109
Mutation Research, 48 (1977) 109--112 © Elsevier/North-HollandBiomedicalPress
Short communication MUTAGENICITY OF NEUTRAL RED
D A N I E L S. L O N G N E C K E R ,
THOMAS
J. C U R P H E Y
and D O U G L A S
S. D A N I E L
Department of Pathology, Dartmouth Medical School, Hanover, N.H. 03 755 (U.S.A.)
(Received May 4th, 1976) (Accepted September 16th, 1976)
Neutral red has long been used as a vital dye in experimental biology to study a wide variety of biological and biochemical processes [2]. In recent years neutral red has been used in the treatment of labial and genital herpes virus II infections in humans [3,5,6,12]. The wisdom of this clinical use has been questioned [ 7,11 ]. We have studied the pancreatic localization of neutral red, evaluating the compound as a possible carrier molecule for carcinogenic moieties in experimental pancreatic carcinogenesis [8]. Both the parent compound (as its free base) and an N-nitrososarcosyl derivative were assayed for mutagenicity in the Ames Salmonella typhimurium bacterial system [1]. The results are recorded here. Neutral red (2-amino-3-methyl-8-dimethylaminophenazine hydrochloride [4]) was obtained commercially (Fisher Certified Biological Stain, Fisher Scientific Company). Examination of this material by thin-layer chromatography (silica gel G, chloroform-methanol 15 : 1) revealed the presence of one major and at least eight minor impurities. A sample of purified neutral red free base was prepared as follows. Neutral red was partitioned between dichloromethane and aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated in vacuo. The resulting dark brown solid was recrystallized from methanol--isopropanol. Thin-layer chromatography showed the presence of only minor amounts of impurities in the recrystallized product. The preparation of the N-nitrososarcosyl derivative of neutral red (Fig. 1) and of its hydrochloride will be described elsewhere. The purified compounds were assayed for mutagenic activity in the Salmonella
~ (CH3)2N ~
N~ - ~ "
CH3
N~.,,.,~'. NHCOCH2N (NO)CH3
Fig. 1. N - n i t r o s o s a r c o s y l N e u t r a l Red.
110
typhimurium reversion system [1] using test strains TA 1535, 1536, 1537 and 1538 which were obtained from Dr. Ames. Increased numbers of revertant colonies were observed when strains TA 1537 and 1538 were incubated with neutral red free base in the presence of microsomal activation system prepared from rat liver (Table I). During another assay it appeared that strain TA 1537 was more sensitive to neutral red mutagenesis than TA 1538. Mutagenicity for neutral red free base, neutral red itself, and for the major impurity in commercial neutral red (thought to be 2-amino3-methyl-8-methylaminophenazine) has been confirmed in this system using strain TA 98 in the presence of a liver activating mixture [9]. These results suggest that neutral red is a frameshift mutagen. The N-nitrososarcosyl derivative of neutral red was consistently inactive as a mutagen in our assays. This suggests either (1) that the side chain may interfere with intercalation of the c o m p o u n d with DNA; or (2) that the amino group may be blocked by the side chain in a way which prevents metabolic activation -- perhaps by N-hydroxylation. McCann has independently suggested that activation at the amino group seems likely on the basis of the behavior of the c o m p o u n d in their tests [9]. In view of the observation that many mutagens detected by the Ames system are carcinogens [10], we believe that evaluation of the carcinogenic potential of neutral red in mammalian bioassay systems should be undertaken.
Acknowledgement This investigation was supported by the Division of Cancer Cause and Prevention, National Cancer Institute (Contracts No. E-73-3274 and E-723296).
TABLE I MUTAGENICITY TYPHIMURIUM
OF
NEUTRAL
Compound
RED
AND
Amount
ITS NITROSOSARCOSAMIDE
FOR
SALMONELLA
R e v e r t a n t s ( m u t a n t s ) per plate a
(~g)
TA 1535
.
TA 1536
TA 1537
TA 1538
7 10 I0 8
11 16 I0 17
9 397 11 14
22 434 22 32
Without activation None N e u t r a l red b N-nitxososarcosyl-neutral red b N-nitrososarcosyl-neutral red c
0 10 I0 10
None N e u t r a l red b N - n i t r o s o s a r c o s y l - n e u t r a l red b N - n i t r o s o s a r c o s y l - n e u t r a l red c
0 10 10 10
10 12 13 14
0 1 0 0
With m i c r o s o m a l a c t i v a t i o n 10 13 10 9
1 0 1 0
a T h e values given are the averages o f 4 r e p l i c a t e plates for e a c h c o m p o u n d t e s t e d . b As t h e free base. c As t h e h y d r o c h l o r i d e .
111
References 1 Ames, B.N., J. McCann, and E. Yamasaki, Methods for detecting carcinogens and mut a ge ns w i t h the S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e m u t a g e n i c i t y test, Mu t a t i on Res., 31 (1975) 3 4 7 ~ 3 6 4 . 2 Barbosa, P. and T.M. Peters, The effects of vital dyes on living organisms with specific reference to m e t h y l e n e blue and neutral red, Histochem. J., 3 (1971) 71--93. 3 Felber, T.D., E.B. Smith, J.M. Knox, C. wallis and J.L. Melnick, P h o t o d y n a m i c inactivation of herpes simplex: rep ort of a clinical trial, J. Amer. Med. Ass., 223 (1973) 289--292. 4 Fernando, J., W.S. Morgan and J.W. Haussar, Structure of neutral red and other 2,8-substituted aminophenazines, J. Org. Chem., 32 (1967) 1120---1123. 5 Friedrich, E.G., Relief for herpes vulvitis, Obstet. Gynecol., 41 (1973) 74--77. 6 Lefebvre, E.B. and E.E. McNellis, P h o t o i n a c t i v a t i o n of herpes simplex, J. Amer. Med. Ass., 224 (1973) 1039. 7 Li, J-L.H., M.A. J e r k o f s k y and F. Rapp, D e m o n s t r a t i o n of oncogenic p o t e n t i a l of m a m m a l i a n cells tr an sformed by DNA-containing viruses following p h o t o d y n a m i e inactivation, Int. J. Cancer, 15 (1975) 190--202. 8 Longnecker, D.S. and T.J. Curphey, Localization of exogenous polycyclic chemicals in the pancreas of rats, Fed eratio n Proceedings, 32 (1973) 826 abs. 9 McCann, J. and B.N. Ames, personal c o m m u n i c a t i o n . 10 McCann, J., E. Choi, E. Yamasaki and B.N. Ames, Detection of carcinogens as mutagens in the S a l m o n e l l a / m i c r o s o m c test: part I, assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.), 72 (1975) 5135--5139. 11 Rapp, F., H-L.H. Li and M. Jerkofsky, Transformation of m a m m a l i a n cells by DNA-contalning viruses following p h o t o d y n a m i c inactivation, Virology, 55 (1973) 339--346. 12 Wanis, C., J.L. Melnick and R.H. Kaufman, Herpes genitalis management, present and predicted, Clin. Obstet. Gynecol., 15 (1972) 939--947.
Mutation Research, 48 (1977) 109--112 © Elsevier/North-HollandBiomedicalPress
Short communication MUTAGENICITY OF NEUTRAL RED
D A N I E L S. L O N G N E C K E R ,
THOMAS
J. C U R P H E Y
and D O U G L A S
S. D A N I E L
Department of Pathology, Dartmouth Medical School, Hanover, N.H. 03 755 (U.S.A.)
(Received May 4th, 1976) (Accepted September 16th, 1976)
Neutral red has long been used as a vital dye in experimental biology to study a wide variety of biological and biochemical processes [2]. In recent years neutral red has been used in the treatment of labial and genital herpes virus II infections in humans [3,5,6,12]. The wisdom of this clinical use has been questioned [ 7,11 ]. We have studied the pancreatic localization of neutral red, evaluating the compound as a possible carrier molecule for carcinogenic moieties in experimental pancreatic carcinogenesis [8]. Both the parent compound (as its free base) and an N-nitrososarcosyl derivative were assayed for mutagenicity in the Ames Salmonella typhimurium bacterial system [1]. The results are recorded here. Neutral red (2-amino-3-methyl-8-dimethylaminophenazine hydrochloride [4]) was obtained commercially (Fisher Certified Biological Stain, Fisher Scientific Company). Examination of this material by thin-layer chromatography (silica gel G, chloroform-methanol 15 : 1) revealed the presence of one major and at least eight minor impurities. A sample of purified neutral red free base was prepared as follows. Neutral red was partitioned between dichloromethane and aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated in vacuo. The resulting dark brown solid was recrystallized from methanol--isopropanol. Thin-layer chromatography showed the presence of only minor amounts of impurities in the recrystallized product. The preparation of the N-nitrososarcosyl derivative of neutral red (Fig. 1) and of its hydrochloride will be described elsewhere. The purified compounds were assayed for mutagenic activity in the Salmonella
~ (CH3)2N ~
N~ - ~ "
CH3
N~.,,.,~'. NHCOCH2N (NO)CH3
Fig. 1. N - n i t r o s o s a r c o s y l N e u t r a l Red.
110
typhimurium reversion system [1] using test strains TA 1535, 1536, 1537 and 1538 which were obtained from Dr. Ames. Increased numbers of revertant colonies were observed when strains TA 1537 and 1538 were incubated with neutral red free base in the presence of microsomal activation system prepared from rat liver (Table I). During another assay it appeared that strain TA 1537 was more sensitive to neutral red mutagenesis than TA 1538. Mutagenicity for neutral red free base, neutral red itself, and for the major impurity in commercial neutral red (thought to be 2-amino3-methyl-8-methylaminophenazine) has been confirmed in this system using strain TA 98 in the presence of a liver activating mixture [9]. These results suggest that neutral red is a frameshift mutagen. The N-nitrososarcosyl derivative of neutral red was consistently inactive as a mutagen in our assays. This suggests either (1) that the side chain may interfere with intercalation of the c o m p o u n d with DNA; or (2) that the amino group may be blocked by the side chain in a way which prevents metabolic activation -- perhaps by N-hydroxylation. McCann has independently suggested that activation at the amino group seems likely on the basis of the behavior of the c o m p o u n d in their tests [9]. In view of the observation that many mutagens detected by the Ames system are carcinogens [10], we believe that evaluation of the carcinogenic potential of neutral red in mammalian bioassay systems should be undertaken.
Acknowledgement This investigation was supported by the Division of Cancer Cause and Prevention, National Cancer Institute (Contracts No. E-73-3274 and E-723296).
TABLE I MUTAGENICITY TYPHIMURIUM
OF
NEUTRAL
Compound
RED
AND
Amount
ITS NITROSOSARCOSAMIDE
FOR
SALMONELLA
R e v e r t a n t s ( m u t a n t s ) per plate a
(~g)
TA 1535
.
TA 1536
TA 1537
TA 1538
7 10 I0 8
11 16 I0 17
9 397 11 14
22 434 22 32
Without activation None N e u t r a l red b N-nitxososarcosyl-neutral red b N-nitrososarcosyl-neutral red c
0 10 I0 10
None N e u t r a l red b N - n i t r o s o s a r c o s y l - n e u t r a l red b N - n i t r o s o s a r c o s y l - n e u t r a l red c
0 10 10 10
10 12 13 14
0 1 0 0
With m i c r o s o m a l a c t i v a t i o n 10 13 10 9
1 0 1 0
a T h e values given are the averages o f 4 r e p l i c a t e plates for e a c h c o m p o u n d t e s t e d . b As t h e free base. c As t h e h y d r o c h l o r i d e .
111
References 1 Ames, B.N., J. McCann, and E. Yamasaki, Methods for detecting carcinogens and mut a ge ns w i t h the S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e m u t a g e n i c i t y test, Mu t a t i on Res., 31 (1975) 3 4 7 ~ 3 6 4 . 2 Barbosa, P. and T.M. Peters, The effects of vital dyes on living organisms with specific reference to m e t h y l e n e blue and neutral red, Histochem. J., 3 (1971) 71--93. 3 Felber, T.D., E.B. Smith, J.M. Knox, C. wallis and J.L. Melnick, P h o t o d y n a m i c inactivation of herpes simplex: rep ort of a clinical trial, J. Amer. Med. Ass., 223 (1973) 289--292. 4 Fernando, J., W.S. Morgan and J.W. Haussar, Structure of neutral red and other 2,8-substituted aminophenazines, J. Org. Chem., 32 (1967) 1120---1123. 5 Friedrich, E.G., Relief for herpes vulvitis, Obstet. Gynecol., 41 (1973) 74--77. 6 Lefebvre, E.B. and E.E. McNellis, P h o t o i n a c t i v a t i o n of herpes simplex, J. Amer. Med. Ass., 224 (1973) 1039. 7 Li, J-L.H., M.A. J e r k o f s k y and F. Rapp, D e m o n s t r a t i o n of oncogenic p o t e n t i a l of m a m m a l i a n cells tr an sformed by DNA-containing viruses following p h o t o d y n a m i e inactivation, Int. J. Cancer, 15 (1975) 190--202. 8 Longnecker, D.S. and T.J. Curphey, Localization of exogenous polycyclic chemicals in the pancreas of rats, Fed eratio n Proceedings, 32 (1973) 826 abs. 9 McCann, J. and B.N. Ames, personal c o m m u n i c a t i o n . 10 McCann, J., E. Choi, E. Yamasaki and B.N. Ames, Detection of carcinogens as mutagens in the S a l m o n e l l a / m i c r o s o m c test: part I, assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.), 72 (1975) 5135--5139. 11 Rapp, F., H-L.H. Li and M. Jerkofsky, Transformation of m a m m a l i a n cells by DNA-contalning viruses following p h o t o d y n a m i c inactivation, Virology, 55 (1973) 339--346. 12 Wanis, C., J.L. Melnick and R.H. Kaufman, Herpes genitalis management, present and predicted, Clin. Obstet. Gynecol., 15 (1972) 939--947.
