Vol. 52 No. 4/2005, 923–926
on-line at: www.actabp.plCommunicationMethylxanthines (ca?eine, pentoxifylline and theophylline) decreasethe mutagenic e?ect of daunomycin, doxorubicin and mitoxantrone
Jacek Piosik1?, Anna Gwizdek-Wi?niewska2, Katarzyna Ulanowska3, Jakub
Ochoci?ski1, Agata Czy?4 and Grzegorz W?grzyn3,51Intercollegiate Faculty of Biotechnology, University of Gda?sk and Medical University of Gda?sk, Gda?sk, Poland;
2Department of Pharmaceutical Technology and Biochemistry, Technical University of Gda?sk, Gda?sk, Poland; 3Department of Molecular Biology, University of Gda?sk, Gda?sk, Poland; 4Laboratory of Molecular Biology (a?liated with the University of Gda?sk), Institute of Biochemistry and Biophysics, Polish Academy ofSciences, Gda?sk, Poland; 5Institute of Oceanology, Polish Academy of Sciences, Gdynia, Poland;
Received: 15 February, 2005; revised: 08 June, 2005; accepted: 24 June, 2005
available on-line: 11 July, 2005Previously performed experiments showed that methylxanthines, especially ca?eine, may protectcells against cytostatic or cytotoxic e?ects of several aromatic compounds. One of the proposedmechanisms of this protection is based on stacking interactions between ? electron systems of polycyclic aromatic molecules. In this work, we demonstrate that ca?eine and other methylxan-thines — pentoxifylline and theophylline — signi?cantly decrease mutagenicity of the antican-cer aromatic drugs daunomycin, doxorubicin and mitoxantrone. The spectrophotometric titration of these aromatic compounds by methylxanthines indicated formation of mixed aggregates. The concentrations of free active forms of the drugs decreased when the concentrations of methylxan-thines increased in the mixture. Therefore, likely methylxanthines may play a role of scavengers of the free active forms of daunomycin, doxorubicin and mitoxantrone.Keywords:
xanthines, daunomycin, doxorubicin, mitoxantrone, Vibrio harveyi
Methylxanthines (MTX), including ca?eine
as well as topotecan and camptothecin (Nahon et
(CAF), pentoxifylline (PTX) and theophylline (TH) al.
, 1993) — topoisomerase I blockers. This protec-
are compounds commonly used worldwide. They
tive action is not only speci?c for aromatic antican-
are components of human diet as well as of sever-
cer drugs but also for other aromatic chemicals of
al drugs (Marks & Kelly, 1973; Medvei et al.
mutagenic activity, e.g. polycyclic aromatic hydro-
Graham, 1978; Williams et al.
, 1978; Samlaska &
carbons (PAHs) (Rothwell, 1974) and acridine muta-
Win?eld, 1994; Tanaka et al.
gens (ICR 170, ICR 191) (Piosik et al.
There are many known biological e?ects
These opposite e?ects can be explained in the
of MTX’s action manifested, among others, by in-
following way. When DNA has already been dam-
creased cytotoxicity or cytostaticity of various com-
aged, the presence of MTX causes a diminution of
pounds, mainly by potentiating the activity of sev-
DNA repair or synthesis (Roberts, 1984; Roberts &
eral DNA damaging agents (Donovan & Dipaolo,
Kotsaki Kovatsi, 1986; Selby & Sancar 1990). There-
1974; Beetham et al.
, 1983; Fingert et al
., 1984; 1986;
fore, the presence of MTX enhances DNA damag-
Labanowska et al.
, 1988; Mourelatos et al.
es. However, if MTX are available before aromatic
Tomita & Tsuchiya, 1989; Boike et al.
, 1990; Petru et
drug administration or are administrated together al.
, 1990; Tanaka et al.
, 1991; Fan et al.
, 1995; Ohsaki
with aromatic drugs, a protective e?ect is observed.et al.
, 1996). On the other hand, it is well known
It was proposed previously that this protective ef-
that MTX also diminish the pharmacological action
fect of MTX is a result of direct stacking interac-
of aromatic drugs such as ellipticine, doxorubicin
tions between MTX and aromatic molecules (Traga-
(DOX) and mitoxantrone (MIT) (Traganos et al.
nos et al.
, 1991b; 1993; Kapuscinski & Kimmel, 1993;
1991a; 1991b), which are topoisomerase II blockers,
Zdunek et al.
, 2000; Kapuscinski et al.
, 2002; Piosik Abbreviations:
CAF, ca?eine; DAU, daunomycin; DOX, doxorubicin; MIT, mitoxantrone; MTX, methylxanthines; PTX,
pentoxifylline; TH, theophylline.
Piosik and others
, 2002; 2003). These interactions are very weak ?nal concentration of 1 mM. A?er cultivation in the
(mixed association constant KAC
about 102 M–1) and liquid medium, 5 · 106 cells were spread onto BOSS
can be disrupted easily. It seems plausible that this agar plates containing neomycin (0.1 mg/ml). Fol-
phenomenon may be used to diminish some side ef-
lowing a 48 h incubation at 30oC, neomycin-resistant
fects associated with the high local concentrations colonies were counted.
of anticancer drugs in the initial phase of their ad-
To estimate the number of spontaneous neo-
ministration (Hande, 1998). Formation of complexes mycin-resistant mutants, analogous experiments
between MTX and aromatic drugs may lead to tem-
were performed in which neither methylxanthines
poral reduction of the concentration of free drug nor drugs were added. The number of such mutants
molecules available for the cells. In this work we in-
(which was between 10 and 20 in all experiments)
vestigated the mutagenic activity of chosen aromatic was then subtracted from the number of mutants in
anticancer drugs and the possible modulation of cultures treated with methylxanthines and/or drugs.
their mutagenic activity by methylxanthines — caf-Light absorption spectrometry.
feine, pentoxifylline and theophylline.
tion spectra were measured using a Beckman DU 650
spectrophotometer connected with a Polystat ther-
mostat constant circulator (25 ± 0.1oC). Two milliliters MATERIALS AND METHODS
of a bu?er (5 mM Hepes, 150 mM NaCl, pH 7.1) con-
taining DAU, DOX or MIT were placed in a quartz Chemicals
. All chemicals used in this work: cuve?e (1 cm light path) and titrated with 5–150 µL
ca?eine (1,2,3-trimethylxanthine, CAF), pentoxifyl-
of CAF, PTX (concentration 0.1 M) or TH (concen-
line (3,7-dimethyl-1-(5-oxohexyl)xanthine, PTX, tren-
tration 0.02 M) dissolved in the bu?er. The spectra
tal), theophylline (1,3-dimethylxanthine, TH), dau-
were measured at 0.5 nm intervals and stored on a
computer disk. The spectra were then corrected for
the absorption of the bu?er, CAF, PTX and TH, and
6,8,11-trihydroxy-1-methoxy-5,12-naphthacenedione, expressed in terms of the molar absorption coe?-
DAU), doxorubicin (14-hydroxydaunomycin, DOX), cient (??, M–1 · cm–1).
and mitoxantrone (1,4-dihydroxy-5,8-bis[[2-[(2-hydrCalculation of association constants.
oxyethyl)amino]ethyl]amino]-9,10-anthracenedi-one, tions of the association constants of DAU, DOX or
MIT) were purchased from Sigma-Aldrich. Their MIT with CAF, PTX and TH were accomplished us-
chemical structures are presented in Fig. 1.
ing statistical thermodynamics of mixed aggregation The Vibrio harveyi mutagenicity assay.
The (Kapuscinski & Kimmel, 1993; Zdunek et al.
assay was performed in liquid medium as described Kapuscinski et al.
, 2002; Piosik et al.
, 2002; 2003). The
previously (Czyz et al.
, 2000; 2002). Brie?y, Vibrio
notations and de?nitions described previously byharveyi
BB7 strain was cultivated in BOSS medium Weller and co-workers (1984) were employed.
(Klein et al.
, 1995) at 30oC. To exponentially grow-
ing cultures, indicated components were added and
cultivation was continued for 3 h. On the basis of RESULTS AND DISCUSSION
preliminary experiments, the following drug concen-
trations were chosen to induce mutagenic but not
Using a previously developed mutagenic-
cytostatic or cytotoxic e?ects: 0.4 µM DAU, 0.03 µM
ity assay (Czyz et al.
, 2000; 2002), we investigated
DOX, 0.6 µM MIT (under these drug concentra-
the e?ects of methylxanthines (CAF, PTX, TH) on
tions signi?cant mutagenic e?ects were observed,
the mutagenic activity of three anticancer drugs —
see Fig. 2, but no signi?cant decrease in bacterial
DAU, DOX and MIT. In all cases a strong mutagenic
growth rate and survival was detected). The methyl-
e?ect of the analyzed drugs was observed (Fig. 2A).
xanthines were added together with the drugs, at a All methylxathines tested, CAF, PTX and TH, had Figure 1. Chemical structures of methylxanthines and anthracycline drugs.
Vol. 52 Methylxanthines decrease the mutagenic e?ect of daunomycin, doxorubicin and mitoxantrone
no signi?cant mutagenic e?ect at the concentrations
Kimmel, 1993; Zdunek et al.
, 2000; Kapuscinski et al.
examined (0.5–2.5 mM). We found that the muta-
2002; Piosik et al.
, 2002; 2003).
genicity of DAU, DOX and MIT was signi?cantly
Based on the data obtained by spectropho-
decreased when they were administrated together tometric titrations and using the thermodynamical
with the methylxanthines (at a ?nal concentration of
model of mixed association (Kapuscinski & Kimmel,
1 mM) (Fig. 2B, C, D). Similar e?ects were observed
1993) we calculated all concentrations of neighbor-
for MTX concentrations ranging from 0.5 to 2.5 mM hoods and the “neighborhood associations” con-
(not shown). Therefore, we conclude that methylxan-
for all systems (Table 1). These results
thines alleviate the mutagenic e?ects of DAU, DOX
show that CAF, as well as PTX and TH, directly
interact with the aromatic drugs through formation
At low concentrations the anticancer drugs of stacking complexes. This causes a decrease in the
DAU, DOX and MIT cause mutagenic e?ects in Vi-
concentrations of the free active form of the drugs brio harveyi
cells. Higher concentrations were toxic when the MTX concentration increases in the mix-
to these bacteria (not shown). At the concentrations ture (not shown). This is in accord with earlier re-
used here, administration of MTX alone exerts no sults which focused on direct interactions between
mutagenic e?ects. It is necessary to underline that
several aromatic compounds, like acridine orange
the examined aromatic drugs were added to the cells (Kapuscinski & Kimmel, 1993), ethidium bromide,
together with MTX in the mixture, and MTX concen-
DAPI (Zdunek et al.
, 2000), DOX, MIT (Piosik et al.
trations were signi?cantly higher than the drug con-
2002), quinacrine mustard with CAF (Kapuscinski
centrations. Nevertheless, in all cases, a considerable et al.
, 2002), and quinacrine mustard, ICR-191, ICR-
decrease in the mutagenicity of the examined aro-
170 (Piosik et al.,
with CAF or PTX. Johnson
matic drugs was observed in the presence of MTX et al.
(2003) also revealed that MTX, theophylline,
(Fig. 2). Therefore, all MTX tested may scavenge the theobromine and ca?eine, can directly interact with
free forms of DAU, DOX and MTX. It is worth not-
intercalated dyes — ethidium bromide and acridine
ing that this process is dynamic and reversible, and orange-e?ecting their de-intercalation from calf thy-
if the MTX concentration is lowered, free molecules mus DNA and reduction of DNA — directed toxic-
of active drugs may re-appear.
Using light absorption spectroscopy we exam-
All examined drugs cause serious side e?ects
ined possible direct interactions between methylxan-
on intravenous or oral administration, e.g. extrava-
thines and DAU, DOX and MIT. It has been demon-
sation and local tissue necrosis (Rudolph & Larson,
strated that interactions between ? electron systems 1987; Richardson & Johnson, 1997; Heitmann et al.
from aromatic rings of aggregating chemicals may 1998; Sla?ery et al.
, 1999; Kassner, 2000; Sparreboom
occur (Traganos et al.
, 1991b; 1993; Kapuscinski & et al.
, 2002) due to their high local concentrations.
Several MTX, including CAF, PTX and TH, are also
used as medicines (Graham, 1978; Samlaska & Win-
?eld, 1994; Shannon, 2004). One might speculate that
the use of suitable combinations of aromatic antican-
cer drugs with MTX could contribute to a temporal
decreases of the high local concentrations of the ac-
tive forms of the drugs and thus limit their undesir-
able side e?ects. This would not necessarily mean a
decrease in the activity of these drugs in the antican-
cer therapy. A decrease in MTX concentration due
to its dilution in the blood would cause an increase
in the amount of free drug that could react further
on. To obtain such an e?ect, it would be necessary
to match suitable proportions of MTX and the drug, Figure 2. E?ects of methylxanthines — ca?eine (CAF),pentoxifylline (PTX) and theophylline (TH) — on muta-genicity of chosen aromatic anticancer drugs: daunomy-Table 1. Mixed association constants calculated for inter-cin (DAU), doxorubicin (DOX) and mitoxantrone (MIT).actions between analyzed compounds
The mutagenicity test was performed as described in
Materials and Methods. E?ects of drugs added alone
Methylxan- Mixed association constants with aromatic
are presented in panel A and e?ects of methylxanthines
± S.E. (M–1)
added alone or together with drugs are presented in pan-
els B (ca?eine), C (pentoxifylline) and D (theophylline).
148.5 ± 4
128.3 ± 10*
356.4 ± 21*
The number of mutants is given a?er subtraction of the
172.7 ± 5
173.0 ± 5
218.2 ± 7
number of spontaneous mutants (10–20 per plate). Aver-
99.9 ± 5
364.5 ± 15
222.7 ± 9
age values of three experiments are presented with error
*Data from other publication (Piosik et al.
Piosik and others
which could be determined on the basis of the cal-
1991b; 1993). On the other hand, there are published
culated mixed association constant, KAC
. We specu- data indicating that CAF and PTX may be toxic at
late that in such a case it would be not necessary to concentrations of 2.24–3.92 mM and 3.17–6.0 mM,
use MTX at concentrations as high as those used in respectively, to several human cell lines (Bohm et al.
this work in the mutagenicity test (total concentra-
2000). This problem requires further detailed studies
tions of MTX between 0.5 and 2.5 mM). Based on if one considers MTX as a potential auxiliary drug
the spectrophotometric data and the results calcu-
to be used in combination with DAU, DOX or MIT.
lated from the thermodynamical–statistical model of
mixed aggregation in aqueous solutions we observed Acknowledgements
that formation of stacking complexes between MTX
and an aromatic drug occurs at signi?cantly lower
This work was supported by the University of
concentrations of MTX. It was also demonstrated Gda?sk (grant no. BW/B000-5-0218-1 and BW/B052-
that at a relatively high concentration (5 mM) CAF 5-0035-2 to J.P.) and by the Ministry of Science and
has no signi?cant e?ect on cell growth or cell cycle
Information Society Technologies (grant No. 2 P04G
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- MATERIALS AND METHODS
- RESULTS AND DISCUSSION
- Figure 1.
- Figure 2.
- Table 1.