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Mutagenicity of glyceryl trinitrate (nitroglycerin) in Salmonella typhimurium
Mutation Research, 298 (1993) 187-195
187
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1218/93/$06.00
MUTGEN 01842
Mutagenicity of glyceryl trinitrate (nitroglycerin) in Salmonella typhimurium Chris M. Maragos a, A.W. Andrews b, Larry K. Keefer a and Rosalie K. Elespuru c Chemistry Section, Laboratory of Comparative Carcinogenesis, b PRl / DynCorp, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702 and c Food and Drug Administration Rockt,ille, MD 20857, USA
(Received 18 March 1992) (Revision received3 August 1992) (Accepted 7 August 1992)
Keywords: Glyceryltrinitrate; Nitroglycerin;Nitric oxide; Sequence analysis; Salmonella typhimurium
Summary The recent finding that the clinical nitrovasodilator, glyceryl trinitrate (GTN), is mutagenic in strain TA1535 has been examined in closer detail, with emphasis on its mechanism of action. GTN increased the number of His + revertants to a maximum of 4 times over background at a GTN dose of 5/~mol/plate. Hamster liver $9 depressed the toxicity of high GTN doses and increased the maximum number of revertants to 5 times over background at 10/~mol/plate. G T N did not cause significant reversion in any of the six other S. typhimurium strains tested (TA1975, TA102, TA1538, TA100, TA100NR, YG1026), although signs of toxicity were observed. Therefore, the mutagenicity of GTN was manifest only in the repair-deficient (uvrB and lacking in pKM101) strain which is responsive to single base changes. Oligonucleotide probe hybridization of TA1535 revertants showed that virtually all of the GTN-induced mutants contained C ~ T transitions in either the first or second base of the hisG46 (CCC) target codon, with a preference for the latter. A similar mutational spectrum was seen previously with a complex of spermine and nitric oxide (NO) which releases nitric oxide. This suggests that NO, which can be derived from GTN via metabolic reduction, may be responsible for GTN's mutagenic action. The known NO scavenger oxymyoglobin did not substantially alter the dose response of GTN, indicating that extracellular NO was not mediating reversion. The data are consistent with the hypothesis that intracellular nitric oxide is responsible for the observed mutations. Salmonella typhimurium
Recently we have reported that glyceryl trinitrate (GTN) is mutagenic in Salmonella typ h i m u r i u m strain TA1535 (Wink et al., 1991). GTN, used commercially as an antianginal agent,
Correspondence: Dr. L. Keefer, NCI-FCRDC, Bldg. 5 3 8 , Room 205E, Frederick, MD 21702, USA.Tel. (301) 846-1467; Fax (301)846-5946.
is believed to reduce blood pressure through the generation of nitric oxide (Schr6r et al., 1991). In addition to having a role in vascular function, nitric oxide (NO) has been implicated in macrophage-mediated cytotoxicity and cytostasis (Hibbs et al., 1988; Marietta, 1988) and was reported to damage DNA and cause mutations in bacterial and mammalian cells (Isomura et al., 1984; Wink et al., 1991; Nguyen et al., 1992; Arroyo ct al.,
188
1992). In the present report we explore several factors influencing GTN-induced mutagenesis to S. typhimurium, in particular those relating to the possible involvement of nitric oxide in its mechanism of action. The identity of sequence changes induced in the hisG46 locus, the responses of various Salmonella strains, the effects of metabolism (by $9 and nitroreductase), and the influence of the nitric oxide scavenger oxymyoglobin were investigated, Materials and methods
Chemicals and reagents
Glyceryl trinitrate (CAS registry number 5563-0) met the requirements of USP XXII for diluted nitroglycerin and was produced in confortuity with current Good Manufacturing Practices for Bulk Pharmaceutical Products. GTN, formulated as 10.1% w / w in lactose, was provided by ICI Specialties (Wilmington, D E 19897). Sublingual tablets containing 0.4 mg GTN (Nitrostat, nitroglycerin USP Lot 22768, expiration May 1993) were produced by Parke-Davis (Morris Plains, NJ). The intravenous formulation was 5 mg G T N / m l in 30% alcohol, 30% propylene glycol, and water, and was manufactured by American Regent Laboratories, Inc. (Shirley, NY; Lot 0229, expiration December 1991). Lactose and sodium nitrite were obtained from Fisher Scientific Chemicals (Springfield, N J). All remaining reagents were of analytical grade or better and were purchased from major suppliers, Oxymyoglobin (MbO 2) was prepared by reduction of ferric myoglobin from horse skeletal muscle (Sigma Chemical Co., St. Louis, MO) as follows. The myoglobin (4.7 g) was dissolved in 23 ml of deionized water and dialyzed against 1 liter of 50 mM ascorbate in phosphate buffered saline (PBS; 0.1 M phosphate, 0.9% NaCI, pH 7.2) at 4°C. After 12 h the solution was dialyzed twice for 30 min against 1 liter of PBS at 4°C and passed through a Millex-GS 22 /zm filter (Millip o r e , Bedford, MA). A portion of this solution w a s diluted, and the M b O 2 c o n t e n t w a s determined from absorbances at 542 and 580 nm (Hewlett Packard 8451A diode array spectrophotometer) using the reported extinction coeffi-
cients of 13.9 and 14.4 m M - I cm t (Antonini and Brunori, 1971). One half milliliter of this solution (or PBS control) was included in the incubation mixture. Bacteria and treatments Salmonella typhimurium strains were obtained
from the sources cited in Table 1 and maintained by the Microbial Mutagenesis Screening Laboratory at the Frederick Cancer Research and Development Center (Frederick, MD). Mutagenicity in the various strains was assessed using the method of Ames et al. (1975) except that 0.2-ml volumes of cell cultures were used (Andrews et al., 1978). Where indicated, liver $9 obtained from hamsters treated with Aroclor 1254 was used at a protein concentration of 3 mg/plate. GTN and lactose were dissolved in dimethyl sulfoxide (DMSO), and tested concurrently with DMSO controls. Methyl methanesulfonate, 2nitronaphthalene, 2-aminoanthracene and sodium azide were included as positive controls. M u t a n t colony analysis
The DNA colony hybridization method of Cebula and Koch (1990) was used without the psoTABLE 1
GENOTYPEAND SOURCE OF Salmonella typhimurium STRAINS Strain
Histidine
uvrB
pKM101 Source
mutation excision repair TA1535
hisG46
TA100
hisG46 hisG428
TA102 TA1538 TA1975
-
B.N. Ames
+
+ +
B.N. Ames B.N. Ames
hisD3052
-
-
hisG46
+
-
-
+ +
B.N. Ames B.N. Ames
Y G 1 0 2 6 hisG46 TA100NR hisG46
-
-
T. Nohmi E. McCoy
All strains have the r f a mutation for partial loss of the lipopolysaccharide (LPS) barrier. Genotypes were reported by Maron and Ames (1983), except for those of YG1026 (Watanabe et al., 1989) and TA100NR (Rosenkranz et al., 1981). TA100NR and YG1026 are both derivatives of TA100. TA100NR is the strain described as TA100FR, selected for resistance to niridazole (E. McCoy, personal communication), and was shown to be deficient in nitroreductase activity. YG1026 contains a nitroreductase gene on a plasmid and overproducesthis enzyme activity.
189
ralen cross-linking step. This step increases probing specificity but is not necessary (Cebula and Koch, personal communication). Mutant colonies were isolated and re-grown on minimal Vogel and Bonner's medium E (Maron and Ames, 1983) without histidine and then in microtiter plates containing nutrient broth. When visually turbid (approximately 4 h), ceils were replica plated onto brain-heart infusion agar in sets of 44 colonies/plate, including 6 positive controls containing the frequently recovered sequence changes. After growth overnight at 37°C, colonies were lifted off the plates by contact with Whatman filter paper #541. Filters were placed in glass dishes containing 0.5 M NaOH/1.5 M NaCI solutions and heated in a microwave oven on high power for 30 sec in order to denature colony DNA, and then rinsed in a buffered solution of 1.0 M Tris (pH 7.2)/2 M NaC1. Air dried filters were probed with a set of six 32p end-labeled 15-mer oligonucleotides containing sequences matching the commonly recovered hisG46 revertants (Fig. 1). Labeled probes (approximately 106 cpm) were added singly to dishes containing prewarmed, buffered solutions and one filter per dish. Incubation and washing temperatures were 47°C, except in the case of probe TC25, which was incubated and washed at 50°C, to maintain stringent hybridization. Filters were dried and autoradiographed using enhancer screens (Fig. 1). The numbers of each type of mutant were determined by hybridization signals from each of the 6 probes,
Results and discussion In the present work only one S. typhimurium strain, TA1535, was responsive to GTN-induced mutagenesis. GTN showed signs of toxicity to all of the strains studied, and was non-mutagenic (histidine reversion less than 2 times background) in strains TA1538, TA100, TA100NR, YG1026, TA1975, and TA102 over the dose range 0.1 to 10 /zmol/plate (Table 2). Lactose, the major component of the GTN mixture tested (89.9% w/w), was non-mutagenic at levels comparable to those present in GTN experiments (Table 2). Two of the clinically useful forms of GTN, sublingual
tablets and an intravenous preparation, were not substantially mutagenic. The latter formulation, distributed as a 5 mg/ml solution and applied to TA1535 plates without dilution at 2.2 /xmol/ plate, caused a 2.5-fold increase over the spontaneous mutant count (Table 3). Results at this dose correlated well with those for the stabilized GTN/lactose formulation at the same dose (Table 2). Sublingual tablets of GTN, at a dose of 1.2 /zmol GTN/plate, caused less than a 2-fold increase in reversion in TA1535 (data not shown). The influences of DNA repair via the Uvr excision pathway and that mediated by the plasmid pKM101 were examined by comparing the response to GTN of strains of different genotypes (Table 1). Strains proficient in either Uvr excision repair (TA1975) or pKM101-mediated repair (TA100, TA100NR, YG1026) or both (TA102) failed to show mutagenesis by GTN, but were at least as susceptible as strain TA1535 to its toxic effects (Table 2). These results suggest that the mutagenic damage was efficiently repaired, while the cytotoxicity was not reduced or was enhanced by DNA repair. Indeed, strain TA102, harboring both repair systems, exhibited the sharpest decline in colony number. It appears that the repair of damage at low doses, coupled with toxicity at higher doses, precludes the detection of GTN-induced mutagenicity in strains proficient in pKM101-mediated repair a n d / o r Uvr excision repair. The influence of metabolism via cytosolic enzymes or nitroreductases was also examined. Hamster $9 was chosen for study because it is the rodent source of choice for activating other mutagens containing N-O bonds (N-nitroso compounds) (Prival et al., 1979). Rat liver cytochrome P450 preparations have been reported to catalyze the release of NO from GTN by reductive denitration (Servent et al., 1989). In our hands, Aroclor-induced hamster $9 did not alter the mutagenic response at GTN doses less than 5 /~mol/plate in TA1535 (Table 3). At higher doses, $9 appeared to reduce toxicity and increase the mutant yield, but not to activate the promutagen significantly (Fig. 2). Nitrite and nitrate, potential products of GTN metabolism, were not overtly toxic to TA1535 at up to 15/zmol/plate, suggesting that neither was responsible for the cytotoxic
190
effects of GTN. The mechanism of G T N toxicity will be the subject of future investigations, The mutagenicity of aromatic nitro compounds in Salmonella is known to depend on resident nitroreductase (NR) activities in the bacteria (Rosenkranz et al., 1981). G T N , although an aliphatic nitrate ester rather than an aromatic nitro compound, is in the same oxidation state and might be a substrate for these enzymes. To determine whether this activity is important for G T N mutagenicity, we obtained bacterial strains either deficient (TA100NR) or over-abundant (YG1026) in nitroreductase. However, the available strains with differences in N R activity were derivatives of a strain (TA100) in which G T N was not mutagenic. Variations in mutagenicity as a
function of N R activity could therefore not bc determined. However, differences in survival were observed. Strain YG1026 (multiple copies of NR) was more sensitive to G T N toxicity than TA100, while a strain with a defective N R (TA100NR) was less sensitive. Therefore, G T N may be a substrate of an S. typhimurium nitroreductase which generates a toxic product. However, the role of this enzyme in G T N mutagenicity cannot be assessed from these experiments. The lack of mutagenicity of G T N in TA1538, a strain sensitive to frameshift mutagens that has the same repair-deficient background as TA1535 (Table 1), suggests that G T N does not induce frameshift mutations. Because of the lack of response of strains harboring pKM101, a negative
:ii 25 GCC
26 TCC
27 CTC
e 28 ACC
29 CAC
S 31 CCC
GTN Fig. 1. (a) Autoradiograms of colony probing reactions for replicated sets of 38 mutants and 6 controls (bottom row) from lactose or GTN-treated S. typhirnuriurn TA1535. Individual probing reactions with 6 different probes are shown, as described in Cebula and Koch (1990). T h e sequences at the hisG46 target providing perfect matches for the six 15-mer oligonucleotide probes (named TC) are G C C (TC25), T C C (TC26), CTC (TC27), A c e (TC28), C A C (TC29) and CCC (TC31); the latter detects mutants with no change in the hisG46 target that presumably contain extragenic suppressor mutations. Slight variations in stringency of different probing reactions were observed. (b) Composite of the probing reactions from (a). Positive signals from each of the 6 probing reactions are compiled into a single composite for each set of mutants (lactose or GTN-treated). 4 sets of GTN-treated mutants and 2 sets of lactose-treated mutants were used to generate the results shown in Table 4.
191
a2
•
....... ..~:~
25 GCC
~!;~i~~% °~
26 TCC
27 CTC
29 CAC
31 CCC
'
28 ACC
Lactose Fig. 1 (continued)
b
(]) O O • • •
O • (]) O O O
0 0 0 0 • • 0 0
Q) 0 (D ~ @ @ • 0
hisG46 locus, encoding proline. His + revertants
(2) O • (2) (I) • O O OOOO0000 [] • • 00000 (~': O • ~ @ Q)
(2) O O O ','.-~)O O O 0000000@ 00 • 0~0 ~ 00 '(~) O • ~ @ (I)
GTN
Lactose
of TA1535 contain three of the six possible single base changes, G C ~ A T , GC--*TA, or G C ~ CG, at the first base pair in the triplet; mutants with GC ~ A T or GC ~ T A at the second base pair also are recovered (Table 4) (Hartman et al., 1986). The third position of the triplet is degenerate, i.e. any sequence change still encodes proline and is not recovered. A fourth sequence change, A T ~ CG, generating an extragenic suppressor (t-RNA anticodon change), is believed to account for His + revertants without any sequence change at the C C C / G G G triplet (Hartman et al., 1986). All of the described sequence changes are recovered among spontaneous revertants (cf. Fig. 1). AT ~ GC and A T ~ TA single base changes do not revert the phenotype of strains harboring this his- allele. In order to determine the nature of GTN-induced mutations against a relatively high background of spontaneous mutants (20-25% of the
O = ~ icrc) ~ = ~ iccc) @ = ~(c~c) • = ~(rcc)
(.~: = 25 (GCC)
(~
= 28 (ACC)
Fig. 1 (continued) result in TA102, which includes A T target sequences, was not informative. Thus, a complete analysis of sequence target preferences could not be made. To explore the nature of GTN-induced sequence changes in TA1535 revertants, the mutant colonies were analyzed (Fig. 1) using the DNA colony hybridization method of Miller and Barnes (1986), as modified by Cebula and Koch (1990).
TA1535 contains a C C C / G G G
target at the
192 TABLE 2 GTN M U T A G E N I C I T Y IN SEVEN S. typhimurium STRAINS ~ TA1535 GTN ( # m o l / p l a t e ) 0 15_+ 3 0.1 13+ 3 1.0 20-+ 4 2.5 33-+ 5 5.0 57-+ 12 7.5 39+ 21 10.0 4-+ 3 12.5 1+ 1 Cellsalone Lactose d NaN3 ~ MMS f 2-NN ~
13-+ 4 10-+ 5 390+253 38_+ 7 258+ 25
TA1975 3+ 3+ 4-+ 4-+ 4+ 0 0 0
2 3 1 2 1
4-+ 2 4-+ 1 4-+ 3 60+_16 4+ 2
TA102 111+ 11 130-+ 2 b 113_+ 15 92+ 1b 12+ 7 2-+ 1 b 0 11 135+ 19 99-+ 38 ND 3181+484 ND
TA1538 15+ 13-+ 12+ 11-+ 3+_ (I 0 ND
TAI00
2 3 2 5 I
97+ 3 85+ 9 91+14 93-+11 85-+ 2 11+13 1+ 1 0
17_+ 4 10_+ 4 13+ 5 26-+ 7 57_+10
97-+16 83-+ 6 707+43 ND ND
TA100NR 146+_ ND ~ 151+ 164-+ 144+ 115+ 40+_ ND
YG1026
7
165+ 181+ 182+ 158+ 36+ 11 0 11
11 10 15 33 18
166+ 11 128+ 7 424-+ 72 3132+_171 157+ 14
17 29 12 15 211
179+ 11 167-+ 4 280_+ 75 2565+330 465+ 20
~ Mean number of revertant colonies per plate + SD in the absence of $9. Underlined values are more than twice background. The zero dose of GTN represents the solvent control. Except as otherwise indicated, number of plates/data point = 3 23. t' Number of replicate plates - 2. ~ ND, not determined. d 70 # m o l / p l a t e . NaN 3, sodium azide, 7.7 nmol/plate. t MMS, methyl methanesulfonate, 9.1 /.,tool/plate. g 2-NN, 2-nitronaphthalene, 30 nmol/plate.
TABLE 3 RESPONSE OF TA1535 A N D TA102 TO A GTN F O R M U L A T I O N USED F O R I N T R A V E N O U S A D M I N I S T R A T I O N 1N HUMANS ~ TA1535
TA102
Without $9
With $9
Without $9
GTN (txmol/plate) 0 (Water) 0.1 0.5 1.0 2.2
12+1 13+6 14-+7 16+5 32+5
13-+2 13-+3 20+6 25_+3 30-+5
159-+21 186-+22 199-+ 19 167-+27 124+50
Cells alone Sodium nitrite b Sodium nitrate d 2-Aminoanthracene ~ Methyl methanesulfonate f
13-+2 26 + 4 18 -+ 6 14 -+ 3 ND
12-+3 ND c 16 + 3 46 -+4 ND
156+ 4 147 + 27 261 + 50 ND 6t5 -+ 70
a b c d
With $9 377+ 405-+ 427+ 392_+ 416-+
31 12 29 15 89
358-+ 21 ND 465 -+ 67 ND 1 136 + 143
Mean number of revertant colonies_+ SD (number of replicate plates = 3). Underlined values are more than twice background. 2.5 /xmol/plate (number of plates = 4). ND, not determined. 10 /xmol/plate. 13 n m o l / p l a t e (number of plates = 3). f 0.9 p~mol/plate (number of plates = 3).
193 TABLE
4
MUTATIONAL
SPECTRA
Treatment
OF his+
Percent
GTN Lactose Untreated
(water)
b
REVERTANTS
(number)
OF S. typhimurium
with codon change
TA1535 a
(CCC + )
CTC
TCC
ACC
CAC
ccc
ccc
Unknown
Total
63 (95) 63 (48) 66 (75)
32 (48) 9 (7) 13 (15)
3 (5) 14 (11) 9 (10)
l(1) 7 (5) 6 (7)
0 (0) 3 (2) 2 (2)
l(1) 4 (3) 2 (2)
0 0 2 (2)
100 (150) 100 (76) 100 (113)
a Sequence determination by colony hybridization (Cebula and Koch, 1990) for GTN- and lactose-treated and untreated TA1535. From three GTN treatment plates (5 pmol GTN/plate) containing 66, 58 and 57 S. typhimurium colonies, 152 colonies were picked. Of the 150 colonies which grew, 143 (95%) contained C + T transitions. 76 colonies were picked from 7 plates exposed to either 56 or 70 pmol lactose/plate (control for GTN formulated with lactose). ’ The analysis of colonies from untreated control plates generated in a separate experiment (Wink et al., 1991) is presented for comparison.
total), the spectrum of mutants arising on the lactose-treated plates was examined in detail (Table 4). From this analysis, the number of each type of sequence change contributed by spontaneous mutants in the GTN-treated sample could be calculated (Table 5). These values were then compared with the numbers of each type of revertant recovered from GTN-treated plates. The results indicated that two of the three sequence changes recovered (GC + TA and AT + CG) from the GTN-treated mutants could be accounted for as spontaneous mutations. The excess sequence changes over background were C + T transitions at either the first or second GC base pair in the triplet, the latter appearing among the greater proportion of mutants.
TABLE
5
SEQUENCE Treatment
GTN Expected
Because the vasodilatory effects of GTN are attributed to the generation of nitric oxide within the smooth muscle cells of the vasculature (Schror et al., 19911, the possible involvement of NO in the mechanism of GTN mutagenicity was examined. NO gas was reported to be mutagenic in Salmonella typhimurium TAlOO (Isomura et al., 19841, and to inhibit DNA synthesis in some cell types, as does GTN (Nakaki et al., 1990). The sequence changes found with GTN in the present report were also found with a nitric oxide-releasing spermine/NO complex [CAS Registry No. 136587-13-81 (Maragos et al., 1991). The latter compound generated only GC -+ AT transitions in TA1.535, predominantly at the middle GC base pair in the triplet (Wink et al., 1991). The similar-
CHANGES Number
IN GTN-INDUCED of colonies
VERSUS
with codon change
SPONTANEOUS
REVERTANTS
OF S. fyphimurium
TA1535
(CCC + 1
CTC
TCC
ACC
CAC
GCC
ccc
Unknown
Total
95
48
5
1
0
1
0
150
4.0
1.8
0.7
1.1
0
from spontaneous 17.6
a 2.6
27.8
a The numbers shown are those expected to arise from spontaneous reversions in the ?-plate GTN sample, as calculated from the lactose control data of Table 4. Lactose plates contained 14, 15, 11, 11, 11, 6 and 10 colonies (total 78 colonies/7 plates) of which 76 colonies (97%) were sampled. GTN plates contained 66, 58 and 57 colonies of which 150 were sampled (83%). The number of spontaneous colonies at each locus, per plate, was calculated as: (total number observed at each locus +0.97)+7 plates. The total number of expected spontaneous colonies at each locus in the GTN sample was calculated by multiplying the number of spontaneous colonies at each locus per plate by 3 (to adjust for the number of GTN plates) and by 0.83 (to adjust for the sampling frequency of GTN-induced colonies).
194 ity o f t h e s e m u t a n t s p e c t r a is c o n s i s t e n t with the h y p o t h e s i s t h a t nitric oxide m e d i a t e s the m u t a genicity of G T N . W h e t h e r this m u t a t i o n a l p a t t e r n is u n i q u e to N O will b e c o m e m o r e c e r t a i n as a d d i t i o n a l c o m p o u n d s a r e tested. N o n e of the 11 m u t a g e n s s t u d i e d by H e n r i k s o n et al. (1992) exh i b i t e d a m u t a t i o n a l p a t t e r n like that of G T N a n d the s p e r m i n e / N O c o m p l e x (uniquely G C - - , A T with a p r e f e r e n c e for the s e c o n d G C b a s e p a i r in the hisG46 G G G / C C C triplet). NM e t h y l - N - n i t r o s o u r e a , for e x a m p l e , e x h i b i t e d a strong ( 8 0 - 9 0 % ) p r e f e r e n c e for G C --* A T transitions in the first b a s e p a i r of t h e triplet. This result suggests t h a t an i n d i r e c t m e c h a n i s m involving N - n i t r o s a t i o n of a m e t h y l u r e a - t y p e comp o u n d , followed by O % m e t h y l a t i o n of g u a n i n e ( S u k u m a r et al., 1983), is an unlikely source of N O - m e d i a t e d m u t a g e n e s i s . A m e c h a n i s m involv-
~m o w ~ ~a
7o
.
.
.
.
.
.
60 50 40 30
o ~
20 10
z
o
, 0
, ' , ' ' 1 2 3 4 5 DOSE ( / z m o l / p l a t e )
' 6
Fig. 3. Mutagenicity of glyceryl trinitrate to S. typhimurium TA1535 in the presence (o) and absence (e) of 3.4 /zmol oxymyoglobin/plate, mean_+SD, number of replicate plates =4.
ing d e a m i n a t i o n of cytosine r e m a i n s the m o s t p l a u s i b l e b a s e d on available data. T o d e t e r m i n e t h e e x t e n t to which N O g e n e r a t e d e x t r a c e l l u l a r l y was r e s p o n s i b l e for G T N m u tagenicity, we t e s t e d t h e i n f l u e n c e of myoglobin, which reacts very r a p i d l y with nitric oxide yielding a stable n i t r o s y l - m y o g l o b i n c o m p l e x ( A n t o n i n i a n d Brunori, 1971). In the p r e s e n t w o r k 3 . 4 / x m o l o x y m y o g l o b i n / p l a t e did not s u b s t a n t i a l l y inhibit
m z o w z
,
,
80
60
4o
o
20
the m u t a g e n i c effect of up to 6 / x m o l G T N / p l a t e (Fig. 3). This i n d i c a t e s that t h e m u t a g e n i c i t y of G T N d o e s n o t result from e x p o s u r e to extracellular nitric oxide. D e s p i t e the w i d e s p r e a d use of glyceryl trinit r a t e as a vasodilator, its m u t a g e n i c i t y to S. typhimurium has, until now, not b e e n t h o r o u g h l y investigated. In p a r t this m a y stem from two factors, the r e q u i r e m e n t for large doses of G T N a n d the lack of susceptibility of s t a n d a r d S. typhimurium t e s t e r strains (i.e., T A 9 8 a n d T A 1 0 0 ) to its m u t a g e n i c action. T h e s e factors suggest t h a t t h e r e may b e c o n s i d e r a b l e variability in the vuln e r a b i l i t y to G T N - i n d u c e d m u t a g e n e s i s . T h e chronic toxicity and c a r c i n o g e n i c i t y o f G T N have b e e n e x a m i n e d in dogs, rats, a n d mice, with h e p a t o c e l l u l a r t u m o r s i n d u c e d in rats (Ellis et al., 1984). F u t u r e investigations in o u r l a b o r a t o r y will f u r t h e r e x p l o r e the effectiveness of G T N as an initiator of carcinogenesis.
o z
' , ' , ' , , , ' , ' , ' , 2 4 6 8 10 12
0
14
DOSE ( / ~ m o l / p l a t e ) Fig. 2. Histidine reversion of S. typhimurium TA1535 following treatment with glyceryl trinitrate in the presence (©) and absence (e) of hamster liver $9. Data were pooled from four experiments, number of replicate plates/data point= 15, mean_+SD. Values for the lactose controls were 10±5 (without $9)and 15_+4 (with $9).
Acknowledgements We Goebel Cebula colony
thank Corinthia Brown and Thomas for assistance with the assays, T h o m a s a n d W a l t e r K o c h for instruction in t h e h y b r i d i z a t i o n assay a n d for oligonu-
c l e o t i d e p r o b e s u s e d in t h e s e e x p e r i m e n t s , a n d C h a r l e s W. Riggs for assistance with t h e statisti-
195
cal analyses. Research was funded in part by the D e p a r t m e n t of Health and H u m a n Services u n der contract number NO1-CO-74102. References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test, Mutation Res., 31,347-364. Andrews, A.W., L.H. Thibault and W. Lijinsky (1978) The relationship between carcinogenicity and mutagenicity of some polynuclear hydrocarbons, Mutation Res., 51, 311318. Antonini, E., and M. Brunori (1971) Hemoglobin and myoglobin in their reactions with ligands, North-Holland Publishing Co., Amsterdam, pp. 19-33. Arroyo, P.L., V. Hatch-Pigott, H.F. Mower and R.V. Cooney (1992) Mutagenicity of nitric oxide and its inhibition by antioxidants, Mutation Res., 281, 193-202. Cebula, T.A., and W.H. Koch (1990) Analysis of spontaneous and psoralen-induced Salmonella typhimurium hisG46 revertants by oligodeoxyribonucleotide colony hybridization: use of psoralens to cross-link probes to target sequences, Mutation Res., 229, 79-87. Ellis, H.V., III, C.B. Hong, C.C. Lee, J.C. Dacre and J.P. Glennon (1984) Subacute and chronic toxicity studies of trinitroglycerin in dogs, rats, and mice, Fund. Appl. Toxicol., 4, 248-260. Hartman, P.E., B.N. Ames, J.R. Roth, W.M. Barnes and D.E. Levin (1986) Target sequences for mutagenesis in Salmonella histidine-requiring mutants, Environ. Mutagen., 8, 631-641. Henrikson, E.N., W.H. Koch and T.A. Cebula (1992) Base substitution specificity of hisG46 reversion in S. typhimurium strain TA100 uniquely characterizes several classes of mutagens, Environ. Molec. Mutagen., 19, supplement 26, 24. Hibbs, J.B., Jr., R.R. Taintor, Z. Vavrin and E.M. Rachlin (1988) Nitric oxide: a cytotoxic activated macrophage effector molecule, Biochem. Biophys. Res. Commun., 157, 87-94. Isomura, K., M. Chikahira, K. Teranishi and K. Hamada (1984) Induction of mutations and chromosome aberrations in lung cells following in vivo exposure of rats to nitrogen oxides, Mutation Res., 136, 119-125. Maragos, C.M., D. Morley, D.A. Wink, T.M. Dunams, J.E. Saavedra, A. Hoffman, A.A. Bove, L. Isaac, J.A. Hrabie and L.K. Keefer (1991) Complexes of -NO with nucleophiles as agents for the controlled biological release of nitric oxide. Vasorelaxant effects, J. Med. Chem., 34, 3242-3247.
Marietta,M.A. (1988) Mammalian synthesis of nitrite, nitrate, nitric oxide, and N-nitrosating agents, Chem. Res. Toxicol., 1,249-257. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Miller, J.K., and W.M. Barnes (1986) Colony probing as an alternative to standard sequencing as a means of direct analysis of chromosomal DNA to determine the spectrum of single-base changes in regions of known sequence, Proc. Natl. Acad. Sci. (U.S.A.), 83, 1026-1030. Nakaki, T., M. Nakayama and R. Kato (1990) Inhibition by nitric oxide and nitric oxide-producingvasodilators of DNA synthesis in vascular smooth muscle cells, Eur. J. Pharmacol., 189, 347-353. Nguyen, T., D. Brunson, C.L. Crespi, B.W. Penman, J.S. Wishnok and S.R. Tannenbaum (1992) DNA damage and mutation in human cells exposed to nitric oxide in vitro, Proc. Natl. Acad. Sci. (U.S.A.), 89, 3030-3034. Prival, M.J., V.D. King and A.T. Sheldon, Jr. (1979) The mutagenicity of dialkyl nitrosamines in the Salmonella plate assay, Environ. Mutagen., 1, 95-104. Rosenkranz, H.S., E.C. McCoy, R. Mermelstein and W.T. Speck (1981) A cautionary note on the use of nitroreductase-deficient strains of Salmonella typhimurium for the detection of nitroarenes as mutagens in complex mixtures including diesel exhausts, Mutation Res., 91, 103-105. Schr6r, K., I. Woditsch and S. F6rster (1991) Generation of nitric oxide from organic nitrovasodilators during passage through the coronary vascular bed and its role in coronary vasodilation and nitrate tolerance, Blood Vessels, 28, 6266. Servent, D., M. Delaforge, C. Ducrocq, D. Mansuy and M. Lenfant (1989) Nitric oxide formation during microsomal hepatic denitration of glyceryl trinitrate: involvement of cytochrome P-450, Biochem. Biophys. Res. Commun., 163, 1210-1216. Sukumar, S., V. Notario, D. Martin-Zanca and M. Barbacid (1983) Induction of mammary carcinomas in rats by nitrosomethylurea involves malignant activation of H-ras-1 locus by single point mutations, Nature, 306, 658-661. Watanabe, M., M. Ishidate, Jr. and T. Nohmi (1989) A sensitive method for the detection of mutagenic nitroarenes: construction of nitroreductase-overproducing derivatives of Salmonella typhimurium strains TA98 and TA100, Mutation Res., 216, 211-220. Wink, D.A., K.S. Kasprzak, C.M. Maragos, R.K. Elespuru, M. Misra, T.M. Dunams, T.A. Cebula, W.H. Koch, A.W. Andrews, J.S. Allen and L.K. Keefer (1991) DNA deaminating ability and genotoxicity of nitric oxide and its progenitors, Science, 254, 1001-1003.
187
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1218/93/$06.00
MUTGEN 01842
Mutagenicity of glyceryl trinitrate (nitroglycerin) in Salmonella typhimurium Chris M. Maragos a, A.W. Andrews b, Larry K. Keefer a and Rosalie K. Elespuru c Chemistry Section, Laboratory of Comparative Carcinogenesis, b PRl / DynCorp, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702 and c Food and Drug Administration Rockt,ille, MD 20857, USA
(Received 18 March 1992) (Revision received3 August 1992) (Accepted 7 August 1992)
Keywords: Glyceryltrinitrate; Nitroglycerin;Nitric oxide; Sequence analysis; Salmonella typhimurium
Summary The recent finding that the clinical nitrovasodilator, glyceryl trinitrate (GTN), is mutagenic in strain TA1535 has been examined in closer detail, with emphasis on its mechanism of action. GTN increased the number of His + revertants to a maximum of 4 times over background at a GTN dose of 5/~mol/plate. Hamster liver $9 depressed the toxicity of high GTN doses and increased the maximum number of revertants to 5 times over background at 10/~mol/plate. G T N did not cause significant reversion in any of the six other S. typhimurium strains tested (TA1975, TA102, TA1538, TA100, TA100NR, YG1026), although signs of toxicity were observed. Therefore, the mutagenicity of GTN was manifest only in the repair-deficient (uvrB and lacking in pKM101) strain which is responsive to single base changes. Oligonucleotide probe hybridization of TA1535 revertants showed that virtually all of the GTN-induced mutants contained C ~ T transitions in either the first or second base of the hisG46 (CCC) target codon, with a preference for the latter. A similar mutational spectrum was seen previously with a complex of spermine and nitric oxide (NO) which releases nitric oxide. This suggests that NO, which can be derived from GTN via metabolic reduction, may be responsible for GTN's mutagenic action. The known NO scavenger oxymyoglobin did not substantially alter the dose response of GTN, indicating that extracellular NO was not mediating reversion. The data are consistent with the hypothesis that intracellular nitric oxide is responsible for the observed mutations. Salmonella typhimurium
Recently we have reported that glyceryl trinitrate (GTN) is mutagenic in Salmonella typ h i m u r i u m strain TA1535 (Wink et al., 1991). GTN, used commercially as an antianginal agent,
Correspondence: Dr. L. Keefer, NCI-FCRDC, Bldg. 5 3 8 , Room 205E, Frederick, MD 21702, USA.Tel. (301) 846-1467; Fax (301)846-5946.
is believed to reduce blood pressure through the generation of nitric oxide (Schr6r et al., 1991). In addition to having a role in vascular function, nitric oxide (NO) has been implicated in macrophage-mediated cytotoxicity and cytostasis (Hibbs et al., 1988; Marietta, 1988) and was reported to damage DNA and cause mutations in bacterial and mammalian cells (Isomura et al., 1984; Wink et al., 1991; Nguyen et al., 1992; Arroyo ct al.,
188
1992). In the present report we explore several factors influencing GTN-induced mutagenesis to S. typhimurium, in particular those relating to the possible involvement of nitric oxide in its mechanism of action. The identity of sequence changes induced in the hisG46 locus, the responses of various Salmonella strains, the effects of metabolism (by $9 and nitroreductase), and the influence of the nitric oxide scavenger oxymyoglobin were investigated, Materials and methods
Chemicals and reagents
Glyceryl trinitrate (CAS registry number 5563-0) met the requirements of USP XXII for diluted nitroglycerin and was produced in confortuity with current Good Manufacturing Practices for Bulk Pharmaceutical Products. GTN, formulated as 10.1% w / w in lactose, was provided by ICI Specialties (Wilmington, D E 19897). Sublingual tablets containing 0.4 mg GTN (Nitrostat, nitroglycerin USP Lot 22768, expiration May 1993) were produced by Parke-Davis (Morris Plains, NJ). The intravenous formulation was 5 mg G T N / m l in 30% alcohol, 30% propylene glycol, and water, and was manufactured by American Regent Laboratories, Inc. (Shirley, NY; Lot 0229, expiration December 1991). Lactose and sodium nitrite were obtained from Fisher Scientific Chemicals (Springfield, N J). All remaining reagents were of analytical grade or better and were purchased from major suppliers, Oxymyoglobin (MbO 2) was prepared by reduction of ferric myoglobin from horse skeletal muscle (Sigma Chemical Co., St. Louis, MO) as follows. The myoglobin (4.7 g) was dissolved in 23 ml of deionized water and dialyzed against 1 liter of 50 mM ascorbate in phosphate buffered saline (PBS; 0.1 M phosphate, 0.9% NaCI, pH 7.2) at 4°C. After 12 h the solution was dialyzed twice for 30 min against 1 liter of PBS at 4°C and passed through a Millex-GS 22 /zm filter (Millip o r e , Bedford, MA). A portion of this solution w a s diluted, and the M b O 2 c o n t e n t w a s determined from absorbances at 542 and 580 nm (Hewlett Packard 8451A diode array spectrophotometer) using the reported extinction coeffi-
cients of 13.9 and 14.4 m M - I cm t (Antonini and Brunori, 1971). One half milliliter of this solution (or PBS control) was included in the incubation mixture. Bacteria and treatments Salmonella typhimurium strains were obtained
from the sources cited in Table 1 and maintained by the Microbial Mutagenesis Screening Laboratory at the Frederick Cancer Research and Development Center (Frederick, MD). Mutagenicity in the various strains was assessed using the method of Ames et al. (1975) except that 0.2-ml volumes of cell cultures were used (Andrews et al., 1978). Where indicated, liver $9 obtained from hamsters treated with Aroclor 1254 was used at a protein concentration of 3 mg/plate. GTN and lactose were dissolved in dimethyl sulfoxide (DMSO), and tested concurrently with DMSO controls. Methyl methanesulfonate, 2nitronaphthalene, 2-aminoanthracene and sodium azide were included as positive controls. M u t a n t colony analysis
The DNA colony hybridization method of Cebula and Koch (1990) was used without the psoTABLE 1
GENOTYPEAND SOURCE OF Salmonella typhimurium STRAINS Strain
Histidine
uvrB
pKM101 Source
mutation excision repair TA1535
hisG46
TA100
hisG46 hisG428
TA102 TA1538 TA1975
-
B.N. Ames
+
+ +
B.N. Ames B.N. Ames
hisD3052
-
-
hisG46
+
-
-
+ +
B.N. Ames B.N. Ames
Y G 1 0 2 6 hisG46 TA100NR hisG46
-
-
T. Nohmi E. McCoy
All strains have the r f a mutation for partial loss of the lipopolysaccharide (LPS) barrier. Genotypes were reported by Maron and Ames (1983), except for those of YG1026 (Watanabe et al., 1989) and TA100NR (Rosenkranz et al., 1981). TA100NR and YG1026 are both derivatives of TA100. TA100NR is the strain described as TA100FR, selected for resistance to niridazole (E. McCoy, personal communication), and was shown to be deficient in nitroreductase activity. YG1026 contains a nitroreductase gene on a plasmid and overproducesthis enzyme activity.
189
ralen cross-linking step. This step increases probing specificity but is not necessary (Cebula and Koch, personal communication). Mutant colonies were isolated and re-grown on minimal Vogel and Bonner's medium E (Maron and Ames, 1983) without histidine and then in microtiter plates containing nutrient broth. When visually turbid (approximately 4 h), ceils were replica plated onto brain-heart infusion agar in sets of 44 colonies/plate, including 6 positive controls containing the frequently recovered sequence changes. After growth overnight at 37°C, colonies were lifted off the plates by contact with Whatman filter paper #541. Filters were placed in glass dishes containing 0.5 M NaOH/1.5 M NaCI solutions and heated in a microwave oven on high power for 30 sec in order to denature colony DNA, and then rinsed in a buffered solution of 1.0 M Tris (pH 7.2)/2 M NaC1. Air dried filters were probed with a set of six 32p end-labeled 15-mer oligonucleotides containing sequences matching the commonly recovered hisG46 revertants (Fig. 1). Labeled probes (approximately 106 cpm) were added singly to dishes containing prewarmed, buffered solutions and one filter per dish. Incubation and washing temperatures were 47°C, except in the case of probe TC25, which was incubated and washed at 50°C, to maintain stringent hybridization. Filters were dried and autoradiographed using enhancer screens (Fig. 1). The numbers of each type of mutant were determined by hybridization signals from each of the 6 probes,
Results and discussion In the present work only one S. typhimurium strain, TA1535, was responsive to GTN-induced mutagenesis. GTN showed signs of toxicity to all of the strains studied, and was non-mutagenic (histidine reversion less than 2 times background) in strains TA1538, TA100, TA100NR, YG1026, TA1975, and TA102 over the dose range 0.1 to 10 /zmol/plate (Table 2). Lactose, the major component of the GTN mixture tested (89.9% w/w), was non-mutagenic at levels comparable to those present in GTN experiments (Table 2). Two of the clinically useful forms of GTN, sublingual
tablets and an intravenous preparation, were not substantially mutagenic. The latter formulation, distributed as a 5 mg/ml solution and applied to TA1535 plates without dilution at 2.2 /xmol/ plate, caused a 2.5-fold increase over the spontaneous mutant count (Table 3). Results at this dose correlated well with those for the stabilized GTN/lactose formulation at the same dose (Table 2). Sublingual tablets of GTN, at a dose of 1.2 /zmol GTN/plate, caused less than a 2-fold increase in reversion in TA1535 (data not shown). The influences of DNA repair via the Uvr excision pathway and that mediated by the plasmid pKM101 were examined by comparing the response to GTN of strains of different genotypes (Table 1). Strains proficient in either Uvr excision repair (TA1975) or pKM101-mediated repair (TA100, TA100NR, YG1026) or both (TA102) failed to show mutagenesis by GTN, but were at least as susceptible as strain TA1535 to its toxic effects (Table 2). These results suggest that the mutagenic damage was efficiently repaired, while the cytotoxicity was not reduced or was enhanced by DNA repair. Indeed, strain TA102, harboring both repair systems, exhibited the sharpest decline in colony number. It appears that the repair of damage at low doses, coupled with toxicity at higher doses, precludes the detection of GTN-induced mutagenicity in strains proficient in pKM101-mediated repair a n d / o r Uvr excision repair. The influence of metabolism via cytosolic enzymes or nitroreductases was also examined. Hamster $9 was chosen for study because it is the rodent source of choice for activating other mutagens containing N-O bonds (N-nitroso compounds) (Prival et al., 1979). Rat liver cytochrome P450 preparations have been reported to catalyze the release of NO from GTN by reductive denitration (Servent et al., 1989). In our hands, Aroclor-induced hamster $9 did not alter the mutagenic response at GTN doses less than 5 /~mol/plate in TA1535 (Table 3). At higher doses, $9 appeared to reduce toxicity and increase the mutant yield, but not to activate the promutagen significantly (Fig. 2). Nitrite and nitrate, potential products of GTN metabolism, were not overtly toxic to TA1535 at up to 15/zmol/plate, suggesting that neither was responsible for the cytotoxic
190
effects of GTN. The mechanism of G T N toxicity will be the subject of future investigations, The mutagenicity of aromatic nitro compounds in Salmonella is known to depend on resident nitroreductase (NR) activities in the bacteria (Rosenkranz et al., 1981). G T N , although an aliphatic nitrate ester rather than an aromatic nitro compound, is in the same oxidation state and might be a substrate for these enzymes. To determine whether this activity is important for G T N mutagenicity, we obtained bacterial strains either deficient (TA100NR) or over-abundant (YG1026) in nitroreductase. However, the available strains with differences in N R activity were derivatives of a strain (TA100) in which G T N was not mutagenic. Variations in mutagenicity as a
function of N R activity could therefore not bc determined. However, differences in survival were observed. Strain YG1026 (multiple copies of NR) was more sensitive to G T N toxicity than TA100, while a strain with a defective N R (TA100NR) was less sensitive. Therefore, G T N may be a substrate of an S. typhimurium nitroreductase which generates a toxic product. However, the role of this enzyme in G T N mutagenicity cannot be assessed from these experiments. The lack of mutagenicity of G T N in TA1538, a strain sensitive to frameshift mutagens that has the same repair-deficient background as TA1535 (Table 1), suggests that G T N does not induce frameshift mutations. Because of the lack of response of strains harboring pKM101, a negative
:ii 25 GCC
26 TCC
27 CTC
e 28 ACC
29 CAC
S 31 CCC
GTN Fig. 1. (a) Autoradiograms of colony probing reactions for replicated sets of 38 mutants and 6 controls (bottom row) from lactose or GTN-treated S. typhirnuriurn TA1535. Individual probing reactions with 6 different probes are shown, as described in Cebula and Koch (1990). T h e sequences at the hisG46 target providing perfect matches for the six 15-mer oligonucleotide probes (named TC) are G C C (TC25), T C C (TC26), CTC (TC27), A c e (TC28), C A C (TC29) and CCC (TC31); the latter detects mutants with no change in the hisG46 target that presumably contain extragenic suppressor mutations. Slight variations in stringency of different probing reactions were observed. (b) Composite of the probing reactions from (a). Positive signals from each of the 6 probing reactions are compiled into a single composite for each set of mutants (lactose or GTN-treated). 4 sets of GTN-treated mutants and 2 sets of lactose-treated mutants were used to generate the results shown in Table 4.
191
a2
•
....... ..~:~
25 GCC
~!;~i~~% °~
26 TCC
27 CTC
29 CAC
31 CCC
'
28 ACC
Lactose Fig. 1 (continued)
b
(]) O O • • •
O • (]) O O O
0 0 0 0 • • 0 0
Q) 0 (D ~ @ @ • 0
hisG46 locus, encoding proline. His + revertants
(2) O • (2) (I) • O O OOOO0000 [] • • 00000 (~': O • ~ @ Q)
(2) O O O ','.-~)O O O 0000000@ 00 • 0~0 ~ 00 '(~) O • ~ @ (I)
GTN
Lactose
of TA1535 contain three of the six possible single base changes, G C ~ A T , GC--*TA, or G C ~ CG, at the first base pair in the triplet; mutants with GC ~ A T or GC ~ T A at the second base pair also are recovered (Table 4) (Hartman et al., 1986). The third position of the triplet is degenerate, i.e. any sequence change still encodes proline and is not recovered. A fourth sequence change, A T ~ CG, generating an extragenic suppressor (t-RNA anticodon change), is believed to account for His + revertants without any sequence change at the C C C / G G G triplet (Hartman et al., 1986). All of the described sequence changes are recovered among spontaneous revertants (cf. Fig. 1). AT ~ GC and A T ~ TA single base changes do not revert the phenotype of strains harboring this his- allele. In order to determine the nature of GTN-induced mutations against a relatively high background of spontaneous mutants (20-25% of the
O = ~ icrc) ~ = ~ iccc) @ = ~(c~c) • = ~(rcc)
(.~: = 25 (GCC)
(~
= 28 (ACC)
Fig. 1 (continued) result in TA102, which includes A T target sequences, was not informative. Thus, a complete analysis of sequence target preferences could not be made. To explore the nature of GTN-induced sequence changes in TA1535 revertants, the mutant colonies were analyzed (Fig. 1) using the DNA colony hybridization method of Miller and Barnes (1986), as modified by Cebula and Koch (1990).
TA1535 contains a C C C / G G G
target at the
192 TABLE 2 GTN M U T A G E N I C I T Y IN SEVEN S. typhimurium STRAINS ~ TA1535 GTN ( # m o l / p l a t e ) 0 15_+ 3 0.1 13+ 3 1.0 20-+ 4 2.5 33-+ 5 5.0 57-+ 12 7.5 39+ 21 10.0 4-+ 3 12.5 1+ 1 Cellsalone Lactose d NaN3 ~ MMS f 2-NN ~
13-+ 4 10-+ 5 390+253 38_+ 7 258+ 25
TA1975 3+ 3+ 4-+ 4-+ 4+ 0 0 0
2 3 1 2 1
4-+ 2 4-+ 1 4-+ 3 60+_16 4+ 2
TA102 111+ 11 130-+ 2 b 113_+ 15 92+ 1b 12+ 7 2-+ 1 b 0 11 135+ 19 99-+ 38 ND 3181+484 ND
TA1538 15+ 13-+ 12+ 11-+ 3+_ (I 0 ND
TAI00
2 3 2 5 I
97+ 3 85+ 9 91+14 93-+11 85-+ 2 11+13 1+ 1 0
17_+ 4 10_+ 4 13+ 5 26-+ 7 57_+10
97-+16 83-+ 6 707+43 ND ND
TA100NR 146+_ ND ~ 151+ 164-+ 144+ 115+ 40+_ ND
YG1026
7
165+ 181+ 182+ 158+ 36+ 11 0 11
11 10 15 33 18
166+ 11 128+ 7 424-+ 72 3132+_171 157+ 14
17 29 12 15 211
179+ 11 167-+ 4 280_+ 75 2565+330 465+ 20
~ Mean number of revertant colonies per plate + SD in the absence of $9. Underlined values are more than twice background. The zero dose of GTN represents the solvent control. Except as otherwise indicated, number of plates/data point = 3 23. t' Number of replicate plates - 2. ~ ND, not determined. d 70 # m o l / p l a t e . NaN 3, sodium azide, 7.7 nmol/plate. t MMS, methyl methanesulfonate, 9.1 /.,tool/plate. g 2-NN, 2-nitronaphthalene, 30 nmol/plate.
TABLE 3 RESPONSE OF TA1535 A N D TA102 TO A GTN F O R M U L A T I O N USED F O R I N T R A V E N O U S A D M I N I S T R A T I O N 1N HUMANS ~ TA1535
TA102
Without $9
With $9
Without $9
GTN (txmol/plate) 0 (Water) 0.1 0.5 1.0 2.2
12+1 13+6 14-+7 16+5 32+5
13-+2 13-+3 20+6 25_+3 30-+5
159-+21 186-+22 199-+ 19 167-+27 124+50
Cells alone Sodium nitrite b Sodium nitrate d 2-Aminoanthracene ~ Methyl methanesulfonate f
13-+2 26 + 4 18 -+ 6 14 -+ 3 ND
12-+3 ND c 16 + 3 46 -+4 ND
156+ 4 147 + 27 261 + 50 ND 6t5 -+ 70
a b c d
With $9 377+ 405-+ 427+ 392_+ 416-+
31 12 29 15 89
358-+ 21 ND 465 -+ 67 ND 1 136 + 143
Mean number of revertant colonies_+ SD (number of replicate plates = 3). Underlined values are more than twice background. 2.5 /xmol/plate (number of plates = 4). ND, not determined. 10 /xmol/plate. 13 n m o l / p l a t e (number of plates = 3). f 0.9 p~mol/plate (number of plates = 3).
193 TABLE
4
MUTATIONAL
SPECTRA
Treatment
OF his+
Percent
GTN Lactose Untreated
(water)
b
REVERTANTS
(number)
OF S. typhimurium
with codon change
TA1535 a
(CCC + )
CTC
TCC
ACC
CAC
ccc
ccc
Unknown
Total
63 (95) 63 (48) 66 (75)
32 (48) 9 (7) 13 (15)
3 (5) 14 (11) 9 (10)
l(1) 7 (5) 6 (7)
0 (0) 3 (2) 2 (2)
l(1) 4 (3) 2 (2)
0 0 2 (2)
100 (150) 100 (76) 100 (113)
a Sequence determination by colony hybridization (Cebula and Koch, 1990) for GTN- and lactose-treated and untreated TA1535. From three GTN treatment plates (5 pmol GTN/plate) containing 66, 58 and 57 S. typhimurium colonies, 152 colonies were picked. Of the 150 colonies which grew, 143 (95%) contained C + T transitions. 76 colonies were picked from 7 plates exposed to either 56 or 70 pmol lactose/plate (control for GTN formulated with lactose). ’ The analysis of colonies from untreated control plates generated in a separate experiment (Wink et al., 1991) is presented for comparison.
total), the spectrum of mutants arising on the lactose-treated plates was examined in detail (Table 4). From this analysis, the number of each type of sequence change contributed by spontaneous mutants in the GTN-treated sample could be calculated (Table 5). These values were then compared with the numbers of each type of revertant recovered from GTN-treated plates. The results indicated that two of the three sequence changes recovered (GC + TA and AT + CG) from the GTN-treated mutants could be accounted for as spontaneous mutations. The excess sequence changes over background were C + T transitions at either the first or second GC base pair in the triplet, the latter appearing among the greater proportion of mutants.
TABLE
5
SEQUENCE Treatment
GTN Expected
Because the vasodilatory effects of GTN are attributed to the generation of nitric oxide within the smooth muscle cells of the vasculature (Schror et al., 19911, the possible involvement of NO in the mechanism of GTN mutagenicity was examined. NO gas was reported to be mutagenic in Salmonella typhimurium TAlOO (Isomura et al., 19841, and to inhibit DNA synthesis in some cell types, as does GTN (Nakaki et al., 1990). The sequence changes found with GTN in the present report were also found with a nitric oxide-releasing spermine/NO complex [CAS Registry No. 136587-13-81 (Maragos et al., 1991). The latter compound generated only GC -+ AT transitions in TA1.535, predominantly at the middle GC base pair in the triplet (Wink et al., 1991). The similar-
CHANGES Number
IN GTN-INDUCED of colonies
VERSUS
with codon change
SPONTANEOUS
REVERTANTS
OF S. fyphimurium
TA1535
(CCC + 1
CTC
TCC
ACC
CAC
GCC
ccc
Unknown
Total
95
48
5
1
0
1
0
150
4.0
1.8
0.7
1.1
0
from spontaneous 17.6
a 2.6
27.8
a The numbers shown are those expected to arise from spontaneous reversions in the ?-plate GTN sample, as calculated from the lactose control data of Table 4. Lactose plates contained 14, 15, 11, 11, 11, 6 and 10 colonies (total 78 colonies/7 plates) of which 76 colonies (97%) were sampled. GTN plates contained 66, 58 and 57 colonies of which 150 were sampled (83%). The number of spontaneous colonies at each locus, per plate, was calculated as: (total number observed at each locus +0.97)+7 plates. The total number of expected spontaneous colonies at each locus in the GTN sample was calculated by multiplying the number of spontaneous colonies at each locus per plate by 3 (to adjust for the number of GTN plates) and by 0.83 (to adjust for the sampling frequency of GTN-induced colonies).
194 ity o f t h e s e m u t a n t s p e c t r a is c o n s i s t e n t with the h y p o t h e s i s t h a t nitric oxide m e d i a t e s the m u t a genicity of G T N . W h e t h e r this m u t a t i o n a l p a t t e r n is u n i q u e to N O will b e c o m e m o r e c e r t a i n as a d d i t i o n a l c o m p o u n d s a r e tested. N o n e of the 11 m u t a g e n s s t u d i e d by H e n r i k s o n et al. (1992) exh i b i t e d a m u t a t i o n a l p a t t e r n like that of G T N a n d the s p e r m i n e / N O c o m p l e x (uniquely G C - - , A T with a p r e f e r e n c e for the s e c o n d G C b a s e p a i r in the hisG46 G G G / C C C triplet). NM e t h y l - N - n i t r o s o u r e a , for e x a m p l e , e x h i b i t e d a strong ( 8 0 - 9 0 % ) p r e f e r e n c e for G C --* A T transitions in the first b a s e p a i r of t h e triplet. This result suggests t h a t an i n d i r e c t m e c h a n i s m involving N - n i t r o s a t i o n of a m e t h y l u r e a - t y p e comp o u n d , followed by O % m e t h y l a t i o n of g u a n i n e ( S u k u m a r et al., 1983), is an unlikely source of N O - m e d i a t e d m u t a g e n e s i s . A m e c h a n i s m involv-
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7o
.
.
.
.
.
.
60 50 40 30
o ~
20 10
z
o
, 0
, ' , ' ' 1 2 3 4 5 DOSE ( / z m o l / p l a t e )
' 6
Fig. 3. Mutagenicity of glyceryl trinitrate to S. typhimurium TA1535 in the presence (o) and absence (e) of 3.4 /zmol oxymyoglobin/plate, mean_+SD, number of replicate plates =4.
ing d e a m i n a t i o n of cytosine r e m a i n s the m o s t p l a u s i b l e b a s e d on available data. T o d e t e r m i n e t h e e x t e n t to which N O g e n e r a t e d e x t r a c e l l u l a r l y was r e s p o n s i b l e for G T N m u tagenicity, we t e s t e d t h e i n f l u e n c e of myoglobin, which reacts very r a p i d l y with nitric oxide yielding a stable n i t r o s y l - m y o g l o b i n c o m p l e x ( A n t o n i n i a n d Brunori, 1971). In the p r e s e n t w o r k 3 . 4 / x m o l o x y m y o g l o b i n / p l a t e did not s u b s t a n t i a l l y inhibit
m z o w z
,
,
80
60
4o
o
20
the m u t a g e n i c effect of up to 6 / x m o l G T N / p l a t e (Fig. 3). This i n d i c a t e s that t h e m u t a g e n i c i t y of G T N d o e s n o t result from e x p o s u r e to extracellular nitric oxide. D e s p i t e the w i d e s p r e a d use of glyceryl trinit r a t e as a vasodilator, its m u t a g e n i c i t y to S. typhimurium has, until now, not b e e n t h o r o u g h l y investigated. In p a r t this m a y stem from two factors, the r e q u i r e m e n t for large doses of G T N a n d the lack of susceptibility of s t a n d a r d S. typhimurium t e s t e r strains (i.e., T A 9 8 a n d T A 1 0 0 ) to its m u t a g e n i c action. T h e s e factors suggest t h a t t h e r e may b e c o n s i d e r a b l e variability in the vuln e r a b i l i t y to G T N - i n d u c e d m u t a g e n e s i s . T h e chronic toxicity and c a r c i n o g e n i c i t y o f G T N have b e e n e x a m i n e d in dogs, rats, a n d mice, with h e p a t o c e l l u l a r t u m o r s i n d u c e d in rats (Ellis et al., 1984). F u t u r e investigations in o u r l a b o r a t o r y will f u r t h e r e x p l o r e the effectiveness of G T N as an initiator of carcinogenesis.
o z
' , ' , ' , , , ' , ' , ' , 2 4 6 8 10 12
0
14
DOSE ( / ~ m o l / p l a t e ) Fig. 2. Histidine reversion of S. typhimurium TA1535 following treatment with glyceryl trinitrate in the presence (©) and absence (e) of hamster liver $9. Data were pooled from four experiments, number of replicate plates/data point= 15, mean_+SD. Values for the lactose controls were 10±5 (without $9)and 15_+4 (with $9).
Acknowledgements We Goebel Cebula colony
thank Corinthia Brown and Thomas for assistance with the assays, T h o m a s a n d W a l t e r K o c h for instruction in t h e h y b r i d i z a t i o n assay a n d for oligonu-
c l e o t i d e p r o b e s u s e d in t h e s e e x p e r i m e n t s , a n d C h a r l e s W. Riggs for assistance with t h e statisti-
195
cal analyses. Research was funded in part by the D e p a r t m e n t of Health and H u m a n Services u n der contract number NO1-CO-74102. References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test, Mutation Res., 31,347-364. Andrews, A.W., L.H. Thibault and W. Lijinsky (1978) The relationship between carcinogenicity and mutagenicity of some polynuclear hydrocarbons, Mutation Res., 51, 311318. Antonini, E., and M. Brunori (1971) Hemoglobin and myoglobin in their reactions with ligands, North-Holland Publishing Co., Amsterdam, pp. 19-33. Arroyo, P.L., V. Hatch-Pigott, H.F. Mower and R.V. Cooney (1992) Mutagenicity of nitric oxide and its inhibition by antioxidants, Mutation Res., 281, 193-202. Cebula, T.A., and W.H. Koch (1990) Analysis of spontaneous and psoralen-induced Salmonella typhimurium hisG46 revertants by oligodeoxyribonucleotide colony hybridization: use of psoralens to cross-link probes to target sequences, Mutation Res., 229, 79-87. Ellis, H.V., III, C.B. Hong, C.C. Lee, J.C. Dacre and J.P. Glennon (1984) Subacute and chronic toxicity studies of trinitroglycerin in dogs, rats, and mice, Fund. Appl. Toxicol., 4, 248-260. Hartman, P.E., B.N. Ames, J.R. Roth, W.M. Barnes and D.E. Levin (1986) Target sequences for mutagenesis in Salmonella histidine-requiring mutants, Environ. Mutagen., 8, 631-641. Henrikson, E.N., W.H. Koch and T.A. Cebula (1992) Base substitution specificity of hisG46 reversion in S. typhimurium strain TA100 uniquely characterizes several classes of mutagens, Environ. Molec. Mutagen., 19, supplement 26, 24. Hibbs, J.B., Jr., R.R. Taintor, Z. Vavrin and E.M. Rachlin (1988) Nitric oxide: a cytotoxic activated macrophage effector molecule, Biochem. Biophys. Res. Commun., 157, 87-94. Isomura, K., M. Chikahira, K. Teranishi and K. Hamada (1984) Induction of mutations and chromosome aberrations in lung cells following in vivo exposure of rats to nitrogen oxides, Mutation Res., 136, 119-125. Maragos, C.M., D. Morley, D.A. Wink, T.M. Dunams, J.E. Saavedra, A. Hoffman, A.A. Bove, L. Isaac, J.A. Hrabie and L.K. Keefer (1991) Complexes of -NO with nucleophiles as agents for the controlled biological release of nitric oxide. Vasorelaxant effects, J. Med. Chem., 34, 3242-3247.
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