Toxicology Letters, 63 (1992) 35-45 0 1992 Elsevier Science Publishers B.V. All rights reserved 0378-4274/92/$ 5.00
35
TOXLET 02786
Mutagenicity of nitroxyl compounds: structure-activity relationships
B. Gallez”, C. De Meesterb, R. Debuyst”, F. Dejehet” and P. Dumont” “Department
of Pharmaceutical
Louvuin,
Brussels,
bDepartment
Cutholic
University
of Louvain,
and Nucleur
Chemistry,
Sciences,
Laboratory
of Pharmaceuticul
Brussels and “Department
Catholic
University
of Medicinal
Sciences, Laboratory
@Louvain,
of Chemistry, Louvain-/a-Xeuve
Chemistry,
Cutholic
of‘Mutagenesis Laboratory
University
oj
und Teratogenesis,
qflnorganic,
Analytical
fBe1gium.l
(Received 23 April 1992) (Revision received 25 June 1992) (Accepted 26 June 1992) Ke,r words: Nitroxyl; Mutagenicity; Toxicity; Stability; Contrast agent; Magnetic resonance imaging SUMMARY Three piperidinoxyl radicals were found to be directly mutagenic in SalmoneNa typhitnurium TA 100, one pyrrolidinoxyl compound had weaker activity. and two other pyrrolidinoxyl derivatives did not produce an increase of the spontaneous revertants. The tester strain TA 100 was selected in preliminary tests for its higher sensitivity compared to TA 98 and TA 102. The mutagenic activity of the three active compounds was abolished by partial reduction with ascorbic acid, suggesting that the mutagenicity was linked to the free radical nature of these compounds, and reduced in the presence of a cofactor supplemented rat liver subcellular fraction. The mutagenicity of the tested compounds was correlated to the resistance of the nitroxyl spin labels to reduction: the more reactive radicals were found to possess higher mutagenic activity.
INTRODUCTION
Nitroxyl free radicals were recently tested as potential contrast enhancers for magnetic resonance imaging. These paramagnetic compounds produce an enhancement of the signal of the imaged tissue mainly by their effects upon spin-lattice relaxation time (T,) of the neighbouring water protons. Although they possess a weaker effect than the metallic complexes, nitroxyl compounds are interesting in consideration of their possible targeting by appropriate vector molecules [ 1,2], and their oxygen-de-
Correspondence to: Bernard Gaflez, Laboratory Mounier 73, B-1200 Brussels, Belgium.
of Medicinal Chemistry. CWIFAiUCL 7340, Avenue
36
pending metab~lisation rate [3--61.These two latter properties should confer on them a place of choice as organ-specific agents and as markers of tissue oxygenation. The development of the techniques of imaging free radicals in aqueous solutions by proton-electron double resonance (PEDRI) also increases the interest for this family of compounds [7,8]. Although many studies concerning relaxivity and stability of the nitroxyls were undertaken, to our knowledge, only three papers dealing with the possible mutagenic activity of these free radicals were published, Afzal et al. [9] demonstrated that 3carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCA) and 2,2,6,6,-tetramethyl-loxido-4-piperidinyl-succinic acid (TES), and their hydroxylamino and amino derivatives did not induce sister chromatid exchanges or mutations at the HGRPT or NalKATPase loci. The same system was used by Gordon et al. [lo] to evaluate the mutagenic activity of [N-( 1-hydroxymethyl-2,3-dihydroxypropyl)-2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxyamide], and no genotoxic effect was noticed. However, Sies and Mehlorn [l l] reported that 4-hydroxy -2,2,6,6-tetramethylpiperidine-1-oxyl and 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-l-oxyl produced a mutagenic effect in ~~~~~~el~~ ty~~i~M~i~?~tester strain TA 104 and that the mutagenicity was dramatically increased in the presence of the superoxide generating system, xanthine oxidaseihypoxanthine. Intrigued by the opposite conclusions of these three studies, we have examined the mutagenic activity of six nitroxyl compounds in Sr&~nellu typhimurium TA 98 and TA 100, strains known to be sensitive to these kind of compounds (the 4-nitroquinoline-~~-oxide was used as positive control for these strains [12]), and TA 102 which is often used for the detection of oxidative mutagens [ 131. The difference in the mutagenicity induced by these nitroxyl compounds led us to correlate the mutagenic activity with the intrinsic reactivity of the free radical species. MATERIALS
AND METHODS
Reagents The chemical structures of the tested compounds and the abbreviations used in this article are presented in Figure 1. All the nitroxyl compounds examined were obtained from Aldrich Company and tested without further pu~fication, except 5-FA, which was synthesized in our laboratory [ 143. Mutagenicity assays These were carried out according to Maron and Ames [ 151 using Salmonella tv~~i~u~j~~ strains TA 98 and TA 102 (tests with 5-COOH and &NH,) and TA 100 (for all compounds). Aliquots of nitroxyl compounds (0.1 ml of a dilution in DMSO corresponding to 0.06, 0.6, 1.5, 3.0, 6.0, 15.0, and 30.0 pmol) were added into the top agar (plate incorporation assay), or into the top agar supplemented with S-9 mix (500 ,~l corre-
37
Carboxy-Proxy1
(5-COOH)
CO-NH-(CH2),
AA
Carbamoyl-Proxy1
I-COOH
5-FA
Tempo
RN
(6)
G
N-00
Amino-Tempo Fig. 1. Chemical
(S-CONH2)
(6-NH2)
structures
of the nitroxyl
Hydroxy-Tempo compounds
(6-OH)
tested in the present
work.
sponding to 50 ~1 of S-9), or were preincubated 1 h at room temperature with a solution of ascorbic acid (1.5 M in phosphate buffer (pH=7.4)) before addition into the top agar. The rat liver subcellular fraction (S 9) was obtained from male Wistar rats pretreated with a PCB mixture, as recommended [12]. The colonies grown on the minimal glucose-agar plates at 37°C were counted after 48 h. Each assay was performed in duplicate with three plates for each concentration. The positive controls were 2-nitrofluorene for TA 98, sodium azide for TA 100, sodium chromate for TA 102, and 2-aminoanthracene when the S-9 mix was used. No solubility problem was noticed with the tested compounds (absence of precipitation in the plates). Stability: reduction rate by ascorbic acid
Two mM solutions of nitroxyl compounds were prepared in a potassium phosphate buffer (KH,PO, 13.6 g/l, pH adjusted at 7.4, glycerol 20% v/v). Five hundred,ul of this solution were added to 500 ~1 of a freshly prepared ascorbate solution (10 mM in the same buffer). The reaction mixture was vortexed for 5 s, and then placed in an EPR flat cell. The EPR scan was immediately initiated. Nitroxyl reduction rates were
38
monitored with a Bruker ER 200 tt EPR spectrometer at room temperature. The evolution of the nitroxyl concentration was monitored by EPR spectroscopy, observing the variation in the signal intensity of the low field peak because the radical ascorbate anion interferes with measurements near the middle nitroxyl peak [16]. The intensity of the signal was estimated using the parameter S,, = w&u, where wIO is the width of the line across one lobe of the derivative curve at height of l/l0 of the amplitude a measured between the baseline and the summit of the peak [ 16,171. Each experiment was repeated at least three times for each compound. The percentage of remaining nitroxyl was calculated by the ratio S,,,/S,,, x 100 where S,,, is the factor S,, at time t and S,,, is the factor S,, extrapolated at time 0 [16]. RESULTS
Mutagenicity assays I. Nitroxyl free radicals. Preliminary experiments were carried out on 5-COOH and 6-NH, using TA 98, TA 100 and TA 102, and indicate no mutagenic effect of 5COOH, but an increase of the revertants number in the presence of 6-NH, with the three strains (Fig. 2). The increase of the revertants was higher in TA 100, and this strain was chosen to test all compounds. The summary of the results of the mutagenicity assays on TA 100 is presented in Figure 3. The typical mean values of the revertants number (TA 100) in the control plates were: 140 for the spontaneous revertants, 380 with the sodium azide (0.2 puglplate), and 750 with the 2-aminoanthracene (1 pug/plate) in the presence of the S-9 mix. The compounds 6-NH, and 6-OH are clearly mutagenic (statistical t-test), and the mutagenic activity clearly follows a dose/effect response. The behaviour of the products 6 and 5-CONH, is more complex. With compound 6, a toxic effect was observed at 30 pmollplate. A weak increase of the number of revertants was observed between 6 and 15 pmollplate (assay repeated three times). For 6, a toxic effect could mask partly the mutagenic activity. Concerning the compound 5-CONH,, an increase of the number of revertants occurred with 30 pmol by plate. This observation was confirmed by repeating the assay with the same amount and a higher concentration of 5-CONH, (45 pmollplate). 2. Hydroxylamines. Nitroxyl radicals are known to be reduced to their corresponding hydroxylamines by ascorbic acid. The preincubation of the nitroxyl compounds with ascorbic acid before addition in the top agar suppressed the mutagenic activity. This observation suggests that the mutagenicity was produced by the free radical species. Incubation in the presence of the S-9 mix did not 3. Incubation with the S-9 mix. produce mutagenicity in the case of the compounds which are not mutagenic alone (5-COOH and 5-FA). In contrast, the presence of the S-9 mix led to a reduction of the number of revertants of the directly mutagenic compounds (6-OH and 6-NH,) (Fig. 4).
39 600 500
-
2 _o a
400-
26
300
-
t 9
200
-
B
100 nc== 0
I .l
600
ma ’
. . . ..~~I
1
.
,
“,
. .”
10
**
.
.
.
.l
,
.
.
.
1
.
,
.
.
10
1
~mol/plate Fig. 2. Ames test results obtained with nitroxyl radicals 5-COOH (top panel) and 6-NH2 (bottom panel) with Salmonella typhimurium TA 98 (o), TA 100 (!li) and TA 102 (0). The symbols indicate a significant difference
between
the number
of spontaneous
nitroxyl
radical
revertants
(statistical
and the number
of revertants
induced
by the
r-test): ‘P
35
TOXLET 02786
Mutagenicity of nitroxyl compounds: structure-activity relationships
B. Gallez”, C. De Meesterb, R. Debuyst”, F. Dejehet” and P. Dumont” “Department
of Pharmaceutical
Louvuin,
Brussels,
bDepartment
Cutholic
University
of Louvain,
and Nucleur
Chemistry,
Sciences,
Laboratory
of Pharmaceuticul
Brussels and “Department
Catholic
University
of Medicinal
Sciences, Laboratory
@Louvain,
of Chemistry, Louvain-/a-Xeuve
Chemistry,
Cutholic
of‘Mutagenesis Laboratory
University
oj
und Teratogenesis,
qflnorganic,
Analytical
fBe1gium.l
(Received 23 April 1992) (Revision received 25 June 1992) (Accepted 26 June 1992) Ke,r words: Nitroxyl; Mutagenicity; Toxicity; Stability; Contrast agent; Magnetic resonance imaging SUMMARY Three piperidinoxyl radicals were found to be directly mutagenic in SalmoneNa typhitnurium TA 100, one pyrrolidinoxyl compound had weaker activity. and two other pyrrolidinoxyl derivatives did not produce an increase of the spontaneous revertants. The tester strain TA 100 was selected in preliminary tests for its higher sensitivity compared to TA 98 and TA 102. The mutagenic activity of the three active compounds was abolished by partial reduction with ascorbic acid, suggesting that the mutagenicity was linked to the free radical nature of these compounds, and reduced in the presence of a cofactor supplemented rat liver subcellular fraction. The mutagenicity of the tested compounds was correlated to the resistance of the nitroxyl spin labels to reduction: the more reactive radicals were found to possess higher mutagenic activity.
INTRODUCTION
Nitroxyl free radicals were recently tested as potential contrast enhancers for magnetic resonance imaging. These paramagnetic compounds produce an enhancement of the signal of the imaged tissue mainly by their effects upon spin-lattice relaxation time (T,) of the neighbouring water protons. Although they possess a weaker effect than the metallic complexes, nitroxyl compounds are interesting in consideration of their possible targeting by appropriate vector molecules [ 1,2], and their oxygen-de-
Correspondence to: Bernard Gaflez, Laboratory Mounier 73, B-1200 Brussels, Belgium.
of Medicinal Chemistry. CWIFAiUCL 7340, Avenue
36
pending metab~lisation rate [3--61.These two latter properties should confer on them a place of choice as organ-specific agents and as markers of tissue oxygenation. The development of the techniques of imaging free radicals in aqueous solutions by proton-electron double resonance (PEDRI) also increases the interest for this family of compounds [7,8]. Although many studies concerning relaxivity and stability of the nitroxyls were undertaken, to our knowledge, only three papers dealing with the possible mutagenic activity of these free radicals were published, Afzal et al. [9] demonstrated that 3carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCA) and 2,2,6,6,-tetramethyl-loxido-4-piperidinyl-succinic acid (TES), and their hydroxylamino and amino derivatives did not induce sister chromatid exchanges or mutations at the HGRPT or NalKATPase loci. The same system was used by Gordon et al. [lo] to evaluate the mutagenic activity of [N-( 1-hydroxymethyl-2,3-dihydroxypropyl)-2,2,5,5-tetramethylpyrrolidine-1-oxyl-3-carboxyamide], and no genotoxic effect was noticed. However, Sies and Mehlorn [l l] reported that 4-hydroxy -2,2,6,6-tetramethylpiperidine-1-oxyl and 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-l-oxyl produced a mutagenic effect in ~~~~~~el~~ ty~~i~M~i~?~tester strain TA 104 and that the mutagenicity was dramatically increased in the presence of the superoxide generating system, xanthine oxidaseihypoxanthine. Intrigued by the opposite conclusions of these three studies, we have examined the mutagenic activity of six nitroxyl compounds in Sr&~nellu typhimurium TA 98 and TA 100, strains known to be sensitive to these kind of compounds (the 4-nitroquinoline-~~-oxide was used as positive control for these strains [12]), and TA 102 which is often used for the detection of oxidative mutagens [ 131. The difference in the mutagenicity induced by these nitroxyl compounds led us to correlate the mutagenic activity with the intrinsic reactivity of the free radical species. MATERIALS
AND METHODS
Reagents The chemical structures of the tested compounds and the abbreviations used in this article are presented in Figure 1. All the nitroxyl compounds examined were obtained from Aldrich Company and tested without further pu~fication, except 5-FA, which was synthesized in our laboratory [ 143. Mutagenicity assays These were carried out according to Maron and Ames [ 151 using Salmonella tv~~i~u~j~~ strains TA 98 and TA 102 (tests with 5-COOH and &NH,) and TA 100 (for all compounds). Aliquots of nitroxyl compounds (0.1 ml of a dilution in DMSO corresponding to 0.06, 0.6, 1.5, 3.0, 6.0, 15.0, and 30.0 pmol) were added into the top agar (plate incorporation assay), or into the top agar supplemented with S-9 mix (500 ,~l corre-
37
Carboxy-Proxy1
(5-COOH)
CO-NH-(CH2),
AA
Carbamoyl-Proxy1
I-COOH
5-FA
Tempo
RN
(6)
G
N-00
Amino-Tempo Fig. 1. Chemical
(S-CONH2)
(6-NH2)
structures
of the nitroxyl
Hydroxy-Tempo compounds
(6-OH)
tested in the present
work.
sponding to 50 ~1 of S-9), or were preincubated 1 h at room temperature with a solution of ascorbic acid (1.5 M in phosphate buffer (pH=7.4)) before addition into the top agar. The rat liver subcellular fraction (S 9) was obtained from male Wistar rats pretreated with a PCB mixture, as recommended [12]. The colonies grown on the minimal glucose-agar plates at 37°C were counted after 48 h. Each assay was performed in duplicate with three plates for each concentration. The positive controls were 2-nitrofluorene for TA 98, sodium azide for TA 100, sodium chromate for TA 102, and 2-aminoanthracene when the S-9 mix was used. No solubility problem was noticed with the tested compounds (absence of precipitation in the plates). Stability: reduction rate by ascorbic acid
Two mM solutions of nitroxyl compounds were prepared in a potassium phosphate buffer (KH,PO, 13.6 g/l, pH adjusted at 7.4, glycerol 20% v/v). Five hundred,ul of this solution were added to 500 ~1 of a freshly prepared ascorbate solution (10 mM in the same buffer). The reaction mixture was vortexed for 5 s, and then placed in an EPR flat cell. The EPR scan was immediately initiated. Nitroxyl reduction rates were
38
monitored with a Bruker ER 200 tt EPR spectrometer at room temperature. The evolution of the nitroxyl concentration was monitored by EPR spectroscopy, observing the variation in the signal intensity of the low field peak because the radical ascorbate anion interferes with measurements near the middle nitroxyl peak [16]. The intensity of the signal was estimated using the parameter S,, = w&u, where wIO is the width of the line across one lobe of the derivative curve at height of l/l0 of the amplitude a measured between the baseline and the summit of the peak [ 16,171. Each experiment was repeated at least three times for each compound. The percentage of remaining nitroxyl was calculated by the ratio S,,,/S,,, x 100 where S,,, is the factor S,, at time t and S,,, is the factor S,, extrapolated at time 0 [16]. RESULTS
Mutagenicity assays I. Nitroxyl free radicals. Preliminary experiments were carried out on 5-COOH and 6-NH, using TA 98, TA 100 and TA 102, and indicate no mutagenic effect of 5COOH, but an increase of the revertants number in the presence of 6-NH, with the three strains (Fig. 2). The increase of the revertants was higher in TA 100, and this strain was chosen to test all compounds. The summary of the results of the mutagenicity assays on TA 100 is presented in Figure 3. The typical mean values of the revertants number (TA 100) in the control plates were: 140 for the spontaneous revertants, 380 with the sodium azide (0.2 puglplate), and 750 with the 2-aminoanthracene (1 pug/plate) in the presence of the S-9 mix. The compounds 6-NH, and 6-OH are clearly mutagenic (statistical t-test), and the mutagenic activity clearly follows a dose/effect response. The behaviour of the products 6 and 5-CONH, is more complex. With compound 6, a toxic effect was observed at 30 pmollplate. A weak increase of the number of revertants was observed between 6 and 15 pmollplate (assay repeated three times). For 6, a toxic effect could mask partly the mutagenic activity. Concerning the compound 5-CONH,, an increase of the number of revertants occurred with 30 pmol by plate. This observation was confirmed by repeating the assay with the same amount and a higher concentration of 5-CONH, (45 pmollplate). 2. Hydroxylamines. Nitroxyl radicals are known to be reduced to their corresponding hydroxylamines by ascorbic acid. The preincubation of the nitroxyl compounds with ascorbic acid before addition in the top agar suppressed the mutagenic activity. This observation suggests that the mutagenicity was produced by the free radical species. Incubation in the presence of the S-9 mix did not 3. Incubation with the S-9 mix. produce mutagenicity in the case of the compounds which are not mutagenic alone (5-COOH and 5-FA). In contrast, the presence of the S-9 mix led to a reduction of the number of revertants of the directly mutagenic compounds (6-OH and 6-NH,) (Fig. 4).
39 600 500
-
2 _o a
400-
26
300
-
t 9
200
-
B
100 nc== 0
I .l
600
ma ’
. . . ..~~I
1
.
,
“,
. .”
10
**
.
.
.
.l
,
.
.
.
1
.
,
.
.
10
1
~mol/plate Fig. 2. Ames test results obtained with nitroxyl radicals 5-COOH (top panel) and 6-NH2 (bottom panel) with Salmonella typhimurium TA 98 (o), TA 100 (!li) and TA 102 (0). The symbols indicate a significant difference
between
the number
of spontaneous
nitroxyl
radical
revertants
(statistical
and the number
of revertants
induced
by the
r-test): ‘P
