Chem. Res. Toxicol. 1994, 7, 559-567
559
Mutagenicity and Cytotoxicity of Naphthoquinones for Ames Salmonella Tester Strains Atsushi Hakura,* Hisatoshi Mochida, Yoshie Tsutsui, and Kiyomi Yamatsu Department of Drug Safety Research, Eisai Co., Ltd., 1-3 Tokodai, 5-chome, Tsukuba-shi, Ibaraki 300-26, Japan Received October 14, 1 9 9 9
The molecular mechanisms involved in quinone cytotoxicity, especially mutagenicity, are still largely unknown. In order to better understand the molecular aspects of the mechanisms of quinone mutagenicity and cytotoxicity, we examined them by using a series of 13simple structural naphthoquinone (NQ) derivatives for 9 Ames Salmonella mutagenicity tester strains in the presence or absence of liver homogenate S9 mix from rats induced with phenobarbital and 5,6-benzoflavone. Most NQs used in this study showed mutagenicity with and/or without S9 mix. The most potent mutagenic NQ was 2,3-dichloro-l,4-NQ,with mutagenicity of 18 induced revertants/nmol/plate for strain TA104 without S9 mix. Among the strains used, TA104, which is sensitive to oxidative mutagens, was the most sensitive to the NQs, and the second most sensitive strain was TA2637, which detects bulky DNA adducts. The relationship of mutagenic potency to the one-electron reduction potential with TA104 suggested that the higher redox potential NQs were more mutagenic than the lower redox potential NQs. Significant reduction of the mutagenicity of 1,4-naphthoquinone without S9 mix was observed in the presence of catalase. Enhancement of the mutagenic potential of the NQs by the pKMlOl plasmid implicated in error-prone repair was also observed. The most cytotoxic NQ was 2,3-dichloro-5,8-dihydroxy1,4-NQ, and the least cytotoxic NQ was P-NQ-4-sulfonic acid potassium salt, a 700-fold range in potency. The cytotoxic effect of the NQs was largely dependent on the structures of their substituents. It was suggested that the higher redox potential NQs were more cytotoxic than the lower redox potential NQs for all of the strains used, in contrast to the mutagenicity of the NQs. The presence of S9 mix decreased the cytotoxic effect of the NQs, the extent of which was also largely dependent on the structures of their substituents and is in accordance with the order of the height of the one-electron reduction potentials. These results indicate that the mutagenicity of NQs in Salmonella typhimurium was due to oxidative damage produced with activated oxygen species such as hydroxy radical and superoxide anion radical, which are generated as a result of the reduction of the NQs, and to bulky NQ-DNA adducts accounting for their electrophilic property, whose contribution was largely dependent on the substituents of NQs. On the other hand, it was suggested that cytotoxicity of NQs for S. typhimurium was mainly derived from oxidative damage.
Introduction Quinones are widely distributed in nature as the biological components of the electron transport chains involved in cellular respiration and photosynthesis, and as environmental pollutants such as diesel exhaust and tobacco smoke (1-3). In addition, quinone metabolites have been proposed as being involved in the toxicity and the carcinogenicity of many different chemicals. Examples include naphthoquinone (NQ),' p-benzoquinone, tertbutylbenzoquinone,and phenylbenzoquinone,which are metabolites of l-naphthol(4,5),benzene (6-91,butylated
* Correspondence should be addreseed to thie author at the Department of Drug Safety Research, Eisai Co.,LM.1-3 Tokodai, 5-chome,Tsukubashi, Ibaraki 300-26, Japan. *Abstract published in Advance ACS Abstracts, June 1, 1994. Abbreviations: NQ, naphthoquinone; p-NQ-4-SOsK, 8-naphthoquinone-4-sulfonic acid potassium salt; lawsone, 2-hydroxy-1,4-naphnaphthazaline, thoquinone; menadione, 2-methyl-1,4-naphthoquinone; 5,&dihy&oxy-l,4naphthoqujnone; 1,2-NQ, 1,2-naphthoquinone;4-amino1,4-NQ, 1,4naphthoquinone;lapa1,2-NQ,4-amino-1,2-naphthoquinone; 2-anilinochol, 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone; plumbagin, 5-hydroxy-2-methyl1,4-NQ,2-anilino-l,4-naphthoquinone; 1,4naphthoquinone;dichlone, 2,bdichloro-1,4naphthoquinone;2,3-diC12,35,8-diOH-1,4-NQ,2,3-dichloro-5,8-dihydroxy-l,4-naphthoquinone; diC1-NT, 2,3-dichloro-l,4,6,8-naphthalenetetrone;SOD, superoxide dismutase; BHA, butylated hydroxyanisole; OPP, o-phenylphenol;XA, xanthine; XAO, xanthine oxidase.
hydroxyanisole (BHA)l (IO),and o-phenylphenol(OPP)' (11, 12), respectively. Compounds containing a quinone nucleus also constitute an important class of anticancer agents such as adriamycin and mitomycin C (1-3). Since humans are exposed to quinones at such a magnitude, and quinone nucleus-containing anticancer agents are important for clinical use, much attention has been paid to the mechanisms of toxicity of quinones in mammalian cells. In general, quinones are likely to be toxic via one of two mechanisms: the production of oxidative stress through redox cycling or the direct arylation of nucleophilic sulfhydryl groups (1-3). One mechanism of cytocidal activity of anticancer agents containing a quinone nucleus is thought to be due to a mechanism involving active oxygen species via flavoprotein reduction enzymes (13-15). Despite the many studies on the cytotoxicityof quinones, the mutagenicity of quinones is much less well studied (3, 16-20). One report focused on the oxygen radicals in mutagenesis by quinones. Thus, Chesis et al. concluded that the mutagenicity of quinones was mainly due to oneelectron reduction of quinones to semiquinones via the formation of 0 2 ' - (superoxide anion radical) and, subsequently, HzO2 (161, while Tikkanen et al. observed that
0893-228~/94/2707-0559$04.50/0 0 1994 American Chemical Society
560 Chem. Res. Toxicol., Vol. 7, No. 4, 1994
Hakura et al.
dichlone (-5)
naphthazalin
2,3-diCI-NT
plumbagin (-135)
OH
0
AK
1.4-NQ
1,2-NQ
(-160)
(-45)
(-50)
menadione or vitamin K3
0
0
ii q
C ,I
-0
h
CH,-CH=C-CH3 OH
4-amino-l,2-NQ
(-240)
0
0
2,3-diCI-5,8-diOH-1,4-NQ
CH3
*NHD lapachol
o=s=o
0
lawsone
I OK
B -NQ-4-SOSK
0
P-anilino-l,4-NQ
Figure 1. Structures of the 13 structurally simple NQs used. The figures in parentheses indicate one-electron reduction potentials E(Q/Q*-)(mV),whose values were obtained in 0.1 M tetraethylammonium perchlorate aqueous solution [HzO:N,N-dimethylformamide = 1:l (v/v)l at 25 "C. Table 1. Characteristics of 9 S. typhimurium TA Strains Used for the Ames Test Tester strain Histidine mutation Strains sensitive to base-pair substitution mutagens TA1535
hisC46
TAloO
hisG46
(proline) -GGG-\+ -ccc-
Repair
(ieucine)
R-factor
uvrB-
-Ra
uvrB-
+R
uvrBuvrB'
-R +R
;GiZ-}
Strains sensitive to frameshift mutagens 8 repetitive -GC- residues TA1538 hisD3052 TA98 hisD3052 -GCGCGCGC+1 (wild) CGCGCGCG-
-+
uvrBuvrB-
-R +R
6 repetitive -GC residues uvrB' CCCCCC-1 (pseudo wild type) -GGGGGG-4 (wild tV!Je) Strains sensitive IO oxidative mutagens hisG428 ochre mutation wild (gluramine) TAlO2 on pAQl plasmid wild type on chromosome uv,-BTA104
+R
TA1537 TA2637
hisC3076 hid3076
not sequenced yet -CC(C).CC- j -1 (wild) -GG(G),GG-
i
(n,O) TA97
hisD6610
+
1+=
}:E::
+R +R
+small deletion + exuaeenic ochre S U D P ~ C S S O ~ a R = plasmid pKM101, in which the muc AB gene implicated in error-prone repair system is encoded.
six naturally occurring NQs, including menadione, were mutagenic in t h e presence of S9 mix for strain TA2637, which is sensitive t o frameshift mutagens (17). This indicates that further study is needed t o elucidate quinone mutagenicity. T o better understand quinone mutagenicity, we examined t h e mutagenicity of a series of 13 simple structural N Q derivatives using 9 Ames Salmonella mutagenicity tester strains with or without r a t liver homogenate S9 mix. T h e Ames test also has t h e potential t o elucidate not only
mutagenic activity but also cytotoxic activity (23,241. In this article, mutagenic as well as cytotoxic data concerning the NQs are summarized, a n d t h e mechanisms of t h e mutagenicity and cytotoxicity of NQs for t h e Ames Salmonella bacterial mutagenicity tester strains are discussed.
Materials and Methods Test Compounds. Caution: The following chemicals are very toxic, and most of them are mutagens. Therefore, they acid were handled with care. fi-Naphthoquinone-4-sulfonic potassium salt (fi-NQ-4-SOa)(1),2-hydroxy-l,Cnaphthoquinone (lawsone) (2), 2-methyl-l,4-naphthoquinone(menadione, or vitamin Ka) (3), and 5,8-dihydroxy-l,4-naphthoquinone (naphthazaline) (4) were purchased from Tokyo Kasei (Tokyo,Japan). 1,2-Naphthoquinone (1,2-NQ) (6),4-amino-l,2-naphthoquinone (4-amino-1,a-NQ) (6), 1,4-naphthoquinone (1,4-NQ) (7), 2-hydroxy-3-(3-methyl-2-butenyl)-l,4-naphthoquinone (lapachol)(S), 2-anilino-l,4-naphthoquinone (2-aniline1,4-NQ)(9), 5-hydroxy2-methyl-1,4-naphthoquinone (plumbagin) (lo), 2,3-dichloro-1,4naphthoquinone (dichlone) (1l),2,3-dichloro-5,8-dihydroxy-1,4naphthoquinone (2,3-diC1-5,8-diOH-1,4-NQ)(12),2,3-dichloro1,4,5,8-naphthalenetetrone(2,s-diCl-NT) (13), and dicoumarol were obtained from Aldrich (Milwaukee, WI). Superoxide dismutase (SOD), catalase, xanthine oxidase (XAO), xanthine (XA), and NADPH were purchased from Sigma (St. Louis, MO). Hydrogen peroxide, N,N-dimethylformamide, and tetraethylammonium perchlorate were obtained from Wako (Osaka, Japan). Figure 1shows the structures of the NQs used in this study. Five-tenths of a milliliter of S9 mix consisted of 0.05 mL of S9 and 0.45 mL of a solution containing cofactors. S9 was the supernatant fraction obtained by centrifugation, at 9OOOg for 10 min, of liver homogenate from male Sprague-Dawley rata pretreated with phenobarbital and 5,6-benzoflavone, and the components of the cofactors were 8 mM MgC12, 33 mM KCl, 5 mM glucose 6-phosphate, 4 mM NADPH, 4 mM NADH, and 100
Chem. Res. Toxicol., Vol. 7, No. 4, 1994 561
Mutagenicity and Cytotoxicity of Quinones
~~
Table 2. Mutagenicity. of 13 Structurally Simple NQs for 9 S. typhimurium TA Strains* in the Absence and in the Presence of S9 Mix bacterial tester strain (induced reve&ts/nmol/plata) compound S9mix TA1535 TAlOO TA1538 TA98 TA1537 TA2637 TA97 TA102 TA104 6.1 18 dichlone ~
2,3-diCl-NT
2,3-diC1-5,7-diOH-1,4-NQ
-
1,2-NQ
naphthazalin 1,4-NQ
plumbagin menadione lawsone
-
-
-
8.1 0.32 1.5 0.19 2.7 0.32
-
0.91 3.9 0.8 0.78 3.8 0.95 0.76 1.9 0.32 1.1 0.94 0.52 0.17
-
-
-
0.56 0.34
-
4-amino-1,P-NQ 2-anilino-1,CNQ
0.68
0.18 0.18
0.18 1.3
