Mutation Research, 281 (1992) 77-80
77
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MUTLET 00612
The protective role of polyphenols in cytotoxicity of hydrogen peroxide Tsutomu Nakayama, Tomoaki Niimi, Toshihiko Osawa and Shunro Kawakishi Department of Food Science and Technology, Faculty of Agriculture, Nagoya University, Chikusa, Nagoya 464-01 (Japan)
(Accepted 23 September 1991)
Keywords: Hydrogenperoxide; Polyphenol;Antioxidant;Active oxygenspecies
Summary A variety of synthetic and natural polyphenols protect mammalian cells from hydrogen peroxide (H202). Cytotoxicity of H 2 0 2 on Chinese hamster V79 cells was assessed with a colony formation assay, and several polyphenols prevented the decrease in the number of colonies caused by H 2 0 2. A study of the structure-activity relationship revealed that affinity of the polyphenols for the cell membrane and the presence of an ortho-dihydroxy moiety in their structure proved essential to this protection.
Active oxygen species, e.g., hydrogen peroxide (H202) , superoxide anion radical, hydroxyl radical and singlet oxygen, have been implicated as mediators of cellular injury in a variety of clinical conditions. In particular, there are many reports describing the cytotoxic effects of H 2 0 2 on mammalian cells, including viability, DNA lesions, mutation, chromosomal aberration and morphological transformation (Mello Filho and Meneghini, 1984, 1985; Cantoni et al., 1989; Nassi-Calo et al., 1989; Shacter et al., 1990; Tachon, 1990; Nakayama et al., 1991). For these cytotoxic effects, a limited number of compounds have been shown to be inhibitors that function in the cells, e.g., o-phenanthroline (a Fe 2+ chelator) and Quin-2 (a Ca 2+ chelator). To the best of our knowledge, there are few reports describing natural products or food additives inhibiting the cyto-
Correspondence: Dr. Tsutomu Nakayama, Department of Food Science and Technology,Facultyof Agriculture,Nagoya University, Chikusa, Nagoya464-01 (Japan).
toxicity of HzO z. Recently we found that N D G A (nordihydroguaiaretic acid), a plant polyphenol and antioxidant used as a food additive, has protective effects against the cytotoxicity of HzO 2 (Nakayama et al., 1991). This discovery prompted us to search for other polyphenols that have similar biological effects. Here we show that some polyphenols with certain structures protect mammalian cells from H 2 0 2.
Materials and methods
Reagents
H 2 0 2 was obtained from Mitsubishi Gas Chemical Co. Ltd. (Tokyo, Japan). Lauryl gallate and gossypol were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Gallic acid, caffeic acid and quercetin were obtained from Nacalai Chemicals Ltd. (Kyoto, Japan). Ferulic acid and methyl gallate were obtained from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan). We prepared methyl esters of caffeic acid and ferulic
78 acid by addition of ether solution of diazomethane to the respective acids. The crude esters were applied to silica gel columns, using h e x a n e / e t h y l acetate (3:1) and h e x a n e / e t h e r (3:2) as the solvent mixture, respectively. The structures of the purified esters were identified by mass spectroscopy and NMR: caffeic acid methyl ester, m / z 194 [M+], IH N M R (TMS, CD3OD), 3.72 (3H), 6.22 (1H), 6.72 (1H), 6.88 (1H), 6.96 (1H), 7.48 (1H); ferulic acid methyl ester, m / z 208 [M+I, 1H N M R (TMS, CD3OD), 3.72 (3H), 3.84 (3H), 6.28 (1H), 6.74 (1H), 7.00 (1H), 7.10 (1H), 7.54 (1H). All other chemicals were reagent grade and were used without further purification.
Colony formation assay Cytotoxic effects of H202 and the protective effects of polyphenols were assessed by the colony formation assay (Nakayama et al., 1991). In our experiments we avoided direct reactions of the antioxidants with H 2 0 2 in the medium in order to evaluate the real effects of polyphenol in the cell or the cell membrane. Chinese hamster V79 cells were seeded in 2 0 0 / 6 0 - m m petri dishes and incubated in minimum essential medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) for 2 h. After changing the medium to M E M free of FBS, we incubated the cells with each polyphenol for 4 h. After washing the cells with HBS (HEPES-buffered saline, p H 7.3), the cells were treated with H202 for 30 min. After culture in M E M supplemented with 10% FBS for 5 days, the number of colonies was counted as described previously (Nakayama et al., 1991). The relative survival fraction was calculated by dividing the number of colonies on treated petri dishes by the number of colonies on untreated petri dishes. Results are expressed as the means and standard deviations (SD) of 4 separately treated cultures. The data were analyzed with Student's t-test, comparing the control (the cells treated only with H 2 0 2) and the groups treated with polyphenol and H 2 0 2. Results and discussion
First, we determined the dose dependence of the cytotoxic effects of antioxidants (data not
,20
'ii 100
100
6O ".g ~
40 ~
40
20
20
0
(a) 0 0.1 1 10 gallic acid (~M)
0 20 40 80 methyl gallate (pM)
120
120
100
100
m o
o
100
177 Y-~
80
,-P-r ///
///
80
**
8 60
40
20
0 0
5
10
20
propyl gallate (gM)
0
50
100
200
lauryl gallate (nM)
Fig. 1. Typical dose effects of gallic acid and its esters on the cytotoxicity of H202. The cells were treated with each polyphenol for 4 h and with H202 (60 p~M)for 30 min as described in Materials and methods. The results are expressed as the mean+SD of 4 separately treated cultures. The data were analyzed with Student's t-test, comparing the group treated with only H202 and the group treated with H202 and the indicated concentration of each polyphenol. a Gallic acid was cytotoxic at concentrations over 40 ~M. * P
77
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MUTLET 00612
The protective role of polyphenols in cytotoxicity of hydrogen peroxide Tsutomu Nakayama, Tomoaki Niimi, Toshihiko Osawa and Shunro Kawakishi Department of Food Science and Technology, Faculty of Agriculture, Nagoya University, Chikusa, Nagoya 464-01 (Japan)
(Accepted 23 September 1991)
Keywords: Hydrogenperoxide; Polyphenol;Antioxidant;Active oxygenspecies
Summary A variety of synthetic and natural polyphenols protect mammalian cells from hydrogen peroxide (H202). Cytotoxicity of H 2 0 2 on Chinese hamster V79 cells was assessed with a colony formation assay, and several polyphenols prevented the decrease in the number of colonies caused by H 2 0 2. A study of the structure-activity relationship revealed that affinity of the polyphenols for the cell membrane and the presence of an ortho-dihydroxy moiety in their structure proved essential to this protection.
Active oxygen species, e.g., hydrogen peroxide (H202) , superoxide anion radical, hydroxyl radical and singlet oxygen, have been implicated as mediators of cellular injury in a variety of clinical conditions. In particular, there are many reports describing the cytotoxic effects of H 2 0 2 on mammalian cells, including viability, DNA lesions, mutation, chromosomal aberration and morphological transformation (Mello Filho and Meneghini, 1984, 1985; Cantoni et al., 1989; Nassi-Calo et al., 1989; Shacter et al., 1990; Tachon, 1990; Nakayama et al., 1991). For these cytotoxic effects, a limited number of compounds have been shown to be inhibitors that function in the cells, e.g., o-phenanthroline (a Fe 2+ chelator) and Quin-2 (a Ca 2+ chelator). To the best of our knowledge, there are few reports describing natural products or food additives inhibiting the cyto-
Correspondence: Dr. Tsutomu Nakayama, Department of Food Science and Technology,Facultyof Agriculture,Nagoya University, Chikusa, Nagoya464-01 (Japan).
toxicity of HzO z. Recently we found that N D G A (nordihydroguaiaretic acid), a plant polyphenol and antioxidant used as a food additive, has protective effects against the cytotoxicity of HzO 2 (Nakayama et al., 1991). This discovery prompted us to search for other polyphenols that have similar biological effects. Here we show that some polyphenols with certain structures protect mammalian cells from H 2 0 2.
Materials and methods
Reagents
H 2 0 2 was obtained from Mitsubishi Gas Chemical Co. Ltd. (Tokyo, Japan). Lauryl gallate and gossypol were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Gallic acid, caffeic acid and quercetin were obtained from Nacalai Chemicals Ltd. (Kyoto, Japan). Ferulic acid and methyl gallate were obtained from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan). We prepared methyl esters of caffeic acid and ferulic
78 acid by addition of ether solution of diazomethane to the respective acids. The crude esters were applied to silica gel columns, using h e x a n e / e t h y l acetate (3:1) and h e x a n e / e t h e r (3:2) as the solvent mixture, respectively. The structures of the purified esters were identified by mass spectroscopy and NMR: caffeic acid methyl ester, m / z 194 [M+], IH N M R (TMS, CD3OD), 3.72 (3H), 6.22 (1H), 6.72 (1H), 6.88 (1H), 6.96 (1H), 7.48 (1H); ferulic acid methyl ester, m / z 208 [M+I, 1H N M R (TMS, CD3OD), 3.72 (3H), 3.84 (3H), 6.28 (1H), 6.74 (1H), 7.00 (1H), 7.10 (1H), 7.54 (1H). All other chemicals were reagent grade and were used without further purification.
Colony formation assay Cytotoxic effects of H202 and the protective effects of polyphenols were assessed by the colony formation assay (Nakayama et al., 1991). In our experiments we avoided direct reactions of the antioxidants with H 2 0 2 in the medium in order to evaluate the real effects of polyphenol in the cell or the cell membrane. Chinese hamster V79 cells were seeded in 2 0 0 / 6 0 - m m petri dishes and incubated in minimum essential medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) for 2 h. After changing the medium to M E M free of FBS, we incubated the cells with each polyphenol for 4 h. After washing the cells with HBS (HEPES-buffered saline, p H 7.3), the cells were treated with H202 for 30 min. After culture in M E M supplemented with 10% FBS for 5 days, the number of colonies was counted as described previously (Nakayama et al., 1991). The relative survival fraction was calculated by dividing the number of colonies on treated petri dishes by the number of colonies on untreated petri dishes. Results are expressed as the means and standard deviations (SD) of 4 separately treated cultures. The data were analyzed with Student's t-test, comparing the control (the cells treated only with H 2 0 2) and the groups treated with polyphenol and H 2 0 2. Results and discussion
First, we determined the dose dependence of the cytotoxic effects of antioxidants (data not
,20
'ii 100
100
6O ".g ~
40 ~
40
20
20
0
(a) 0 0.1 1 10 gallic acid (~M)
0 20 40 80 methyl gallate (pM)
120
120
100
100
m o
o
100
177 Y-~
80
,-P-r ///
///
80
**
8 60
40
20
0 0
5
10
20
propyl gallate (gM)
0
50
100
200
lauryl gallate (nM)
Fig. 1. Typical dose effects of gallic acid and its esters on the cytotoxicity of H202. The cells were treated with each polyphenol for 4 h and with H202 (60 p~M)for 30 min as described in Materials and methods. The results are expressed as the mean+SD of 4 separately treated cultures. The data were analyzed with Student's t-test, comparing the group treated with only H202 and the group treated with H202 and the indicated concentration of each polyphenol. a Gallic acid was cytotoxic at concentrations over 40 ~M. * P
