Mutagenesis vol.12 no.6 pp.411—415, 1997
Inhibition of potassium dichromate mutagenicity by todralazine
Kazimierz Gasiorowski1, Katarzyna Szyba,
Dorota Wozniak and Bogdan Gulanowski
Medical University, Department of Basic Medical Sciences,
14th Kochanowskiego Str., 51-601 Wroclaw, Poland
Todralazine, an antihypertensive drug of the hydrazinophthalazine group, markedly decreased the mutagenic activity
of potassium dichromate in standard bacterial tests. At the
highest todralazine dose tested inhibition of potassium
dichromate mutagenic activity by ~90% in the Ames test
and up to 100% (complete) inhibition in the Bacillus subtilis
rec" assay was observed. Spectrophotometric analyses
proved that todralazine induced reduction of Cr(VI) to
Cr(III) and complexation of Cr(III) ions. These spectrophotometric results may be a presumptive explanation of
the observed mutagenic activity decrease, as it is known
that Cr(m) is poorly transported across cell membranes
and therefore is not mutagenic to bacterial cells. We
perceive our experiments as an example of attempts which
should lead to an effective reduction in chromium genotoxic
and carcinogenic activity in exposed individuals.
Introduction
Chromium is a widely used industrial chemical, finding application in steel and metal finishes, alloyed cast iron and welding
and also in chromate plating, paint production, leather tanning
and wood treatment (IARC, 1990). The acute toxicity, chronic
toxicity, neurotoxicity, reproductive toxicity, genotoxicity and
carcinogenicity of chromium are already well documented (see
for example Costa et al, 1984, Cohen et al, 1993; Von Burg
and Liu, 1993). This transition metal can exist in various
oxidation states ranging from -2 to +6, but- in the human
environment states 3 and 6 are the most predominant oxidation
states of chromium compounds emitted in the workplaces.
The chromate ion (CrO4)"2, the dominant form of Cr(VI) in
neutral aqueous solution, can readily cross cellular membranes
via non-specific anion carriers, while Cr(IH) is poorly transported across cell membranes (Danielsson et al, 1982; De
Flora and Wetterhahn, 1989; Wetterhahn and Hamilton, 1989;
Snow, 1992). The differences in membrane transport may
explain the differences in genotoxicity of these two forms. It has
been well documented that Cr(VI) compounds are mutagenic in
a number of bacterial systems (Baker et al, 1984; Zakour and
Glickman, 1984; De Flora et al, 1990), while most Cr(m)
compounds are not mutagenic in those systems (Langerwerf
et al, 1985; De Flora et al, 1990; Gulanowski et al, 1994).
DNA damaging effects were observed with Cr(Vr), which is
readily taken up by cells in culture, followed by intracellular
reduction to lower oxidation states accompanied by chromiuminduced DNA damage, including predominantly single-strand
breaks, DNA-protein cross-links and chromium—DNA adducts
(Standeven and Wetterhahn, 1989; Paustenbach et al, 1996).
For chromium compounds the induction of DNA damage and
mutagenic potential are consistent with their carcinogenic
potential (Hartwig, 1995). Among mechanisms of chromium
genotoxic activity, induction of genotoxic free radicals seems
to be important. The most probable source of reactive oxygen
species in response to chromium salts could be redox cycling
of the Cr(III)/Cr(n) and Cr(VI)/Cr(V) pairs (Shi and Dalai,
1992; Shi et al, 1993; Bagchi et al, 1995; Sugden et al,
1994). The reactive oxygen species may lead to a variety of
damaging effects, contributing to the toxic and genotoxic
manifestations of chromium cations (De Flora et al, 1990;
Feig et al, 1994; Wetterhahn and Dudek, 1996).
As chromium belongs to a family of widespread metals it
could be impossible to eliminate it completely from the
human environment. An alternative way to decrease toxic and
genotoxic effects of chromium would be blockage of Cr(VI)/
Cr(V) and Cr(ni)/Cr(II) transitions. This could be achieved
by stabilizing chromium VI and III by complexing them with
different ligands. For instance, we previously showed that
copper(II) chromate and dichromate complexes with polypyridines exhibited a markedly lower mutagenic activity than
the reference compounds, potassium dichromate and sodium
chromate (Szyba et al, 1992).
Currently we have commenced experiments on the influence
of todralazine upon the mutagenicity of chromium dichromate.
Todralazine is a hydralazinophthalazine-derived drug currently
used in the treatment of arterial hypertension. It is well
documented that some hydralazinophthalazines, such as hydralazine, dihydralazine and endralazine, are genotoxic in several
short-term mutagenicity test systems (Williams et al, 1980;
De Flora et al, 1982; Chlopkiewicz et al, 1995a). We found
that in the Ames test hydralazine exhibited strong mutagenic
activity and dihydralazine also exerted a significant genotoxic
effect, whereas todralazine had no mutagenic activity on
Salmonella typhimurium TA100 both in the presence and
absence of S9 fraction (Gasiorowski et al, 1993). Previously
we proved that todralazine markedly decreased the mutagenic
activity of several direct and indirect acting mutagens in
the Ames test (Gasiorowski et al, 1993, 1994a,b, 1995).
Todralazine also revealed a strong inhibitory impact on free
radical release caused by benzo[a]pyrene and by the tumor
promoter phorbol myristate acetate in human granulocyte
cultures (Gasiorowski et al, 1997). The marked antimutagenic
activity of todralazine and especially strong inhibitory influence
upon free radical release by standard mutagens in human
granulocyte cultures were the main premises for undertaking
studies on the influence of todralazine upon Cr(VI) mutagenicity in bacterial tests. The results are presented in this paper.
Material and methods
Chemicals
Todralazine-HCl (analytical grade) was kindly supplied by Polfa (Poland).
The preparation of bacterial growth media was carried out with Oxoid Nutrient
'To whom correspondence should be addressed. Tel: +48 71 486024; Fax: +48 71 215729
© UK Environmental Mutagen Society/Oxford University Press 1997
411
K.Gaslorowski el al
NH-NH-C-OCyHt
Tbble L Mutagenicity testing of todralazine m the Ames test
Tested compound
Reversion coefhcienr
TA98
-S9
Fig. 1. The general structural formula of todralazine.
Broth no. 2 (Oxoid, UK), Difco Bacto Nutrient Broth and Difco Agar (Difco,
USA). Potassium dichromate (K^C^O,) as well as reagents used for buffers
and media preparation were obtained from POCH (Poland).
Mutagenicity assays
The influence of todralazine on the mutagenic activity of chromium(Vl) was
assayed by two bacterial methods routinely used for mutagenicity testing, i.e.
the Ames test and Bacillus subtilis rec~ assay.
The Ames test was performed by the plate incorporation assay (Maron and
Ames, 1983) with a S.typhimurium TA100 tester strain, kindly supplied by
Prof. B.N.Ames. K2Cr2O7 was used at a dose of 75 nmol/plate and todralazine
in the range of doses 10-500 (ig/plate. Both compounds were dissolved in
water and added simultaneously to the tester mixtures after preincubation for
1.5 h at 37°C ( r ^ h), or without preincubation (t0). The mixtures contained
0. 1 ml K2Cr2O7 solution, 0. 1 ml todralazine solution, 0. 1 ml overnight
culture of S.typhimurium TA100 cells, 0. 5 ml phosphate buffer, pH 7. 4, and
2 ml top agar. After vigorous mixing, the samples were poured onto minimal
glucose agar plates, with three plates for each sample. The experiments were
repeated three times. The cytotoxicity of tested compounds was assayed with
S.typhimurium in accordance with Maron and Ames (1983). Briefly, for
determination of the surviving cells, 0. 1 ml each tested mixture diluted to
10""6 was poured onto nutrient agar plates. The number of revertants and
viable cells was counted after incubation for 2 days at 37°C The number of
surviving cells was expressed as viable cells per plate (X106).
The rec" assay test was performed according to Kada and co-workers (Kada
et al., 1984) with the use of Bacillus subtilis H17 n»c+and M45 rec' strains.
In the test todralazine was used at the same dose range as in the Ames test,
while the K2Cr2O7 dose was higher at 250 nmol/disc.
A mixture of both compounds was dropped onto a filter paper disc (diameter
9 mm) placed on plates with solid cultures of H17 rec+ and M45 rec~ strains
in bacterial growth medium, pH 7. 4. The plates were incubated at 37°C
overnight. The mutagenicity of tester mixtures was expressed as the differences
between the inhibition zones of rec" and rec+ strains and the presented results
were the means of n = 6 plates/observation point.
Spectrophotometry
The absorption spectra were recorded with a M40 spectrophotometer (Carl
Zeiss, Jena, Germany). The spectral analyses were performed in the wavelength
range 400-900 nm. Incubations of the samples were carried out in a 0. 05 M
sodium phosphate buffer, pH 7. 4, at 37°C for 1.5, 24 and 48 h respectively.
The samples contained K2Cr2O7 at a concentration of 0. 4 mM and todralazine
in the concentration range 50-1250 ug/ml in consecutive samples. The
concentrations of tested compounds in different samples are given in the
figure legends.
Statistical anatysis
Regression equations were calculated according to standard statistical procedures (see Bourke et al., 1985)
Results
The general structural formula of todralazine is given in
Figure 1.
As may be seen in Figure 1, todralazine could be a good
coordinating agent since it possess two groups of donors: a
nitrogen in chain and ring as well as a carbonyl group in
chain. We determined the toxicity levels of K2Cr2O7 and
todralazine to bacterial cells. The results proved that todralazine
in the range of tested doses (10-500 |Xg/plate) did not influence
the toxicity of K 2 Cr 2 0 7 (at a dose of 75 nmol/plate) to bacterial
cells. Therefore we chose the above doses of the tested
compounds for the experiments with S.typhimurium strains.
412
Todralazine (ug/ml)
500
1000
2000
NQNO (0.5 ug/ml)
Mitomycin C (0.1 ug/ml)
2AF (5 ug/ml)
TA100
+S9
1.19
1.06
1.00
11.48
1.16
1.29
1.13
TA102
-S9
+S9
-S9
+ S9
1.02
1.09
0.99
6.74
1.14
1.22
1.01
0.96
0.82
0.87
1.09
1.18
1.08
3.12
16.06
4.94
"Reversion coefficient was calculated following the formula: rc = (revertant
numbers per test plate)/[revertant numbers per control plate (spontaneous)].
400
350
300
250
200
150
100
50
100
200
300
400
500
Todralazine dose (ng/ptate)
Fig. 2. Influence of todralazine on the mutagenicity of potassium
dichromate in the Ames test with S.typhimurium strain TA100. The
regression lines describe the results obtained with samples containing
K2Cr2<>7 plus todralazine added to the Ames test without preincubation
(upper line) and preincubated for 1.5 h (lower line).
We assessed mutagenicity of todralazine in the Ames test
with S.typhimurium tester strains TA98, TA100 and TA102.
The results were calculated in proportion to the spontaneus
revertant numbers and are presented as reversion coefficients
in Table I. We also give positive controls, i.e. results obtained
with the standard mutagens recommended by Ames for the
particular bacterial strain (Maron and Ames, 1983). As the
tested compounds were dissolved in water the negative controls
closely approximated the spontaneous revertant numbers. We
counted the following revertant numbers (x ± SD, n = 6):
TA98, 31 ± 8. 5; TA100, 146 ± 7. 6; TA102, 254 ± 2. 12.
As may be seen in Table I, todralazine did not exhibit
mutagenic activity in the range of tested doses (0. 5-2 mg/
plate), either in the presence or absence of the S9 promutagenactivating fraction; the calculated reversion coefficient of ~ 1.0
means that the results obtained with todralazine were very
similar to the spontaneous revertant numbers estimated for
each S.typhimurium tester strain.
Results of the experiments on the influence of todralazine
upon K 2 Cr 2 0 7 mutagenicity in the Ames test with a S.typhimurium strain TA100 are presented in Figure 2.
Todralazine antimutagenic action on Cr(VI)
N
5
€
400
900
WAVELENGHT (nm)
100
200
300
Todralazine dose (ng/ptate)
Fig. 3. Impact of todralazine upon the mutagenicity of potassium
dichromate in the B.subtilis rec" assay. The regression lines describe the
result obtained with mixtures of ^ C ^ O / and todralazine added to the test
without preincubation (upper line) and preincubated for 1.5 h (lower line).
The results are expressed by regression lines and regression
equations calculated for the data obtained in the samples
without preincubation (%, upper regression line) and after
1.5 h preincubation of K2Cr2O7 with todralazine (fj j h. lower
regression line). As may be seen in Figure 2, the preincubation
procedure caused a marked inhibition of potassium dichromate
mutagenicity (by even >90% at the highest todralazine dose
tested), whereas the samples without preincubation exhibited
a noticeably lower decrease in mutagenicity (by ~25%).
The impact of todralazine on the mutagenicity of K2Cr2O7
(250 nmol/disc) in the B.subtilis rec~ assay is shown in Figure 3.
As may be noticed from Figure 3, inhibition of potassium
dichromate mutagenicity by todralazine is very strong in this
test. For the highest todralazine dose tested we estimated the
decrease to be ~40% in the samples without preincubation
(*o) and complete inhibition (up to 100%) in the samples
preincubated for 1.5 h before addition to bacterial cells (t, 5 h ).
The calculated regression equations could be depicted by
straight line dose-effect relations with a very high correlation.
To estimate the direct chemical interactions of potassium
dichromate with todralazine we undertook spectrophotometric
analyses of the samples containing todralazine and K2Cr207
incubated together in phosphate buffer, pH 7. 4, for 1.5, 24
and 48 h. The bulk of the absorption spectra of potassium
dichromate (0. 4 mM) incubated with different concentrations
of todralazine in phosphate buffer, pH 7. 4, are presented in
Figure 4. The spectra of samples incubated for 1.5 h are
presented in Figure 4A and those incubated for 24 h are shown
in Figure 4B.
As may be seen in Figure 4, incubation with todralazine
caused marked changes in potassium dichromate absorption.
The main differences are revealed at 560 and 690 nm, where
distinct absorption peaks appear. These absorption peaks are
not observed in the control samples, containing potassium
dichromate incubated in phosphate buffer, pH 7. 4, without
todralazine. This effect strongly depends on the concentration
of todralazine; the absorption peaks at 560 and 690 nm are
B
400
900
WAVELENGHT (nm)
Fig. 4. The absorption spectra of the samples containing potassium
dichromate (0. 4 mM) and todralazine at different concentrations
(50-1250 |i.g/ml) incubated in phosphate buffer, pH 7. 4, for 1.5 (A) and
24 h (B). Todralazine concentration in the samples: 0 (line 1), 50 )ig/ml
(line 2), 250 ug/ml (line 3), 500 ilg/ml (line 4) and 1250 ug/ml (line 5).
highest at a todralazine concentration of 1250 (i.g. As may be
seen in Figure 4B, the absorption peaks at 560 and 690 nm
are markedly higher after the 24 h incubation than those after
1.5 h incubation presented in Figure 4A. We also recorded the
absorption spectra of samples incubated for 48 h (data not
shown) and found that there were no differences between 24
and 48 h absorption spectra.
Discussion
Chromium(VI) compounds are known to be genotoxic, mutagenic and carcinogenic, producing lung cancer in humans and
causing tumor formation in laboratory animals (see De Flora
and Wetterhahn, 1989; Wetterhahn and Hamilton, 1989; De
Flora et ai, 1990). The chromium(VI) ion is not genotoxic
per se. The 'uptake-reduction' model postulates that Cr(VI)
easily enters cells through non-specific anion transport channels
and undergoes intracellular reduction to the more stable Cr(Tn)
species, producing reactive Cr(V) and Cr(TV) intermediates
with concommitant generation of reactive oxygen species and
other free radical species (Wetterhahn and Dudek, 1996). The
oxidation properties of the CrO42" ion and its structural
similarity to biologically important inorganic anions, such as
413
K.GasiorowskJ et al
SO42 and PO42 , seem to be responsible for chromate interactions with cell components (Cieslak-Golonka, 1995). For
instance, it was demonstrated in EPR spectra that Cr(V), the
main intracellular reduction product of Cr(VI), might interact
with phosphate moieties in DNA molecules (Sugden and
Wetterhahn, 1996).
Reduction of Cr(VI) to the more stable Cr(III) species
provides genotoxic chromium intermediates Cr(V) and Cr(rV)
with a concomitant generation of reactive oxygen species and
other free radicals (Wetterhahn and Dudek, 1996; Kadiiska
et al, 1994).
The physicochemical properties of Cr(VT), e.g. the pHdependent equilibria, redox and coordination properties as well
as thermodynamic and kinetic stability, of various chromium
oxidation states play a key role in the interactions of Cr(Vr)
in living systems (Cieslak-Golonka, 1995).
As the intracellular transitions of Cr(VI) are multidirectional
and depend on, difficult to predict, discreet intracellular conditions, attempts at obtaining stable complexes of chromium
ions with various organic ligands appear to be an alternative
way to decrease its genotoxic activity. We showed that todralazine markedly decreases the mutagenic effect of potassium
dichromate in standard bacterial tests. The diminution of
mutagenic activity was markedly stronger after 1.5 h preincubation as compared with non-preincubated samples (t0). The
effect was dependent on the todralazine dose and could be
described by the regression equations given in Figures 2 and
3. The results confirm the antimutagenic activity of todralazine,
which was previously observed by us in the cases of several
direct and indirect acting mutagens with the Ames test
(Gasiorowski et al, 1994a,b, 1995).
The B.subtilis rec assay showed a stronger todralazine
effect as compared with that of the Ames test with S.typhimurium strain TA100. The B.subtilis rec~ assay is recommended
for detection of metal-induced mutagenicity, as being more
sensitive to metal-induced mutations than is the Ames test
(Nishioka, 1975; Nikaido and Vaara, 1985; Kuroda et al,
1991). There are several explanations for the higher sensitivity
of the rec" assay in detection of metal-induced mutations. For
instance, it is well established that some mutagens, including
metal compounds, do not penetrate the cell walls of Gramnegative bacteria (among which is S.typhimurium) but enter
those of Gram-positive bacteria (which include B.subtilis)
(Nikaido and Vaara, 1985). The other possible explanation
suggests that the mutagenic range of many metal compounds
is closely related to the toxic range. The range of Cr(VI)
concentrations causing mutations in S.typhimurium is very
close to the toxic range (Nishioka, 1975; Petrilli and De
Flora, 1977) but it is not the decisive limitation disturbing
mutagenicity assessment in the B.subtilis rec system
(Nishioka, 1975). In our experiments we were able to test a
dose of potassium dichromate three times higher in the
B.subtilis rec assay than in the Ames test.
It is possible that in the case of higher concentrations of
tested compounds both mutagenic and antimutagenic activity
become more distinct. In fact, we observed that the antimutagenic effect of todralazine against potassium dichromate mutagenicity was ~ 10-20% stronger in the rec" assay than in the
Ames test.
It should also be stressed that todralazine did not exhibit
any mutagenic activity in standard bacterial tests. This conclusion can be drawn from the results presented in Table I and
414
these results are in agreement with data found in the literature
(Gasiorowski et al, 1993; Chlopkiewicz et al, 1995b).
As 1.5 h preincubation of K 2 Cr 2 0 7 with todralazine noticeably enhanced the impact on potassium dichromate mutagenicity, it was important to assess the direct chemical
interaction of the tested compounds. To solve this problem,
we started from analyses of absorption spectra of potassium
dichromate incubated with various concentrations of todralazine and for various periods of time (1.5, 24 and 48 h). As
the todralazine absorption spectrum extends from 200 to
350 nm (Gasiorowski et al, 1994b), the absorption spectrum
range from 400 to 900 nm given in this paper reflects the
changes in potassium dichromate absorption. We found that
two new peaks emerged after incubation of the compounds:
at 560 and 690 nm. According to the data found in the literature
(Lever, 1984), these two peaks would be perceived as belonging
to a d-d transition in the octahedral Cr(IH) environment. These
absorption peaks reflect the appearance of Cr(ITI) in the
incubation mixture. The chemical kinetics of the reaction are
rather slow, the absorption spectra stabilize during a 24 h
incubation, although after 1.5 h incubation the above absorption
peaks are already well distinguished. We interpret these changes
in absorption spectra as a hint that todralazine induces the
reduction of Cr(VI) to Cifni) and also complexes the Cr(III)
ions. These effects strongly depend on the todralazine dose.
It is well established that Cr(III) and the majority of
Cr(ni) complexes are poorly transported across cell membranes
(Danielsson et al, 1982; De Flora and Wetterhahn, 1989;
Wetterhahn and Hamilton, 1989), therefore Cr(III) compounds
are not mutagenic in the standard bacterial tests (Langerwerf
et al, 1985; De Flora et al., 1990; Gulanowski et al, 1994).
This is in agreement with our bacterial test results, in which
a marked decrease in the mutagenic activity of chromium
compounds was accompanied by the appearance of CiflU) in
the tested samples, as was proved by spectrophotometric
analyses.
The ability of todralazine to form complexes with Cr(in)
may also be beneficial for preventing the genotoxic activity
of this part of Cr(IH) complexes, which could possibly penetrate
the cell. The complexes with todralazine stabilize Cr(IH) and
can prevent the Cr(IH)/Cr(II) transitions which are suggested
to be a possible source of chromium-induced genotoxic radicals
(Shi et al, 1993; Sugden et al, 1994; Bagchi et al, 1995).
The results presented in this paper can be conceived of as
a model study suggesting an alternative way to diminish
chromium-induced genotoxicity. The compounds generating
fast and effective reduction to Cr(HI), which is known to
penetrate the cell poorly, should be considered to be an
effective mechanism reducing chromium genotoxicity and
carcinogenicity in exposed individuals. Todralazine is an
example of such a compound and a search for other compounds
which adopt this mechanism seems to be worth further investigation.
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Received on March 17, 1997; accepted on July 11, 1997
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