Mechanisms of DNA damage induced by morin, an inhibitor of amyloid β-peptide aggregation

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25

Figure legends

Fig. 1. Autoradiograms of 32P-5’-end-labeled DNA fragments incubated with morin in

the presence of Cu(II).

Reaction mixtures contained the 32P-5’-end-labeled 328 bp fragment, 100 M/base calf

thymus DNA, the indicated concentrations of morin, and 20 M CuCl2 in 4 mM sodium

phosphate buffer (pH 7.8) containing 5 M DTPA. After incubation at 37 ºC for 5 hr,

the DNA fragment was treated A) with, or B) without, piperidine and electrophoresed

on a polyacrylamide gel.

Fig. 2. Effects of scavengers on morin-induced DNA damage in the presence of

Cu(II).

Reaction mixtures contained the 32P-5’-end-labeled 328 bp fragment, 100 M/base calf

thymus DNA, 50 M morin, each scavenger or bathocuproine, and 20 M CuCl2 in 4

mM sodium phosphate buffer (pH 7.8) containing 5 M DTPA. After incubation at 37

ºC for 5 hr, the DNA fragment was treated with piperidine and electrophoresed on a

polyacrylamide gel. The concentration of each scavenger and bathocuproine were as

follows: 0.8 M ethanol (EtOH), 0.1 M mannitol, 0.1 M sodium formate, 1.0 M

methional, 30 U catalase, 50 μM bathocuproine, and 30 U superoxide dismutase (SOD).

Fig. 3. Site specificity of morin-induced DNA cleavage in the presence of Cu(II).

Reaction mixtures contained the 32P-5’-end-labeled A) 147 bp fragment or B) 309 bp

fragment, 100 M/base calf thymus DNA, 500 M morin, and 20 M CuCl2 in 4 mM

sodium phosphate buffer (pH 7.8) containing 5 M DTPA. After incubation at 37 ºC for

5 hr, the DNA fragment was treated with piperidine and electrophoresed on a

polyacrylamide gel. Abbreviations indicate each DNA base, A: adenine, T: thymine,

G: guanine, C: cytosine.

Fig. 4. Formation of 8-oxodG in calf thymus DNA induced by morin in the presence

of Cu(II).

Calf thymus DNA fragments of 100 μM/base were incubated with indicated

concentrations of morin in the presence of 20 μM CuCl2 in 4 mM sodium phosphate

buffer (pH 7.8) containing 5 M DTPA at 37ºC for 1 hr. After ethanol precipitation, the

26

DNA was digested to nucleosides with nuclease P1 and calf intestine phosphatase, then

analyzed with an HPLC-ECD. In order to clarify the mechanism of autoxidation,

dissolved oxygen in the reaction mixture containing morin and Cu(II) was removed by

bubbling nitrogen gas for 1 min (hypoxic conditions), after which 8-oxodG was

determined. Results are expressed as mean of values obtained from two independent

experiments.

Fig. 5.

Analysis of reaction products by morin in the presence of Cu(II).

(A) HPLC chromatograms of oxidized products of morin after reaction at 37 ºC for 21

hr. Reaction mixture consisted 500 µM of morin and (a) 0 µM, (b) 20 µM, or (c) 200 µM of

CuCl2 in 4 mM sodium phosphate buffer (pH 7.8) containing 5 M DTPA. (B) 1H- and

13

C-NMR spectra of the major oxidized product (Rt 14.8 min) were recorded in

DMSO-d6 at 20 ºC. In the 1H-NMR chart, integral values corresponding each proton

were indicated under the scale bar of chemical shift. (C) Estimated chemical structure of

the oxidized product generated by morin and Cu(II).

Fig. 6. A possible mechanism of oxidative DNA damage induced by morin in the

presence of Cu(II).

27

Fig. 1

A) Piperidine (+)

B) Piperidine (-)

500

200

100

50

20

500

200

100

50

20

Morin (μM)

+ SOD

+ Bathocuproine

+ Catalase

+ Methional

+ Sodium formate

+ Mannitol

+ EtOH

Morin + Cu(II)

Control

Fig. 2

Fig. 3

A) DNA fragment of 147 bp (p16)

10

Intensity

(5’)

AA

AAG

GA

AA

AC

(3’)

GG

GC

9910

9920

9900

9890

9880

B) DNA fragment of 309 bp (p16)

Intensity

40

20

(5’)

C C

T AT

C GG C

CG

G C

CC

A C GC T

CC

T C

C G A TC G

G G A A G

C T T TC

GTC

T G

(3’)

TT

9500

9510

9520

9530

9540

9550

9560

Nucleotide number

Fig. 4

180

8-oxodG/dG (x100000)

160

140

120

normal conditions

100

80

-hypoxic conditions

60

40

20

100

200

300

400

500

Morin (μM)

B-1)

Fig. 5

1H-NMR

in DMSO-d6, 600 MHz

A)

DMSO

uV

mV

95000

85000

H2O

5-CH

14.8

min

90000

90

morin

I I

80000

80

75000

70000

70

3-CH

6-CH

65000

4-OH

60000

60

55000

COOH

35000

30000

30

(c)

25000

20000

20

(a)

5000

12

10

'-r''-r'y

0.52

10000

10

14

(b)

15000

2-OH

. .

ppm

1.03

1.00

5.6 8.0 12.5

min min min

1.01

40

40000

1.00

45000

0.90

50000

50

-5000

0.0

5.0

10.0

10

15.0

15

20.0

20

25.0

25

30.0

30

35.0

35

Retention time (Rt)

40.0

40

min

45

B-2)

min

13C-NMR

in DMSO-d6, 150 MHz

DMSO

C)

OH

HO

1-C

6-CH

4-C, 2-C

5-CH

OH

3-CH

~ I~

COOH

2,4-dihydroxybenzoic acid

200

180

160

140

120

100

80

60

40

20

ppm

Fig. 6

reactive oxygen

species

[Cu(I)OOH]

H2O2

O2・- O2

HO 2’

HO

1’

O 2

6’

OH

3’

H2O2

OH

4’

HO

Cu(II) Cu(I)

5’

ー亡

oxidative

DNA damage

HO

HO

OH

HO

O2 O2・-

OH

Morin

OH

OH

para-quinone methide

H2O

HO

OH

HO

OH

OH

HO

HO

HO

OH

OH

OH O

OH

OH

2,4-dihydroxybenzoic acid

...