1. Art and Diseño

Toxicology Letters 144 (2003) 289 /294
www.elsevier.com/locate/toxlet
Spin labeled antioxidants protect bacteria against the toxicity
of alkylating antitumor drug CCNU
Vesselina Gadjeva a,*, Grozdanka Lazarova b, Antoaneta Zheleva a
a
Department of Chemistry and Biochemistry, Medical Faculty, Thracian University, 11 Armeiska Str., 6000 Stara Zagora, Bulgaria
b
Department of Microbiology, Medical Faculty, 11 Armeiska Str., Thracian University, 6000 Stara Zagora, Bulgaria
Received 11 December 2002; received in revised form 5 May 2003; accepted 5 May 2003
Abstract
We have studied the toxic effect of the alkylating antitumor drug N ?-cyclohexyl-N -(2-chloroethyl)-N -nitrosourea
(lomustine, CCNU) on Escherichia coli (E. coli ) and Staphylococcus aureus (S. aureus ) strains, alone and in presence of
oxygen radical-scavenging substances [Vitamin E, stable nitroxyl radical 2,2,6,6-tetramethylpiperidine-N -oxyl (TMPO),
and spin labeled (nitroxyl free radical moiety containing) analogues of CCNU] and compared with that of the
alkylating antitumor drug 5-(3,3-dimethyltriazene-1-yl)-imidazole-4-carboxamide (dacarbazine, DTIC). All spin
labeled compounds tested were almost no toxic at doses of 50 /500 mM/ml, whereas the alkylating antitumor drug
CCNU showed toxicity in a dose dependent manner. Even low doses of spin labeled nitrosoureas provided protection
against the toxicity caused by the antitumor drug CCNU alone. The lowest toxicity against E. coli and S. aureus were
achieved when 500 mM/ml of CCNU was combined with 200 mM/ml of spin labeled nitrosourea N -[N? -(2-chloroethyl)N ?-nitrosocarbamoyl]-glycine amid of 2,2,6,6-tetramethyl-4-aminopiperidine-1-oxyl (SLCNUgly). A combination of
TMPO with vitamin E completely abolished the toxicity of CCNU. Endogenous formation of oxygen radicals and their
possible involvement in CCNU toxicity towards the bacteria strains tested have been also discussed.
# 2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: CCNU; DTIC; Spin labeled; Antioxidants; E. coli ; S. aureus
1. Introduction
2-Chloroethylnitrosoureas and triazenes are alkylating chemotherapeutic agents. Some of them,
such as N ?-cyclohexyl-N -(2-chloroethyl)-N -nitrosourea (CCNU) and 5-(3,3-dimethyltriazene-1-yl)imidazole-4-carboxamide (DTIC) are used for
* Corresponding author. Tel.: /359-42-600-879; fax: /35942-600-005.
E-mail address: [email protected] (V. Gadjeva).
treatment of human cancers, mainly lymphomas,
gliomas, a few solid tumors and melanomas
(Comis, 1976; Carter et al., 1988). Unfortunately,
the clinical efficacy of these drugs is limited
because they show delayed and cumulative hematological toxicity (Gnewuch and Sosnovsky, 1997).
Reduced toxicity and increased antineoplastic
properties were achieved when nitroxyl (aminoxyl)
groups, such as 2,2,6,6-tetramethylpiperidine-N oxyl (TMPO) were introduced into the chemical
structure of certain antitumor drugs (Sosnovsky,
0378-4274/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/S0378-4274(03)00234-0
290
V. Gadjeva et al. / Toxicology Letters 144 (2003) 289 /294
1992; Gnewuch and Sosnovsky, 1997). This finding encouraged us to synthesize a number of spin
labeled analogues of the anticancer drugs CCNU
and DTIC. These compounds showed advantages
over CCNU and DTIC, having lower toxicity and
higher anticancer activity against some experimental tumor models (Raikov et al., 1993; Zheleva et
al., 1995, 1996b). Using electron spin resonance
(ESR) we have shown that spin labeled nitrosoureas, spin labeled triazenes and their precursor
2,2,6,6-tetramethyl-4-aminopiperidine-1-oxyl (4amino TMPO) can scavenge superoxide anion
(+ O2) and exhibit high superoxide scavenging
activity (SSA) (Gadjeva et al., 1994). Moreover,
we have demonstrated a light dependent nitric
oxide (+ NO) generation from the antitumor drug
CCNU (Zheleva et al., 1997). Recently, because of
these facts we have made an assumption that a
probable in vivo generation of the highly toxic
ONOO and + OH by CCNU might contribute to
its severe toxicity, while the lower toxicities of spin
labeled analogues of CCNU are possibly due to
their excellent SSA (Zheleva and Gadjeva, 2001).
Great problems for all clinically used nitrosoureas
are adverse reactions on the gastrointestinal tract
such as diarrhea and mucositis (Mc Clay and Mc
Clay, 1996). Bearing in mind these adverse effects
of nitrosourea drugs and also to try to explain the
possible mechanism of nitrosourea bacterial toxicity we studied in vitro the influence of the
antitumor drug CCNU on survival of Escherichia
coli (E. coli ) and Staphylococcus aureus (S.
aureus ) alone and in presence of oxygen radicalscavenging substances.
(SLCNUgly) and 1-ethyl-1-nitroso-3-[4-(2,2,6,6tetramethylpiperidine-1-oxyl)]-urea (SLENU),
spin labeled triazene 2,2,6,6-tetramethyl-4-[3-(4chloro-2-methylphenyl)-1-methyl-2-tiazenyl]-piperidyl-1-oxyl (SLTA) and nonlabeled glycine
nitrosourea N -[N ?-(2-chloroethyl)-N ?-nitrosocarbamoyl]-glycine amid of cyclohexylamine
(ChCNUgly) used for this study were synthesized
as previously described (Raikov et al., 1993;
Zheleva et al., 1995, 1996a). The chemical structures of the compounds used in this study are
shown in Fig. 1.
2.2. Bacterial strains
The bacterial strains used in this study included
both a Gram negative bacterium (E. coli ATTC
25922) and Gram positive bacterium (S. aureus
ATTC 53l54), purchased from Becton Dickinson
Microbiology Systems, Cockeysville, USA.
2. Materials and methods
2.1. Compounds tested
The antitumor drugs CCNU (Lomustine) and
DTIC (Dacarbazine) were purchased from BristolMyers Squibb Co. (Wallingford, CT, USA). The
free stable nitroxyl radical TMPO was purchased
from Aldrich Chemical Co. (Milwaukee, USA).
The spin labeled nitrosoureas N -[N?-(2-chloroethyl)-N ?-nitrosocarbamoyl]-glycine amid of
2,2,6,6-tetramethyl-4-aminopiperidine-1-oxyl
Fig. 1. Chemical structures of the antitumor drugs CCNU
(Lomustine), DTIC (Dacarbazine), nonlabeled ChCNUgly
nitrosourea and spin labeled antioxidants used in this study.
V. Gadjeva et al. / Toxicology Letters 144 (2003) 289 /294
291
2.3. Microbiological assay
Fresh overnight cultures of either E. coli or S.
aureus were diluted in normal saline to obtain a
bacterial suspension of 5/105 colony-forming
units CFU/ml. Bacterial suspensions at a final
concentration 50 CFU/ml were exposed to the test
compounds (50 /500 mM/ml) in phosphate buffered saline (PBS), pH 7.4, incubated 2 h at 37 8C
and then spread on agar. The plates were incubated overnight at 37 8C and cell survival was
calculated as %CFU versus control.
2.4. Statistical analysis
The results are reported as mean9/S.D. Statistical analysis was performed with Student’s t-test
and multiple regression analysis. P B/0.05 was
considered statistically significant.
3. Results
Effects of the three nonlabeled compounds (do
not contain a nitroxyl radical moiety), CCNU,
DTIC and nonlabeled glycine chloroethylnitrosourea (ChCNUgly) on survival of E. coli and S.
aureus strains were compared with those of spinlabeled chloroethyl nitrosourea (SLCNUgly), spin
labeled ethyl nitrosourea (SLENU), spin labeled
triazene (SLTA) and also to that of the free stable
nitroxyl radical TMPO (Fig. 2A, B). As is seen, the
nitroxyl radical TMPO and all studied spin labeled
compounds were almost no toxic for E. coli and S.
aureus at doses of 50 /500 mM/ml whereas CCNU,
DTIC and ChCNUgly showed toxicity in a dose
dependent manner.
The cell survival of E. coli and S. aureus after
CCNU or DTIC treatment at different doses alone
and in combination with SLCNUgly is presented
in Table 1. The highest percent of survival for E.
coli was achieved when 500 mM/ml of CCNU was
combined with 200 mM/ml of SLCNUgly. At this
combination CCNU toxicity effect was significantly counteracted (mean 88.0 vs. 35.3%, P B/
0.001, t-test) and reached levels close to those of
SLCNUgly administrated alone (mean 88.0 vs.
99.3%, P /0.05, t-test) (see Table 1). It is also
Fig. 2. Cell survival of E. coli ATCC 25 922 and S. aureus after
treatment with antitumor drugs CCNU and DTIC, spin labeled
nitrosoureas SLENU, SLCNUgly and spin labeled triazene
SLTA. Values represent the mean of three experiments. The
S.E.M. associated with the data of this figure are within 6% of
the presented values.
evident, that for both bacteria strains there was
almost no effect when SLCNUgly was added to
DTIC (see Table 1).
Comparisons between cell survival of E. coli
after treatment with CCNU (500 mM/ml) alone
and in combination with spin labeled antioxidants
and the well known antioxidant vitamin E (200
mM/ml) are shown in Table 2. Cell survival for E.
coli was not increased when CCNU was combined
with
nonlabeled
chloroethyl
nitrosourea
ChCNUgly (mean 38.5 vs. 35.3%, P /0.05, t test). However, when bacteria were exposed to
CCNU in combination with spin labeled ethyl
nitrosourea SLENU (200 mM/ml) there was protection against the toxicity of CCNU and survival
was almost the same as that of the bacteria coincubated with CCNU plus spin labeled chloroethyl nitrosourea SLCNUgly (mean 90.0 vs.
88.0%, respectively, P /0.05, t -test). It should
V. Gadjeva et al. / Toxicology Letters 144 (2003) 289 /294
292
Table 1
Effect of SLCNUgly on the bacterial cell survival in the presence of CCNU and DTIC
Cytostatic (mM/ml)
Cell survival (% CFU) cytostatic
P l/
Cell survival (% CFU) cytostatic/SLCNU gly (200 mM/ml)
E. coli
PBS (n/8)
CCNU 50.0
CCNU 100.0
CCNU 500.0
DTIC 50.0
DTIC 100.0
DTIC 500.0
100
75.79/19.1
65.09/15.0
35.39/14.2
79.79/19.5
35.29/13.4
20.19/15.0
/0.05
99.39/3.1
/0.05
/0.05
36.79/15.1
23.79/7.0
S. aureus
PBS (n/8)
CCNU 50.0
CCNU 100.0
CCNU 500.0
DTIC 50.0
DTIC 100.0
DTIC 500.0
100
75.79/19.1
50.09/20.0
35.39/24.2
85.29/20.4
55.29/20.4
50.19/15.0
/0.05
100.39/9.1
B/0.01
B/0.01
65.49/5.8
69.39/11.2
/0.05
/0.05
56.79/9.1
53.79/7.0
B/0.001 86.49/5.8 (*P /0.05)
B/0.001 88.09/19.2 (*P /0.05)
Cell growth (% CFU from the control); n , number of the experiments; *P (in comparison with the effect of SLCNUgly
administrated alone).
also be mentioned that addition of TMPO plus
vitamin E to CCNU provided better protection
against CCNU bacterial toxicity than they did
individually. Moreover, this combination completely abolished the toxic effect of CCNU (mean
99.8% (result is not presented) vs. 80.1 and 70.2%).
As is seen SLTA, spin labeled analogue of DTIC
decreases CCNU bacterial toxicity in a consider-
able degree (mean 78.19/9.0, see Table 2), while
there is no effect on DTIC bacterial toxicity (result
is not presented).
Interestingly, a definitive positive correlation
existed between SSA (previously reported, Gadjeva et al., 1994) of the antioxidants used in this
study and cell survival of E. coli after treatment
with CCNU in combinations with the same
antioxidants (R /0.805, P /0.029, Correlation
analysis).
Table 2
Effect of spin labeled antioxidants and vitamin E on the
bacterial cell survival of E. coli in the presence of CCNU
Compound
a
SSA activity ks
(/105 M/s)
b
Cell survival (%CFU)
CCNU/antioxidant
CCNU
(alone)
ChCNUgly
SLENU
SLCNUgly
SLTA
TMPO
Vitamin E
0
35.39/14.2
0
3.1
4.0
2.7
5.0
0.16
38.59/12.2
90.09/14.7
88.09/19.2
78.19/9.0
80.19/10.9
70.29/9.8
a
Note comments on its evaluation made in Gadjeva et al.
(1994).
b
Cell growth of E. coli ATCC 25 922 after treating with
CCNU (500 mM/ml) alone and in combinations with antioxidants (200 mM/ml).
4. Discussion
Toxicity mediated by reactive drugs may often
occur through one or both of two main mechanisms. These are: (1) production of activated drug
metabolites and (2) production of reactive oxygen
species (ROS), often involving redox cycling. It is
known that chloroethyl nitrosourea derivatives
and DTIC exhibit in vivo activity by alkylation
of nucleic acids and proteins (Wheeler et al., 1974;
Babson and Reed, 1978; Lucas and Huang, 1982).
Our present results clearly demonstrated that,
both CCNU and DTIC were toxic to the bacterial
models tested in a dose dependent manner. Be-
V. Gadjeva et al. / Toxicology Letters 144 (2003) 289 /294
cause both of these drugs are alkylating agents,
one might suppose alkylation is involved in the
mechanism of the drug toxicity towards the both
bacteria strains. However, in the present instance,
we find that the bactericidal effects of CCNU were
counteracted to a considerable degree by the spin
labeled nitrosoureas and triazenes, while there was
almost no effect when these compounds were
added to DTIC. It is obvious that CCNU and
DTIC exhibit their toxicity to the bacteria by
different ways. Our present findings show that the
main event involved in CCNU toxicity is probably
due to the generation of ROS. This conclusion is
based on the following: (1) the different behavior
of both nitrosoureas SLCNUgly and its nonlabeled analogue ChCNUgly; (2) the effect of
SLENU (without alkylating activity) on the survival of the bacterial models; (3) the different
behavior of spin labeled triazene SLTA after its
addition to CCNU or DTIC and (4) the influence
of the three nitrosoureas on CCNU toxicity.
SLCNUgly and SLENU possess excellent SSA
while ChCNUgly has no SSA activity (see Table
2).
Under
our experimental
conditions,
SLCNUgly and SLENU counteracted the toxicity
of CCNU to a considerable degree, while
ChCNUgly alone (possesses no nitroxide) exhibited almost the same toxicity as CCNU and did
not affect the toxicity after incubation with
CCNU. Another indirect proof for involvement
of ROS in CCNU toxicity was the complete
prevention of that toxicity by adding of two
typical antioxidants */TMPO and vitamin E
(both possessing high SSA).
Possible involvement of ROS in CCNU toxicity
under our experimental conditions is supported
also by several other studies. Acikgoz et al. (1995)
demonstrated enhanced membrane lipid peroxidation caused by the antitumor drug CCNU. It is
well known that increased production of lipid
peroxides might due to increased levels of ROS
(Esterbauer et al., 1992; de Zwart et al., 1999). On
the basis of our formerly reported light dependent
+
NO generation from CCNU, we have proposed
that the in vivo formation of ONOO and + OH
contribute to the severe toxicity of this drug
(Zheleva and Gadjeva, 2001). It seems very likely,
during the incubation of the bacteria strains with
293
CCNU, the formation of these species is involved
in the drug toxicity. Based on ESR studies, we
have established that clinically used nitrosourea
drug CCNU and triazene DTIC could not
scavenge.O2 while their spin-labeled nitrosourea
and triazene derivatives and their precursor 4amino-TMPO, could successfully scavenge.O2,
exhibiting high SSA. Well-expressed SSA of the
spin-labeled compounds was attributed to the
presence of nitroxide in their structures. We also
showed that the mechanism of SSA activity was a
result of a redox cycling between nitroxide and its
corresponding hydroxylamine according to the
following equations (Gadjeva et al., 1994):
Kr
NO+ + O
2 H 0 NOH
K0
+
NOH + O
2 H 0 NO H2 O2
Based on the above mentioned equations and
our formerly demonstrated light dependent + NO
generation from antitumor drug CCNU we try to
explain the nontoxic effect of the spin labeled
nitrosoureas and prevention of the toxicity towards the bacteria by adding the latter compounds
to CCNU under our experimental conditions by
the following hypothesis: Since all nitrosoureas
possess nitroso groups in their structures during
their incubation with the bacteria strains they
could generate + NO. This latter might react
with.O2 to form ONOO and + OH by the
following reactions (Freeman, 1994):
However, during alone or together incubation
of spin labeled nitrosoureas with CCNU and
bacteria, the nitroxyl free radical moiety (+ N /
O+ ) incorporated only in the spin labeled compounds might successfully compete with + NO
(generated by any of the nitrosourea) for scaven-
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V. Gadjeva et al. / Toxicology Letters 144 (2003) 289 /294
ging of + O2 and that could prevent formation of
high toxic ONOO and + OH species.
Having in mind that spin labeled 2-chloroethylnitrosoureas exhibited in vivo high antitumor
effects against different tumor models and possessed antioxidant properties like those of the
typical antioxidants as Vitamin E and TMPO,
we consider that new combination chemotherapy
schemes containing lower dose of the high toxic
CCNU plus proper spin labeled nitrosoureas such
as SLCNUgly may decrease the adverse effects of
CCNU on the gastrointestinal tract probably by
scavenging + O2. Further detailed analyses are in
progress to clarify whether spin labeled nitrosoureas might counteract hemotoxicity of this class
antitumor drugs.
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