocin. TLC anal
ysis showed that for all 3 analogs this effect was due to
reduction of the radioactive fraction that remained at the origin.
Glucosamine was associated with enhanced accumulation of
intact drug and of the bicyclic urethan decomposition product.
Unlabeled chlorozotocin was allowed to decompose in PBS
for 4 hr at 37°. A 5-fold excess of this incubation mix and a
50-fold excess of the bicyclic urethan decomposition product
each failed to inhibit uptake of glucose-labeled chlorozotocin
by the cells.
Effect of Metabolic Inhibitors on the Uptake of Chlorozo
tocin. The effect of several metabolic inhibitors on the uptake
of total radioactivity by cells treated with glucose- or chloro
ethyl-labeled chlorozotocin was examined (Table 2). The con
centration of inhibitor was the highest that could be used
without producing loss of cell viability as detected by trypan
blue dye exclusion. CCCP, an uncoupler of oxidative phosphorylation, inhibited uptake of total radioactivity by cells
treated with either glucose- or chloroethyl-labeled chlorozoto
cin. Uptake of radioactivity by cells treated with glucose-la
beled drug was also inhibited by phlorizin and phloretin, 2
glucose transport inhibitors; these compounds produced no
inhibition in the presence of the chloroethyl-labeled compound;
on the contrary, enhanced uptake was observed with phloretin.
The effect of those agents which inhibited uptake of total
radioactivity was studied further using TLC (Table 3). CCCP
inhibited the appearance of origin counts and of the bicyclic
urethan decomposition product in cells treated with glucoselabeled chlorozotocin, and, conversely, the level of intact drug
was increased. All 3 major radioactive fractions were inhibited
in cells treated with phlorizin; phloretin suppressed accumula
tion of origin counts and the bicyclic urethan derivative but not
the parent compound. In cells treated with chloroethyl-labeled
chlorozotocin, CCCP inhibited accumulation of the radioactive
compounds at the origin and solvent front and increased the
amount of intact drug.
Effect of Sodium Ion on the Uptake of Chlorozotocin.
Since carrier-mediated transport of glucose is sodium depend
ent (5), the effect of sodium on chlorozotocin uptake was
investigated. Uptake of radioactivity in sodium-poor medium
was 78 ±4 and 54 ±3% of control uptake for cells incubated
with ring and chloroethyl-labeled chlorozotocin, respectively.
TLC analysis of cell lysates showed that sodium concentration
did not affect uptake of intact drug (Table 4). In cells exposed
to glucose-labeled chlorozotocin, sodium depletion reduced
CANCER
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Uptake and Decomposition
of Chlorozotocin
Table 1
Effect of Chlorozotocin, glucose, and glucosamine on the accumulation of intact Chlorozotocin and its metabolites in
L5178Y cells
Cells (4 x 106/ml) were incubated with 0.1 mM glucose-labeled Chlorozotocin in the presence of 5 mM unlabeled
Chlorozotocin, 10 mM glucose, or 10 mM glucosamine for 10 min at 37°. The accumulation of intact drug was
determined by TLC as described in the text. The data presented are the amount of radioactivity accumulated at different
zones on TLC in the presence of inhibitor as a percentage of radioactivity appearing in the absence of inhibitor. Results
were analyzed statistically by the 2-tailed t test.
Radioactivity(%
of
control)InhibitorChlorozotocin
Chlorozotocin
total
activity91
±8a' b
95 ±e"
Glucose
101 ±7bTotal
Glucosamine[2-chloroethyl-U-"C]-
urethan114±
activity70
±5°
±4d
6"
63 ±3d
94 ±126
74 ±4a
75±6d[g/ucose-1-Origin76
126 ± 5CIntact
54 ±5"'4C]ChlorozotocinBicyclic
drug113
±6"
102
7*1 ±
37 ±6C
Mean ±S.E. of 4 determinations.
b Not significant.
cp<0.01.
d p < 0.001.
Table 2
Effect of metabolic inhibitors on uptake of Chlorozotocin by L5178Y
lymphoblasts in vitro
Cells were preincubated at 37°for 15 min with the inhibitor, 0.1 mM glucoseor chloroethyl-labeled
Chlorozotocin was added to the cell suspension, and
incubation was continued for 2 min before uptake of radioactivity was determined
as described in the text and previously (8, 9). The data are expressed as uptake
of radioactivity per 106 cells in the presence of metabolic inhibitor as a percentage
of control uptake in the absence of inhibitor. Results were statistically evaluated
by the 2-tailed f test.
-14C]Chlorozo-tocinInhibitorNaCN(0.1
[g/ucose-1
4C]-Chlorozotocin%
Table 4
Effect of sodium ion depletion on the accumulation of radioactive compounds
from Chlorozotocin in L51 78Y cells in vitro
Cells were incubated for 10 min in 0.1 mM glucose- or chloroethyl-labeled
Chlorozotocin in Hank' balanced salt solution or in sodium-poor medium consist
ing of Hanks' balanced salt solution with Tris replacing NaCI in isomolar propor
tions, with a Na* concentration of 5 mEq/liter. Radioactive decomposition
compounds derived from Chlorozotocin were separated by TLC. The data are
presented as the amount of radioactivity appearing in cells incubated in sodiumdepleted medium as a percentage of radioactivity accumulated by cells incubated
in Hanks' balanced salt solution. Results were evaluated by the 2-tailed t test.
control)ChlorozotocinGlucose
mw)Ouabain
mM)IAA(0.1
(0.25
mM)POMB(0.1
mM)CCCP
UM)HgCI2
(50
fiM)Phlorizin
(1
mM)Phloretin
(1
(0.1 mM)%
control88
of
3a101±
Radioactivity
(% of
control103
of
891 ±
front30
urethan89
drug97
594 ±
390 ±
±2°Intact ±1d
71 ±
699 ±
labeled
45 ±36Bicyclic
91 ±6dSolvent ±1b
09
1063±
762 ±
Chloroethyl labeledOrigin63±1a'b
4100 ±
699 ±
Mean ±S.E. of 4 determinations.
475 ±
292 ±
6 p < 0.001.
252 ±
3129 ±
c p < 0.05.
± 4PNSbNSNSNS<0.001NS<0.001<0.001[2-chloroethyl-U-i
±6PNSNSNSNS<0.001NSNS<0.02
Not significant.
Mean ±S.E. of 4 to 12 determinations.
1NS, not significant; IAA, iodoacetic acid; POMB, p-hydroxymercuribenzoate.
Table 3
Effect of metabolic inhibitors on the accumulation of intact Chlorozotocin and its
metabolites in L5178Y cells
Cells were preincubated with inhibitor for 15 min, 0.1 mM glucose- or chloro
ethyl-labeled Chlorozotocin was added to the cell suspension, and the incubation
was continued for another 10 min. The radioactive compounds in the cell lysate
were separated by TLC on silica gel plates developed in chloroform/methanol
(2/1). The data are expressed as accumulation of radioactivity in different
fractions in the presence of inhibitor as a percentage of radioactivity accumulated
in the absence of inhibitor. Results were evaluated by the 2-tailed i test.
Inhibitor[
glucose- 1-' 4C]Chlorozotocin
CCCP
Phlorizin
Phloretin
[2-ch/oroefhy/-U-'
urethan66
57 ±4b
19±4b30
Mean ±S.E. of 4 determinations.
p < 0.001.
°p < 0.02.
d Not significant.
NOVEMBER
±8°
60 ±2b
60 ±8°
68 ±2b
57 ±5CIntact
113
6d1 ±
"CJ-
Chlorozotocin
CCCPOrigin29±4a'b
drug1
±1bBicyclic
accumulation of the bicyclic urethan derivative and of origin
counts. In cells exposed to chloroethyl-labeled drug, sodium
depletion inhibited accumulation of radioactive compounds at
both the origin and solvent front.
DISCUSSION
A time course demonstrated that L5178Y cells treated with
chloroethyl-labeled Chlorozotocin accumulated radioactivity at
a greater rate than cells exposed to glucose-labeled drug.
However, TLC showed that uptake of intact drug was identical
in cells treated with either form of Chlorozotocin; furthermore,
the distribution ratio, of intact drug was less than unity, sug
gesting that uptake probably was not an active process.
In a study of the temperature dependence of Chlorozotocin
uptake, a do of 1.7 was obtained for uptake of intact drug
over the temperature interval from 27 to 37°. Although it is
difficult to distinguish between simple diffusion and facilitated
diffusion on the basis of Q10 values alone, the Oi0 for simple
diffusion is generally considered to approximate unity. How
ever, since diffusion probably involves breaking of hydrogen
bonds between the substrate and solvent, as well as thermal
54 ±5bSolventfront7±2b
1980
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3953
H-Y. P. Lam et al.
movement of parts of the molecules that constitute the barrier,
do values of 2 or greater may occur (4).
TLC analysis of cell contents following treatment with either
glucose- or chloroethyl-labeled chlorozotocin showed that the
fraction of radioactivity remaining at the origin constituted a
higher percentage of total radioactivity than the corresponding
fraction in the incubation medium (Chart 3). In the origin frac
tion, at least 70% of the radioactivity was TCA soluble. These
TCA solubility and Chromatographie findings suggested that
the origin fraction probably contains small polar metabolites
derived from chlorozotocin.
The possibility that the origin fraction within the cell might
arise by active transport of degradation products from the
medium was excluded by the finding that uptake of glucoselabeled chlorozotocin was not blocked by either degraded
chlorozotocin or by the cyclic urethan derivative. The difference
in uptake of total radioactivity observed with the glucose- and
chloroethyl-labeled
compounds may be due to alkylation of
macromolecules by the chloroethyl moiety.
Uptake of chloroethyl-labeled chlorozotocin was not altered
by an excess of unlabeled chlorozotocin, glucose, or glucosamine (Table 1). TLC analysis revealed that uptake of intact
chlorozotocin labeled in the glucose moiety was also not in
hibited by these 3 compounds. The finding that chlorozotocin
uptake was not saturable and did not display evidence of
chemical specificity suggested that uptake occurs by simple
diffusion.
However, unlabeled chlorozotocin and the simple analogs
glucose and glucosamine all inhibited uptake of total radioac
tivity in cells treated with glucose-labeled drug, and this effect
was due entirely to a reduction of the origin fraction, which
contains the small polar metabolites (Table 1). These findings
suggested that chlorozotocin, glucose and glucosamine inter
fere with chlorozotocin metabolism, specifically blocking for
mation of those metabolites arising from the glucose moiety.
Uptake of total radioactivity by cells treated with glucoseand chloroethyl-labeled
chlorozotocin was inhibited by the
metabolic antagonist CCCP. With glucose-labeled drug, this
inhibition was due to reduced formation of the origin fraction,
which includes polar metabolites, and of the bicyclic urethan
derivative (Table 3). With chloroethyl-labeled
chlorozotocin,
the inhibition represented a marked reduction of both the origin
fraction and that at the solvent front, which contained the
nonpolar metabolites. These findings together with the en
hanced accumulation of intact drug suggested that CCCP
blocked chlorozotocin metabolism. The observation that, with
either glucose- or chloroethyl-labeled chlorozotocin, uptake of
intact drug was not inhibited by CCCP also suggested that
chlorozotocin uptake occurs by simple diffusion.
Since chlorozotocin contains a glucose moiety, a study was
undertaken to determine whether chlorozotocin uptake is me
diated by the transport carrier for glucose. Phlorizin and phloretin, known inhibitors of glucose transport, reduced uptake of
total radioactivity by cells exposed to glucose-labeled but not
to chloroethyl-labeled
chlorozotocin (Table 2). TLC analysis
showed that phlorizin but not phloretin inhibited uptake of intact
chlorozotocin, while both inhibitors suppressed accumulation
of the bicyclic urethan derivative and of origin counts (Table
3). The significance of these observations is unclear, because
the mechanism whereby phlorizin and phloretin inhibit glucose
transport is not completely understood. Phlorizin has been
3954
shown to inhibit a number of energy-supplying enzyme reac
tions which are not directly involved with sugar metabolism
(11, 14), and, there is evidence that this agent may induce
structural changes in mitochondrial membranes (12). Thus,
interactions with enzymes and membranes may explain the
observed effects on chlorozotocin uptake and metabolism.
In summary, evidence that chlorozotocin uptake in L5178Y
cells occurs by simple diffusion was that the cell/medium
distribution ratio of drug never exceeded unity, uptake was
relatively temperature insensitive, sodium independent, and
unimpeded by metabolic inhibitors, the uptake process was
not saturable, and chemical specificity was not demonstrated.
This study does not exclude the possibility of an alternate
transport mechanism at lower drug concentrations.
Evidence for metabolism of chlorozotocin in L51 78Y cells
was the finding of differential rates of uptake of radioactivity in
cells treated with glucose- and chloroethyl-labeled
chlorozo
tocin and the formation of radioactive decomposition products.
In cells treated with glucose-labeled chlorozotocin, TCA-soluble polar metabolite(s) and a bicyclic urethan decomposition
product were observed. In cells treated with chloroethyl-la
beled chlorozotocin, nonpolar decomposition products, which
migrated at the solvent front, were noted, as well as polar
metabolites.
The formation of small polar metabolites from glucose-la
beled chlorozotocin was inhibited by unlabeled chlorozotocin,
glucose, and glucosamine, suggesting that chlorozotocin me
tabolism was saturable and chemically specific. Metabolism of
both glucose- and chloroethyl-labeled
drug was impeded by
CCCP and Na+ depletion. Finally phlorizin and phloretin in
hibited metabolism of the glucose-labeled but not the chloro
ethyl-labeled moiety. The properties of saturability, chemical
specificity, sensitivity to metabolic inhibitors, and sodium de
pendence suggested that chlorozotocin metabolism is enzymemediated.
ACKNOWLEDGMENTS
We thank Dr. Asher Begleiter tor helpful discussions and Dorothy Faulkner for
typing the manuscript.
REFERENCES
1. Anderson, T., McMenamin, M. G., and Schein, P. S. Chlorozotocin, 2-[3-(2chloroethyl)-3-nitrosoureido]-D-glucopyranose,
an antitumor agent with
modified bone marrow toxicity. Cancer Res., 35: 761-765, 1975.
2. Begleiter, A., Lam. H-Y. P., and Goldenberg, G. J. Mechanism of uptake of
nitrosoureas by L5178Y lymphoblasts in vitro. Cancer Res.. 37: 10221027. 1977.
3. Busse. D.. Elsas. L. J., and Rosenberg L. E. Uptake of D-glucose by renal
tubule membrane. J. Biol. Chem.. 247: 1188-1193.
1972.
4. Christensen. H. N. Biological Transport, pp. 107-111. London: Benjamin
Inc., 1975.
5. Crane, R. K. Na*-dependent transport in the intestine and other animal
tissues. Fed. Proc., 24: 1000-1006.
1965.
6. Fox, P. A., Panasci, L. C., and Schein, P. S. Biological and biochemical
properties of 1-(2-chloroethyl)-3-(/i
-glucopyranosyl)-1-nitrosourea,
a nitrosourea with reduced bone marrow toxicity. Cancer Res., 37: 383-387,
1977.
7. Gardner. J. D.. Brown. M. S., and Laster. L. The columnar epithelial cell of
the small intestine: digestion and transport. N. Engl. J. Med.. 283: 11961202. 1970.
8. Goldenberg, G. J.. Lam. H-Y. P.. and Begleiter, A. Active carrier-mediated
transport of melphalan by two separate amino acid transport systems in
LPC-1 plasmacytoma cells in vitro. J. Biol. Chem., 254 1057-1064, 1979.
9. Goldenberg. G. J.. Vanstone. C. L., Israels, L, G., Use, D., and Bihler, I.
Evidence for a transport carrier of nitrogen mustard in nitrogen mustardsensitive and -resistant L5178Y lymphoblasts. Cancer Res.. 30: 22852291. 1970.
CANCER
RESEARCH
VOL. 40
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1980 American Association for Cancer Research.
Uptake and Decomposition
10. Herr, R. R., Jahnke, H. K., and ArgoudeMs, A. D. The structure of streptozotocin. J. Am. Chem. Soc., 89: 4808-4809,
1967.
11. Keller, D. M., and Lotspeich, W. D. Some effects of phlorizin on the
metabolism of mitochondria. J. Biol. Chem., 234. 987-990, 1959.
12. Keller, D. M., and Lotspeich, W. D. Effect of phlorizin on the osmotic behavior
of mitochondria in isotonic sucrose. J. Biol. Chem., 234. 991-994. 1959.
13. Lefevre, P. G. Sugar transport in the red blood cell: structure activity
relationships in substrates and antagonists. Pharmacol. Rev., )3: 39-70.
1961.
14. Lotspeich, W. D., and Keller, D. M. A study of some effects of phlorizin on
the metabolism of kidney tissue in vitro. J. Biol. Chem., 222. 843-853,
NOVEMBER
of Chlorozotocin
1956.
15. Nagourney, R. A., Fox, P.. and Schein, P. S. A comparison of the biological
and biochemical properties of 1-(4-amino-2-methyl-pyrimidin-5-yl)-methyl3-<2-chloroethyl)-3-nitrosourea
and 2-(3-(2-chloroethyl)-3-nitrosoureido)-Dglucopyranose. Cancer Res., 38. 65-68, 1978.
16. Panasci, L., Fox, P., Wooley. P.. and Schein, P. S. Pharmacologie studies
of non-myelosuppressive nitrosourea antitumor agents. Clin. Res.. 24: 380A,
1976.
17. Stein, W. D. The Movement of Molecules across Cell Membranes, pp. 266308. New York: Academic Press, Inc., 1967.
1980
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1980 American Association for Cancer Research.
3955
Uptake and Decomposition of Chlorozotocin in L5178Y
Lymphoblasts in Vitro
Hing-Yat Peter Lam, Michael M. Talgoy and Gerald J. Goldenberg
Cancer Res 1980;40:3950-3955.
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