Mutation Research, 298 (1992) 17-23
17
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1218/92/$05.00
MUTGEN 01822
Assessment of the genotoxic potential of riboflavin and lumiflavin B. Effect of light Hema Kale, P. Harikumar, S.B. Kulkarni, P.M. Nair and M.S. Netrawali Food Technology and Enzyme Engineering Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India
(Received 28 January 1992) (Revision received 13 May 1992) (Accepted 20 May 1992)
Keywords: Ribofavin; Lumiflavin;Photomutagenicity;Singlet oxygen;Superoxide radicals
Summary On exposure to visible light, riboflavin and lumiflavin produced reactive oxygen species such as singlet oxygen and superoxide radicals. The reaction was found to be time- and concentration-dependent. Both riboflavin and lumiflavin, upon illumination, showed mutagenic response in the u m u test as well as in the Ames/Salmonella assay with Salmonella typhimurium TA102. The mutagenic response was partially abolished by superoxide dismutase while sodium azide did not have any effect. No mutagenicity was observed if the compounds were not illuminated. The results suggested the involvement of superoxide radicals in light-induced mutagenicity of riboflavin as well as lumiflavin.
Exposure of chemicals to light can lead to generation of reactive oxygen species (Vuillaume, 1987) which in turn can induce mutagenic i n t e r actions (Hassan and Moody, 1984; Joenje, 1989). Riboflavin, commonly used as a colourant in milk products, icecreams, candies, etc., and as a fortificant in bread preparations, has been shown to generate reactive oxygen species (Srivastava et al., 1986). There has also been indirect evidence to indicate that when exposed to light, riboflavin induces genotoxic (Bradley and Sharkey, 1977;
Correspondence: Dr. P. Harikumar, Food Technology and Enzyme Engineering Division, Bhabha Atomic Research Centre, Trombay, Bombay400 085, India.
Griffin et al., 1981) and cytotoxic interactions (Misra et al., 1987; Misra, 1990; Wang, 1975). However, the precise mechanism by which riboflavin induces toxic effects in the presence of light is not well understood. In the present investigation the i n f u e n c e of visible light on the mutagenic potential of riboflavin and its stable photodegradation product lumiflavin was studied.
Materials and methods Riboflavin which was found to be chromatographically pure was a kind gift from Glindia Ltd. (Bombay, India). Lumiflavin, nalidixic acid, mitomycin C, L-histidine. HC1, biotin, p-nitrophenylphosphate (PNPP), O-nitrophenyl-/3-D-galacto-
18 pyranoside (ONPG), nitroblue tetrazolium (NBT), nitroblue diformazan, N,N'-dimethyl-p-nitrosoaniline (RNO), superoxide dismutase of bovine erythrocytes (SOD), sodium azide, trishydroxymethyl aminomethane (Tris) and sodium dodecyl sulfate (SDS) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Methylene blue and metanil yellow were obtained from Aldrich Chemical Company (Milwaukee, WI, USA). All other chemicals and solvents were of A R grade and solvents were distilled before use. Tester strain Salmonella typhimurium TA102 was provided by Prof. Bruce N. Ames (University of California, Berkeley, CA, USA). Salmonella typhimurium TA1535/pSK1002 was obtained from Dr. Yoshimitsu Oda (Osaka Prefectural Institute of Public Health, Nakamachi-1, Japan). Characteristics of the strains were confirmed by the procedure of Maron and Ames (1983). Working stock cultures of TA102 and T A 1 5 3 5 / pSK1002 were stored on nutrient agar slants and Luria agar slant respectively and held at 0-4°C until use.
Mutagenicity assays (1) Umu test. The general screening of mutagenicity of riboflavin and iumiflavin was carried out using the umu test (Oda et al., 1985). The system consisted of 0-100 /xg/ml of test compounds, 2.5 ml of exponentially growing cells (OD~,00 adjusted to 0.25-0.3) and 33 mM phosphate buffer, p H 7.4. The mixture was incubated at 37°C for 180 min and was centrifuged at 3020 × g for 10 rain. The pellet was washed 3 times with 0.1 M phosphate buffer, pH 7.4, resuspended in 3 ml of the same buffer and 00600 was determined. 0.2 ml of this suspension was added to 1.8 ml of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCI and 1 mM MgSO4, pH 7) followed by 50/~1 SDS (0.1%) and 10 p.l chloroform. After mixing it thoroughly, 0.2 ml O N P G solution (4 m g / m l in 0.1 M phosphate buffer, pH 7) was added to the reaction mixture and incubated at 28°C for 20 rain. The reaction was stopped by the addition of 1 ml of 1 M Na2CO 3 and the absorbances at 420 nm and 550 nm were determined. The activity of /3-galactosidase was calculated according to Miller (1972).
(2) Ames / Salmonella assay. The liquid preincubation procedure was adopted for determining the mutagenicity of riboflavin and lumiflavin (Maron and Ames, 1983). The incubation mixture consisting of 0.2 ml of a 16-18-h-old culture of tester strains (Salmonella typhimurium TA100, TA98 and TA97a), 0-100 /~g/ml test compounds and 0.1 ml PBS, pH 7.4, was incubated at 37°C for 30 rain and centrifuged at 3020 × g for 10 rain. The pellet was resuspended in 0.4 ml of 0.1 M PBS, pH 7.4. To this suspension 2 ml molten soft agar was added, mixed rapidly and spread immediately on preset minimal agar plates. Effect of light on mutagenicity To study the effect of light on the mutagenic potential of riboflavin and lumiflavin in the umu test and the Ames/Salmonella assay, identical procedures were followed except that the incubation mixture was exposed to light as detailed below. Illumination of samples for umu test An incubation mixture consisting of 0-100 ~ g / m l test compounds, 2.5 ml of exponentially growing cells and 33 mM phosphate buffer, pH 7, was held at 37°C for 30 min and then exposed to visible light in a petri plate covered with a lid, using a Philips 'Ultraphill' MLU, 300 W, 220-240 V, F.28 sunlamp with a glass filter (0.5 mm thickness). The filter was used to isolate specific effects of the radiation regime at 360-400 nm and to avoid possible effects of stray UV light. A black-ray J221 UV intensity meter which was designed and calibrated at 365 nm with a standard 365-nm detector was used to determine the energy of emitted light. For examining the effect of light in the Ames/Salmonella assay a similar procedure was followed except that the incubation mixture consisted of 0.2 ml of exponentially growing cells and 0.1 ml of phosphate-buffered saline, pH 7.4. Quantitation of reactit:e oxygen species Singlet oxygen (IO 2) was quantitated using a calorimetric procedure (Kraljick and Mohisni, 1978). The optical density at 440 nm of a 10-ml aliquot of RNO solution in 0.1 M phosphate buffer, pH 7.0, containing 1 mM histidine was
19
0.0
400
i
0.6
g
o.~ 0.2
/
,::" "'"
3oo" -
,,,,
g20c-
,~"
°
1oo- .,~;~,
0
I0 20 30 0 I0 20 30 ILLUMINATION (MIN) ILLUMINATION (MIN) Fig. 1. Effect of illumination time on the formation of (A) singlet oxygen and (B) superoxide radicals from riboflavin and lumifavin. B, Riboflavin; [3, riboflavin + quencher; A, lumiflavin; zx, lumiflavin + quencher.
procedure of Korycka-Dahl and Richardson (1978). A 10-ml aliquot o f 1.67 m M n i t r o b l u e t e t r a z o l i u m in 0.1 M c a r b o n a t e buffer, p H 10, was i l l u m i n a t e d at 8 J / m 2 in t h e p r e s e n c e of riboflavin or lumiflavin. T h e p r e c i p i t a t e of nitrob l u e d i f o r m a z a n was s e p a r a t e d by c e n t r i f u g a t i o n at 3020 × g for 10 rain, w a s h e d once with distilled w a t e r a n d dissolved in 3 ml of d i m e t h y l form-
a d j u s t e d to 1.2-1.4 on B a u s c h - L o m b s p e c t r o p h o t o m e t e r . T h e r e a c t i o n mixture was i l l u m i n a t e d with a s u n l a m p at 8 J / m 2 in the p r e s e n c e of riboflavin o r lumiflavin a n d t h e d e c r e a s e in optical d e n s i t y at 440 n m was d e t e r m i n e d . 1 m M s o d i u m a z i d e was u s e d to q u e n c h singlet oxygen (Srivastava et al., 1985). S u p e r o x i d e r a d i c a l s w e r e q u a n t i t a t e d by t h e
& o.e-
A
400-- B
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-
aE 0-6
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,~ o.4
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100--
//~t¢ /P•/
;;l(
r -~ 10 20 30 0 10 20 30 C O N C E N T R A T I O N (juglml) CONCENTRATION (./ug / ml) Fig. 2. Effect of concentration on the formation of (A) singlet oxygen and (B) superoxide radicals from ribofavin and lumiflavin, l , Riboflavin; rq, riboflavin + quencher; A, lumiflavin; zx, lumiflavin + quencher. 0
20 amide. T h e absorbance was d e t e r m i n e d at 360 nm. Superoxide radicals were q u e n c h e d by using 40 u n i t s / m l of S O D (Srivastava et al., 1985). Results and discussion
D a t a p r e s e n t e d in this p a p e r shows that both riboflavin and lumiflavin cause mutagenicity in the presence of visible light due to production of reactive oxygen species. As shown in Figs. 1 and 2, riboflavin and its p h o t o d e g r a d a t i o n p r o d u c t lumiflavin p r o d u c e singlet oxygen ( 1 0 2) and superoxide radicals ( 0 2 ) . G e n e r a t i o n of both these reactive oxygen species was f o u n d to be dependent on time of exposure (Fig. 1) and concentration of the c o m p o u n d s (Fig. 2). W h e n illumination time was increased from 0 to 30 min O D at 440 nm c h a n g e d from 0 to 0.3 for riboflavin and from 0 to 0.8 for lumiflavin. I n c o r p o r a t i o n of sodium azide in the above reaction system was found to q u e n c h the singlet oxygen formation by riboflavin by 86%. T h e c o r r e s p o n d i n g value for lumiflavin was f o u n d to be 81%. As the concentration of riboflavin and lumiflavin was increased, formation of singlet oxygen was also found to increase in a d o s e - d e p e n d e n t manner. Lumiflavin was f o u n d to be more efficient in generating singlet oxygen than riboflavin.
In addition to singlet oxygen, riboflavin and lumiflavin generated superoxide radicals. Riboflavin (1 # M ) p r o d u c e d 0.0011 ~ M superoxide radicals while 1 p~M lumiflavin f o r m e d 0.0094 /~M superoxide radicals within 30 rain of illumination. H e r e again lumiflavin was found to be more efficient than riboflavin in forming 0 2 . Superoxide dismutase was found to quench superoxide radical formation by 88% and 90% for riboflavin and [umiflavin respectively. These resuits clearly show that both riboflavin and lumiflavin generate singlet oxygen and superoxide radicals on exposure to visible light. Such interactions of the vitamin with electromagnetic radiations have been shown by Korycka-Dahl and R i c h a r d s o n (1980) and Srivastava et al. (1986). Lumiflavin has also been shown to be photo-excited to the activated state. However, there is no direct evidence on the formation or quantitative assessment of singlet oxygen and superoxide radicals on exposure to visible light. Activated oxygen species such as 10 2 and 0 2 as well as hydrogen peroxide, hydroxyl radicals, etc., are known to contribute towards the degradation of D N A , R N A , proteins, lipids and other target molecules (Krinsky, 1977; Linding and Rodgers, 1981; Joshi, 1985; A d e l m a n et al., 1988). T h e y also act a s , g e n o t o x i n s (Hsie et al., 1986;
TABLE 1 PHOTOMUTAGENICITY OF RIBOFLAVIN AND LUMIFLAVIN WITH THE UMU TEST Compound (~g/ml)
/3-Galactosidase activity (Units/OD6o o) 1
2
3
4
0
112.00_+ 15.87
110.10_+17.23
115.60+ 17.23
110.90_+ 5.43
Riboflavin 25 50 100
110.23_+ 7.88 116.80 +_28.70 118.50 + 7.85
120.90_+ 9.10 126.80 -+ 2.30 221.20 -+14.80
120.60_+ 1 0 . 5 0 162.50 -+52.60 223.90 _+20.36
110.70_+35.19 140.20 + 52.80 156.40 _+39.60
Lumiflavin 25 50 100
110.50 _+ 4.76 117.30 _+ 6.39 128.20 -+39.00
179.30 + 19.90 200.10 _+20.10 246.00 -+24.20
180.90 _+19.00 201.00 +_37.30 248.10 -+43.39
123.80 + 20.00 142.60 + 40.20 159.00 _+29.50
Values represent mean -+ SD of 3 independent experiments. 1, Ambient light conditions; 2, illuminated for 5 rain at 20 j/m2; 3, illumination in the presence of sodium azide; 4, illumination in the presence of superoxide dismutase.
21
Spitz et al., 1988) and tumour promoters (Weitzman et al., 1985; Vuillaume et al., 1987). In the case of riboflavin also, photooxidation of DNA (Speck et al., 1976; Korycka-Dahl and Richardson, 1980; Alvi et al., 1984), purines and pyrimidines (Uehara et al., 1966; Kuratomi and Kobayashi, 1977), proteins and amino acids (Byrom and Turnbull, 1967), has been indicated. Riboflavin induces cytotoxic effects in Paramecium (Misra et al., 1987), Tetrahymena (Misra et al., 1990) as well as in human and animal cells in tissue culture (Bradley and Sharkey, 1977). It has also been shown to cause mutations in microorganisms (Webb et al., 1967) and in cultured mammalian cells (Bradley and Sharkey, 1977; Webb and Malina, 1977; Griffin et al., 1981). However, these studies do not provide evidence for the involvement of either reactive oxygen species or one or more of the photodegradation products of riboflavin. Data presented in Tables 1 and 2 confirm the earlier observations of the genotoxic potential of riboflavin on exposure to light. Additionally, they also demonstrate the mutagenic response of lumiflavin, and the involvement of superoxide radicals in the mutagenicity of these compounds. Table 1 presents the data on the evaluation of the compounds in the u m u test. Riboflavin and lumiflavin caused an increase in/3-galactosidase activity to 2 and 2.2 times respectively following exposure to
visible light. In the presence of sodium azide the mutagenic response was unaltered while in the presence of superoxide dismutase it was reduced to 1.4 times for both riboflavin and lumiflavin. Table 2 incorporates data on the photomutagenicity of both compounds evaluated using the Ames/Salmonella assay with Salmonella typ h i m u r i u m TA102. In the presence of illuminated riboflavin and lumiflavin the histidine revertants of TA102 increased by 2.2 and 3 times respectively. This photoactivation was inhibited by superoxide dismutase while sodium azide did not exert any influence. A relatively better response in the Ames/Salmonella assay than in the u m u test could be explained on the basis of the generalised nature of the u m u test (Oda et al., 1985) and the specificity of Salmonella typhimurium strain TA102 towards oxidative mutagens (Levin et al., 1982). Our results on the non-interfering nature of singlet oxygen are in agreement with earlier reports (Dahl et al., 1987, 1988). Singlet oxygen is reactive towards cellular products other than DNA and is consequently not involved in bacterial DNA damage (Straight and Spike, 1985; Dahl et al., 1987). The data show the partial involvement of superoxide radicals in the mutagenic response. Such radical species have been shown to contribute to mutagenicity (Fiala et al., 1987). Photogenerated O 2 has been shown to undergo secondary reactions to form hydrogen
TABLE 2 P H O T O M U T A G E N I C I T Y OF RIBOFLAVIN A N D LUMIFLAVIN W I T H T H E A M E S / S A L M O N E L L A ASSAY Compound (/xg/ml)
Histidine revertants/plate 1
2
3
4
0
220.80 _+37.80
219.10 + 26.20
220.70 + 45.80
240.22 + 31.68
Ribofavin 25 50 100
229.00 +_26.50 218.04 + 23.35 217.50 _+28.20
301.00 + 37.10 410.70 + 33.20 503.90 + 41.40
301.20 _+33.50 410.60 + 37.30 504.00 + 23.80
231.20 + 13.07 260.80 _+28.80 334.20 _+43.00
Lumiflavin 25 50 100
224.50 + 62.60 320.10 + 23.30 221.60 + 7.36
320.60 + 29.80 496.80 + 56.20 657.30__. 41.17
328.10 + 25.80 509.20 + 46.30 637.10 _+32.40
241.20 _+43.30 365.30 + 28.10 390.20 + 18.50
Values represent mean + SD of 3 independent experiments. 1, Ambient light; 2, illuminated for 5 min at 20 J / m 2 ; 3, illumination in the presence of sodium azide; 4, illumination in the presence of superoxide dismutase.
22 p e r o x i d e a n d h y d r o x y l r a d i c a l s ( F r i d o v i c h , 1977) w h i c h in t u r n p l a y a r o l e in t h e g e n o t o x i c i t y o f p h o t o s e n s i t i v e c o m p o u n d s ( A l e j a n d r e - D u r a n et al., 1987; F i a l a et al., 1987; C a r i s o n et al., 1988; D e b u s et al., 1988). T h u s it is p o s s i b l e t h a t s u c h s e c o n d a r y r e a c t i v e s p e c i e s m a y also p l a y a r o l e in t h e m u t a g e n i c i t y i n d u c e d by r i b o f l a v i n a n d l u m i flavin. T h e s t r o n g e r m u t a g e n i c r e s p o n s e o f l u m i flavin o b s e r v e d by us is in a g r e e m e n t w i t h t h e r e p o r t s o f K u r a t o m i a n d K o b a y a s h i (1976) w h o have demonstrated t h a t l u m i f l a v i n was t h e s t r o n g e s t p h o t o s e n s i t i s e r a m o n g v a r i o u s flavin compounds. The effectiveness of lumiflavin could b e a t t r i b u t e d to its ability to f o r m s e m i q u i n o n e t y p e f r e e r a d i c a l s by r e d u c t i o n o f r i b o f l a v i n by visible light ( K u r a t a m i a n d K o b a y a s h i , 1977). T h e r e s u l t s p r e s e n t e d in this p a p e r t h u s c l e a r l y i n d i c a t e t h a t r i b o f l a v i n o n e x p o s u r e to v i s i b l e light g e n e r a t e s g e n o i n t e r a c t i v e l u m i f l a v i n as w e l l as s u p e r o x i d e r a d i c a l s . In this c o n t e x t t h e o b s e r v a t i o n o f C o u n s e l l et al. (1981) t h a t r i b o f l a v i n in v a r i o u s f o o d p r o d u c t s s u c h as i c e c r e a m s , f l o u r c o n f e c t i o n e r y , s a l a d d r e s s i n g , etc., is s u s c e p t i b l e to d e g r a d a t i o n w h e n e x p o s e d to light is r e l e v a n t . T h u s it is i m p e r a t i v e t h a t c a u t i o n h a s to b e e x e r c i s e d in h a n d l i n g , p r o c e s s i n g a n d s t o r a g e o f f o o d p r o d u c t s c o n t a i n i n g r i b o f l a v i n , in o r d e r to p r e v e n t t h e i r e x p o s u r e to light.
References Adelman, R., R.L. Saul and B.N. Ames (1988) Oxidative damage to DNA: relation to species metabolic rate and life span, Proc. Natl. Acad. Sci. USA, 85, 2706-2708. Alejandre-Duran, E., A. Alonso-Moraga and C. Pueyo (1987) Implication of active oxygen species in the direct acting mutagenicity of tea, Mutation Res,, 188, 251-257. Alvi, N.K., N.S. Akmad, S. Ahmad and S.M. Hadi (1984) Effect of riboflavin and light on the secondary structure of DNA, Chem.-Biol. Interact., 48, 367-376. Bradley, M.O., and N.A. Sharkey (1977) Mutagenicity and toxicity of visible fluorescent light to cultured mammalian cells, Nature, 266, 724-726. Byrom, P., and T.H. Turnbull (1967) Excited states of flavin coenzymes. II. Anaerobic oxidation of amino acids by excited riboflavin derivatives, Photochem. Photobiol., 6, 125-131. Carison, J., E.H. Berglin, R. Claesson, M.B.K. Edland and S. Persson (1988) Catalase inhibition by sulfide- and hydrogen peroxide-induced mutagenicity in Salmonella typhimurium TA102, Mutation Res., 202, 59-64. Counsell, J.N., G.S. Jeffries and C.J. Knewstubb (1981) in:
J.N. Counsell (Ed.), Natural Colours for Food and Other Uses, Applied Science Publishers, London, pp. 123-151. Dahl, T.A., W.R. Midden and P.E. Hartman (1987) Pure singlet oxygen cytotoxicity for bacteria, Photochem. Photobiol., 46, 345-352. Dahl, T.A., W.R. Midden and P.E. Hartman (1988) Pure exogenous singlet oxygen: nonmutagenicity in bacteria, Mutation Res., 201, 127-136. Debus, R.J., B.A. Barry, G.T. Babcock and L. Mclntosh (1988) Site directed mutagenesis identifies a tyrosine radical involved in the photosynthetic oxygen-evolving system, Proc. Natl. Acad. Sci. USA, 85, 427-430. Fiala, E.S., C.C. Conaway, W.T. Biles and B. Johnson (1987) Enhanced mutagenicity of 2-nitropropane nitronate with respect to 2-nitropropane - possible involvement of free radical species, Mutation Res., 179, 15-22. Fridovich, I. (1977) in: O. Haiyashi and K. Asada (Eds.), Biochemical and Medical Aspects of Active Oxygen, University Park Press, Baltimore, MD. Griffin, F.M., G. Ashland and R.L. Cipizzi (1981) Kinetics of phototoxicity of Fischer's medium for L5178Y leukemic cells, Cancer Res., 41, 2241-2248. Hassan, H.M., and C.S. Moody (1984) Determination of the mutagenicity of oxygen free radicals using microbial systems, Methods Enzymol., 105, 254-263. Hise, A.W., L. Recca, D.S. Katz, C.Q. Lee, M. Wagner and R.L. Schenley (1986) Evidence for reactive oxygen species inducing mutations in mammalian cells, Proc. Natl. Acad. Sci. USA, 83, 191-197. Joenje, H. (1989) Genetic toxicology of oxygen, Mutation Res., 219, 193-208. Joshi, P.C. (1985) Comparison of the DNA damaging property of photosensitized riboflavin via singlet oxygen (IO 2) and superoxide radical (O 2) mechanisms, Toxicol. Lett., 26, 211-217. Korycka-Dahl, M., and T. Richardson (1978) Photogeneration of superoxide anion in serum of bovine milk and in model systems containing riboflavin and amino acids, J. Dairy Sci., 61,400-407. Korycka-Dahl, M., and T. Richardson (1980) Photodegradation of DNA with fluorescent light in the presence of riboflavin and photoprotection by flavin triplet state quenchers, Biochim. Biophys. Acta, 610, 229-234. Kraljic, I., and E.S. Mohisni (1978) A new method for the detection of singlet oxygen in aqueous solutions, Photochem. Photobiol., 28, 577-581. Krinsky, N.I. (1977) Singlet oxygen in biological systems, Trends Biochem. Sci., 2, 35-38. Kuratomi, K., and Y. Kobayashi (1976) Photodynamic action of lumiflavin on the template DNA of RNA polymerase, FEBS Len., 72, 295-298. Kuratomi, K., and Y. Kobayashi (1977) Studies on the interactions between DNA and flavins, Biochim. Biophys. Acta, 476, 207-217, Levin, D.E., M. Hollstein, M.F. Christman, E.A. Schwiers and B.N. Ames (1982) A new Salmonella strain (TA102) with AT base pairs at the site of mutation detects oxidative mutagens, Proc. Natl. Acad. Sci. USA, 79, 7445-7449.
23 Linding, B.A., and M.A.J. Rodgers (1981) Rate parameters for the quenching of singlet oxygen by water soluble and lipid soluble substrates in aqueous and micellar systems, Photochem. Photobiol., 33, 627-634. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Miller, J. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Misra, R.B., V. Sundaraman and P.C. Joshi (1987) Riboflavin induced phototoxicity to Paramecium, J. Exp. Biol., 25, 194-201. Misra, R.B., L.P. Srivastava and P.C. Joshi (1990) Phototoxic effects of riboflavin in Tetrahymena thermophila, Indian J. Exp.. Biol., 28, 858-861. Oda, Y., S. Nakamura, I. Oki, T. Kato and H. Shinagawa (1985) Evaluation of the new system (umu test) for the detection of environmental mutagens and carcinogens, Mutation Res., 147, 219-229. Speck, W.T., S. Rosenkranz and H.S. Rosenkranz (1976) Further observation on the photooxidation of DNA in the presence of riboflavin, Biochim. Biophys. Acta, 435, 39-44. Spitz, D.R., G.C. Li, M.L. McCormick, Y. Sun and L.W. Oberley (1988) Stable H 2 0 2 resistant variants of Chinese hamster fibroblasts demonstrate increase in catalase activity, Radiat. Res., 114, 114-124. Srivastava, L.P., R.B. Misra and P.C. Joshi (1985) Photosensi-
tized generation of singlet oxygen and superoxide radicals by selected dyestuffs, food additives and their metabolites, Photochem. Photobiophys., 11, 129-137. Straight, R.C., and J.D. Spikes (1985) Photosensitized oxidation of biomolecules, in: A.A. Frimer (Ed.), Singlet Oxygen IV, CRC Press, Boca Raton, FL, pp. 91-143. Uehara, K., T. Mizoguchi and S. Hosami (1966) Photooxidation of adenine and its nucleotides in the presence of riboflavin, J. Biochem., 59, 550-555. Vuillaume, M. (1987) Reduced oxygen species mutation induction and cancer initiation, Mutation Res., 186, 43-72. Wang, R.J. (1975) Lethal effects of daylight fluorescent light on human cells in tissue culture medium, Photochem. Photobiol., 21,373-375. Webb, R.B., and M.M. Malina (1977) Mutagenesis in E. coli by visible light, Science, 156, 1104-1105. Webb, R.B., M.M. Malina and D.F. Benson (1967) Action spectrum for mutagenesis by visible light in Escherichia coli, Genetics, 56, 594-595. (Abstracts of the papers presented at the 1967 meeting of the Genetic Society of India, 31 Aug.-2 Dec.) Weitzman, S.A., G. Weitberg, E.P. Clark and E.P. Stossel (1985) Phagocytes as carcinogens: malignant transformation produced by human neutrophils, Science, 227, 12311233.
17
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1218/92/$05.00
MUTGEN 01822
Assessment of the genotoxic potential of riboflavin and lumiflavin B. Effect of light Hema Kale, P. Harikumar, S.B. Kulkarni, P.M. Nair and M.S. Netrawali Food Technology and Enzyme Engineering Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India
(Received 28 January 1992) (Revision received 13 May 1992) (Accepted 20 May 1992)
Keywords: Ribofavin; Lumiflavin;Photomutagenicity;Singlet oxygen;Superoxide radicals
Summary On exposure to visible light, riboflavin and lumiflavin produced reactive oxygen species such as singlet oxygen and superoxide radicals. The reaction was found to be time- and concentration-dependent. Both riboflavin and lumiflavin, upon illumination, showed mutagenic response in the u m u test as well as in the Ames/Salmonella assay with Salmonella typhimurium TA102. The mutagenic response was partially abolished by superoxide dismutase while sodium azide did not have any effect. No mutagenicity was observed if the compounds were not illuminated. The results suggested the involvement of superoxide radicals in light-induced mutagenicity of riboflavin as well as lumiflavin.
Exposure of chemicals to light can lead to generation of reactive oxygen species (Vuillaume, 1987) which in turn can induce mutagenic i n t e r actions (Hassan and Moody, 1984; Joenje, 1989). Riboflavin, commonly used as a colourant in milk products, icecreams, candies, etc., and as a fortificant in bread preparations, has been shown to generate reactive oxygen species (Srivastava et al., 1986). There has also been indirect evidence to indicate that when exposed to light, riboflavin induces genotoxic (Bradley and Sharkey, 1977;
Correspondence: Dr. P. Harikumar, Food Technology and Enzyme Engineering Division, Bhabha Atomic Research Centre, Trombay, Bombay400 085, India.
Griffin et al., 1981) and cytotoxic interactions (Misra et al., 1987; Misra, 1990; Wang, 1975). However, the precise mechanism by which riboflavin induces toxic effects in the presence of light is not well understood. In the present investigation the i n f u e n c e of visible light on the mutagenic potential of riboflavin and its stable photodegradation product lumiflavin was studied.
Materials and methods Riboflavin which was found to be chromatographically pure was a kind gift from Glindia Ltd. (Bombay, India). Lumiflavin, nalidixic acid, mitomycin C, L-histidine. HC1, biotin, p-nitrophenylphosphate (PNPP), O-nitrophenyl-/3-D-galacto-
18 pyranoside (ONPG), nitroblue tetrazolium (NBT), nitroblue diformazan, N,N'-dimethyl-p-nitrosoaniline (RNO), superoxide dismutase of bovine erythrocytes (SOD), sodium azide, trishydroxymethyl aminomethane (Tris) and sodium dodecyl sulfate (SDS) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Methylene blue and metanil yellow were obtained from Aldrich Chemical Company (Milwaukee, WI, USA). All other chemicals and solvents were of A R grade and solvents were distilled before use. Tester strain Salmonella typhimurium TA102 was provided by Prof. Bruce N. Ames (University of California, Berkeley, CA, USA). Salmonella typhimurium TA1535/pSK1002 was obtained from Dr. Yoshimitsu Oda (Osaka Prefectural Institute of Public Health, Nakamachi-1, Japan). Characteristics of the strains were confirmed by the procedure of Maron and Ames (1983). Working stock cultures of TA102 and T A 1 5 3 5 / pSK1002 were stored on nutrient agar slants and Luria agar slant respectively and held at 0-4°C until use.
Mutagenicity assays (1) Umu test. The general screening of mutagenicity of riboflavin and iumiflavin was carried out using the umu test (Oda et al., 1985). The system consisted of 0-100 /xg/ml of test compounds, 2.5 ml of exponentially growing cells (OD~,00 adjusted to 0.25-0.3) and 33 mM phosphate buffer, p H 7.4. The mixture was incubated at 37°C for 180 min and was centrifuged at 3020 × g for 10 rain. The pellet was washed 3 times with 0.1 M phosphate buffer, pH 7.4, resuspended in 3 ml of the same buffer and 00600 was determined. 0.2 ml of this suspension was added to 1.8 ml of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCI and 1 mM MgSO4, pH 7) followed by 50/~1 SDS (0.1%) and 10 p.l chloroform. After mixing it thoroughly, 0.2 ml O N P G solution (4 m g / m l in 0.1 M phosphate buffer, pH 7) was added to the reaction mixture and incubated at 28°C for 20 rain. The reaction was stopped by the addition of 1 ml of 1 M Na2CO 3 and the absorbances at 420 nm and 550 nm were determined. The activity of /3-galactosidase was calculated according to Miller (1972).
(2) Ames / Salmonella assay. The liquid preincubation procedure was adopted for determining the mutagenicity of riboflavin and lumiflavin (Maron and Ames, 1983). The incubation mixture consisting of 0.2 ml of a 16-18-h-old culture of tester strains (Salmonella typhimurium TA100, TA98 and TA97a), 0-100 /~g/ml test compounds and 0.1 ml PBS, pH 7.4, was incubated at 37°C for 30 rain and centrifuged at 3020 × g for 10 rain. The pellet was resuspended in 0.4 ml of 0.1 M PBS, pH 7.4. To this suspension 2 ml molten soft agar was added, mixed rapidly and spread immediately on preset minimal agar plates. Effect of light on mutagenicity To study the effect of light on the mutagenic potential of riboflavin and lumiflavin in the umu test and the Ames/Salmonella assay, identical procedures were followed except that the incubation mixture was exposed to light as detailed below. Illumination of samples for umu test An incubation mixture consisting of 0-100 ~ g / m l test compounds, 2.5 ml of exponentially growing cells and 33 mM phosphate buffer, pH 7, was held at 37°C for 30 min and then exposed to visible light in a petri plate covered with a lid, using a Philips 'Ultraphill' MLU, 300 W, 220-240 V, F.28 sunlamp with a glass filter (0.5 mm thickness). The filter was used to isolate specific effects of the radiation regime at 360-400 nm and to avoid possible effects of stray UV light. A black-ray J221 UV intensity meter which was designed and calibrated at 365 nm with a standard 365-nm detector was used to determine the energy of emitted light. For examining the effect of light in the Ames/Salmonella assay a similar procedure was followed except that the incubation mixture consisted of 0.2 ml of exponentially growing cells and 0.1 ml of phosphate-buffered saline, pH 7.4. Quantitation of reactit:e oxygen species Singlet oxygen (IO 2) was quantitated using a calorimetric procedure (Kraljick and Mohisni, 1978). The optical density at 440 nm of a 10-ml aliquot of RNO solution in 0.1 M phosphate buffer, pH 7.0, containing 1 mM histidine was
19
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I0 20 30 0 I0 20 30 ILLUMINATION (MIN) ILLUMINATION (MIN) Fig. 1. Effect of illumination time on the formation of (A) singlet oxygen and (B) superoxide radicals from riboflavin and lumifavin. B, Riboflavin; [3, riboflavin + quencher; A, lumiflavin; zx, lumiflavin + quencher.
procedure of Korycka-Dahl and Richardson (1978). A 10-ml aliquot o f 1.67 m M n i t r o b l u e t e t r a z o l i u m in 0.1 M c a r b o n a t e buffer, p H 10, was i l l u m i n a t e d at 8 J / m 2 in t h e p r e s e n c e of riboflavin or lumiflavin. T h e p r e c i p i t a t e of nitrob l u e d i f o r m a z a n was s e p a r a t e d by c e n t r i f u g a t i o n at 3020 × g for 10 rain, w a s h e d once with distilled w a t e r a n d dissolved in 3 ml of d i m e t h y l form-
a d j u s t e d to 1.2-1.4 on B a u s c h - L o m b s p e c t r o p h o t o m e t e r . T h e r e a c t i o n mixture was i l l u m i n a t e d with a s u n l a m p at 8 J / m 2 in the p r e s e n c e of riboflavin o r lumiflavin a n d t h e d e c r e a s e in optical d e n s i t y at 440 n m was d e t e r m i n e d . 1 m M s o d i u m a z i d e was u s e d to q u e n c h singlet oxygen (Srivastava et al., 1985). S u p e r o x i d e r a d i c a l s w e r e q u a n t i t a t e d by t h e
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r -~ 10 20 30 0 10 20 30 C O N C E N T R A T I O N (juglml) CONCENTRATION (./ug / ml) Fig. 2. Effect of concentration on the formation of (A) singlet oxygen and (B) superoxide radicals from ribofavin and lumiflavin, l , Riboflavin; rq, riboflavin + quencher; A, lumiflavin; zx, lumiflavin + quencher. 0
20 amide. T h e absorbance was d e t e r m i n e d at 360 nm. Superoxide radicals were q u e n c h e d by using 40 u n i t s / m l of S O D (Srivastava et al., 1985). Results and discussion
D a t a p r e s e n t e d in this p a p e r shows that both riboflavin and lumiflavin cause mutagenicity in the presence of visible light due to production of reactive oxygen species. As shown in Figs. 1 and 2, riboflavin and its p h o t o d e g r a d a t i o n p r o d u c t lumiflavin p r o d u c e singlet oxygen ( 1 0 2) and superoxide radicals ( 0 2 ) . G e n e r a t i o n of both these reactive oxygen species was f o u n d to be dependent on time of exposure (Fig. 1) and concentration of the c o m p o u n d s (Fig. 2). W h e n illumination time was increased from 0 to 30 min O D at 440 nm c h a n g e d from 0 to 0.3 for riboflavin and from 0 to 0.8 for lumiflavin. I n c o r p o r a t i o n of sodium azide in the above reaction system was found to q u e n c h the singlet oxygen formation by riboflavin by 86%. T h e c o r r e s p o n d i n g value for lumiflavin was f o u n d to be 81%. As the concentration of riboflavin and lumiflavin was increased, formation of singlet oxygen was also found to increase in a d o s e - d e p e n d e n t manner. Lumiflavin was f o u n d to be more efficient in generating singlet oxygen than riboflavin.
In addition to singlet oxygen, riboflavin and lumiflavin generated superoxide radicals. Riboflavin (1 # M ) p r o d u c e d 0.0011 ~ M superoxide radicals while 1 p~M lumiflavin f o r m e d 0.0094 /~M superoxide radicals within 30 rain of illumination. H e r e again lumiflavin was found to be more efficient than riboflavin in forming 0 2 . Superoxide dismutase was found to quench superoxide radical formation by 88% and 90% for riboflavin and [umiflavin respectively. These resuits clearly show that both riboflavin and lumiflavin generate singlet oxygen and superoxide radicals on exposure to visible light. Such interactions of the vitamin with electromagnetic radiations have been shown by Korycka-Dahl and R i c h a r d s o n (1980) and Srivastava et al. (1986). Lumiflavin has also been shown to be photo-excited to the activated state. However, there is no direct evidence on the formation or quantitative assessment of singlet oxygen and superoxide radicals on exposure to visible light. Activated oxygen species such as 10 2 and 0 2 as well as hydrogen peroxide, hydroxyl radicals, etc., are known to contribute towards the degradation of D N A , R N A , proteins, lipids and other target molecules (Krinsky, 1977; Linding and Rodgers, 1981; Joshi, 1985; A d e l m a n et al., 1988). T h e y also act a s , g e n o t o x i n s (Hsie et al., 1986;
TABLE 1 PHOTOMUTAGENICITY OF RIBOFLAVIN AND LUMIFLAVIN WITH THE UMU TEST Compound (~g/ml)
/3-Galactosidase activity (Units/OD6o o) 1
2
3
4
0
112.00_+ 15.87
110.10_+17.23
115.60+ 17.23
110.90_+ 5.43
Riboflavin 25 50 100
110.23_+ 7.88 116.80 +_28.70 118.50 + 7.85
120.90_+ 9.10 126.80 -+ 2.30 221.20 -+14.80
120.60_+ 1 0 . 5 0 162.50 -+52.60 223.90 _+20.36
110.70_+35.19 140.20 + 52.80 156.40 _+39.60
Lumiflavin 25 50 100
110.50 _+ 4.76 117.30 _+ 6.39 128.20 -+39.00
179.30 + 19.90 200.10 _+20.10 246.00 -+24.20
180.90 _+19.00 201.00 +_37.30 248.10 -+43.39
123.80 + 20.00 142.60 + 40.20 159.00 _+29.50
Values represent mean -+ SD of 3 independent experiments. 1, Ambient light conditions; 2, illuminated for 5 rain at 20 j/m2; 3, illumination in the presence of sodium azide; 4, illumination in the presence of superoxide dismutase.
21
Spitz et al., 1988) and tumour promoters (Weitzman et al., 1985; Vuillaume et al., 1987). In the case of riboflavin also, photooxidation of DNA (Speck et al., 1976; Korycka-Dahl and Richardson, 1980; Alvi et al., 1984), purines and pyrimidines (Uehara et al., 1966; Kuratomi and Kobayashi, 1977), proteins and amino acids (Byrom and Turnbull, 1967), has been indicated. Riboflavin induces cytotoxic effects in Paramecium (Misra et al., 1987), Tetrahymena (Misra et al., 1990) as well as in human and animal cells in tissue culture (Bradley and Sharkey, 1977). It has also been shown to cause mutations in microorganisms (Webb et al., 1967) and in cultured mammalian cells (Bradley and Sharkey, 1977; Webb and Malina, 1977; Griffin et al., 1981). However, these studies do not provide evidence for the involvement of either reactive oxygen species or one or more of the photodegradation products of riboflavin. Data presented in Tables 1 and 2 confirm the earlier observations of the genotoxic potential of riboflavin on exposure to light. Additionally, they also demonstrate the mutagenic response of lumiflavin, and the involvement of superoxide radicals in the mutagenicity of these compounds. Table 1 presents the data on the evaluation of the compounds in the u m u test. Riboflavin and lumiflavin caused an increase in/3-galactosidase activity to 2 and 2.2 times respectively following exposure to
visible light. In the presence of sodium azide the mutagenic response was unaltered while in the presence of superoxide dismutase it was reduced to 1.4 times for both riboflavin and lumiflavin. Table 2 incorporates data on the photomutagenicity of both compounds evaluated using the Ames/Salmonella assay with Salmonella typ h i m u r i u m TA102. In the presence of illuminated riboflavin and lumiflavin the histidine revertants of TA102 increased by 2.2 and 3 times respectively. This photoactivation was inhibited by superoxide dismutase while sodium azide did not exert any influence. A relatively better response in the Ames/Salmonella assay than in the u m u test could be explained on the basis of the generalised nature of the u m u test (Oda et al., 1985) and the specificity of Salmonella typhimurium strain TA102 towards oxidative mutagens (Levin et al., 1982). Our results on the non-interfering nature of singlet oxygen are in agreement with earlier reports (Dahl et al., 1987, 1988). Singlet oxygen is reactive towards cellular products other than DNA and is consequently not involved in bacterial DNA damage (Straight and Spike, 1985; Dahl et al., 1987). The data show the partial involvement of superoxide radicals in the mutagenic response. Such radical species have been shown to contribute to mutagenicity (Fiala et al., 1987). Photogenerated O 2 has been shown to undergo secondary reactions to form hydrogen
TABLE 2 P H O T O M U T A G E N I C I T Y OF RIBOFLAVIN A N D LUMIFLAVIN W I T H T H E A M E S / S A L M O N E L L A ASSAY Compound (/xg/ml)
Histidine revertants/plate 1
2
3
4
0
220.80 _+37.80
219.10 + 26.20
220.70 + 45.80
240.22 + 31.68
Ribofavin 25 50 100
229.00 +_26.50 218.04 + 23.35 217.50 _+28.20
301.00 + 37.10 410.70 + 33.20 503.90 + 41.40
301.20 _+33.50 410.60 + 37.30 504.00 + 23.80
231.20 + 13.07 260.80 _+28.80 334.20 _+43.00
Lumiflavin 25 50 100
224.50 + 62.60 320.10 + 23.30 221.60 + 7.36
320.60 + 29.80 496.80 + 56.20 657.30__. 41.17
328.10 + 25.80 509.20 + 46.30 637.10 _+32.40
241.20 _+43.30 365.30 + 28.10 390.20 + 18.50
Values represent mean + SD of 3 independent experiments. 1, Ambient light; 2, illuminated for 5 min at 20 J / m 2 ; 3, illumination in the presence of sodium azide; 4, illumination in the presence of superoxide dismutase.
22 p e r o x i d e a n d h y d r o x y l r a d i c a l s ( F r i d o v i c h , 1977) w h i c h in t u r n p l a y a r o l e in t h e g e n o t o x i c i t y o f p h o t o s e n s i t i v e c o m p o u n d s ( A l e j a n d r e - D u r a n et al., 1987; F i a l a et al., 1987; C a r i s o n et al., 1988; D e b u s et al., 1988). T h u s it is p o s s i b l e t h a t s u c h s e c o n d a r y r e a c t i v e s p e c i e s m a y also p l a y a r o l e in t h e m u t a g e n i c i t y i n d u c e d by r i b o f l a v i n a n d l u m i flavin. T h e s t r o n g e r m u t a g e n i c r e s p o n s e o f l u m i flavin o b s e r v e d by us is in a g r e e m e n t w i t h t h e r e p o r t s o f K u r a t o m i a n d K o b a y a s h i (1976) w h o have demonstrated t h a t l u m i f l a v i n was t h e s t r o n g e s t p h o t o s e n s i t i s e r a m o n g v a r i o u s flavin compounds. The effectiveness of lumiflavin could b e a t t r i b u t e d to its ability to f o r m s e m i q u i n o n e t y p e f r e e r a d i c a l s by r e d u c t i o n o f r i b o f l a v i n by visible light ( K u r a t a m i a n d K o b a y a s h i , 1977). T h e r e s u l t s p r e s e n t e d in this p a p e r t h u s c l e a r l y i n d i c a t e t h a t r i b o f l a v i n o n e x p o s u r e to v i s i b l e light g e n e r a t e s g e n o i n t e r a c t i v e l u m i f l a v i n as w e l l as s u p e r o x i d e r a d i c a l s . In this c o n t e x t t h e o b s e r v a t i o n o f C o u n s e l l et al. (1981) t h a t r i b o f l a v i n in v a r i o u s f o o d p r o d u c t s s u c h as i c e c r e a m s , f l o u r c o n f e c t i o n e r y , s a l a d d r e s s i n g , etc., is s u s c e p t i b l e to d e g r a d a t i o n w h e n e x p o s e d to light is r e l e v a n t . T h u s it is i m p e r a t i v e t h a t c a u t i o n h a s to b e e x e r c i s e d in h a n d l i n g , p r o c e s s i n g a n d s t o r a g e o f f o o d p r o d u c t s c o n t a i n i n g r i b o f l a v i n , in o r d e r to p r e v e n t t h e i r e x p o s u r e to light.
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