**4. Coffee and 8-oxo-Gua / OGG1**

Coffee has been a quite popular beverage in many parts of the world for a long time. However, its effects on human health are not well understood. Some studies suggested that coffee consumption had preventive properties for metabolic diseases, such as type 2 diabetes [40, 41], or cancers, such as hepatocellular carcinoma [42, 43] and colorectal cancer [44, 45], through its content of potentially antimutagenic substances [46]. These findings led to the hypothesis that coffee consumption lowers the risk of some types of cancers.

On the other hand, some studies provided negative conclusions for the effects of coffee consumption on human health. A large cohort study in Sweden and Japan indicated that coffee consumption was not associated with the risk of colorectal cancer [47, 48]. Furthermore, another cohort study indicated that coffee consumption increased the risk of certain cancers, such as gastric cancer [49]. These carcinogenic actions of coffee were supported by evidence that coffee contained numerous substances, such as glyoxal, methylglyoxal, ethylglyoxal, propylglyoxal, diacetyl, and acetol, with potentially genotoxic and mutagenic properties [46, 49-51]. Although the polyphenols in beverages, such as green tea, black tea, and coffee, are antioxidant substances, a recent study indicated the production of hydrogen peroxide, a harmful molecule for living organisms, by polyphenol-rich beverages [52]. Therefore, although numerous studies have been performed, the effects of coffee consumption on human health have remained undefined.

Food Factors and Oxidative DNA Damage / DNA Repair Systems 551

To address this issue, we focused on the effects of instant coffee consumption on the generation of 8-oxo-Gua. In our previous work, we analyzed the 8-oxo-Gua level and the 8 oxo-Gua repair system in the livers of mice fed with / without a 0.1% instant coffee solution. In addition, we employed an autoclaved diet (low vitamin diet; LV diet) to maintain half of the mice under low vitamin conditions. We found that instant coffee consumption does not alter the 8-oxo-Gua generation level and *OGG1* mRNA expression, although it prevents lowvitamin diet-induced 8-oxo-Gua production [53] (Fig. 3). Our study suggested that daily instant coffee consumption may provide more beneficial effects than detrimental effects. However, we have not examined other types of DNA damage. To clarify the role of coffee

Urinary 8-oxo-Gua levels are often analyzed, especially as a marker of oxidation [54, 55]. Among the urinary biomarkers of oxidative stresses, 8-oxo-Gua is possibly the most studied. We previously investigated the effects of age, smoking, dietary factors, and other life style factors on urinary 8-oxodeoxyguanosine (8-oxo-dG) levels [56]. Urine samples were collected from healthy employees in a steel-manufacturing company, after obtaining informed consent. The mean level of urinary 8-oxo-dG (μg / g creatinine) in the 361 male subjects was 4.20 ± 1.47 (Table 1). The relationships between 16 categorical lifestyle factors and urinary 8-oxo-dG levels were analyzed by ANOVA. The results revealed that the urinary 8-oxo-dG level was significantly negatively related to fruit consumption (*P* = 0.03) and physical activity (*P* = 0.03). It is noteworthy that, in terms of fruit consumption, the urinary 8-oxo-dG levels of the "rarely" and "two or three times per week" groups were significantly higher than those of the "everyday" group (*P* = 0.03). The results of the Scheffe's test also indicated that fruit consumption significantly reduced the urinary 8-oxodG level. Besides urinary analyses, a diet rich in fruit and vegetables was also reportedly effective in the reduction of oxidative stresses [57]. These data indicated that fruit

consumption on human health, further studies should be performed.

consumption and physical activity reduce oxidative stress generation.

slightly deficient sufficient

once a week twice a week

sometimes rarely

moderate easy

no pattern

moderation every time

size of a meal full stomach every time

mostly regular regular

sleep deficient

fatigue always

rhythm irregular

refreshing difficult

holiday little or none

**variables category n % urinary 8-oxo-dG** *P* 

10 181 170

2 51 308

18 256 87

66 215 80

13 255 93

24 198 139 2.8 50.1 47.1

0.6 14.1 85.3

5.0 70.9 24.1

18.3 59.6 22.2

3.6 70.6 25.8

6.6 54.8 38.5 4.37 ± 0.25 4.19 ± 0.12 4.20 ± 0.11

5.41 ± 1.20 3.96 ± 0.21 4.23 ± 0.08

3.66 ± 0.32 4.17 ± 0.09 4.39 ± 0.17

4.05 ± 0.19 4.20 ± 0.10 4.30 ± 0.16

4.40 ± 0.38 4.13 ± 0.09 4.33 ± 0.17

3.87 ± 0.27 4.29 ± 0.11 4.12 ± 0.11 0.53

1.00

0.86

0.25

0.48

0.85

**5. Urinary 8-oxo-Gua level and food consumption** 

Fig. 2. (A) The experimental protocol. A: control [ND / water] for 10 months; B: [ND / water] for the first 2 months and [0.06% 3'-MeDAB / water] for the last 8 months; C: [ND / alcohol] for 10 months; D: [ND / alcohol] for the first 2 months and [0.06% 3'-MeDAB / alcohol] for the last 8 months; E: [ND / alcohol] for the first 2 months, [0.06% 3'-MeDAB / alcohol] for the next 4 months, and [0.06% 3'-MeDAB / water] for the last 4 months; F: [ND / water] for the first 2 months, [3'-MeDAB / water] for the next 4 months, and [3'-MeDAB / alcohol] for the last 4 months. ND: normal diet, DAB: 3'-MeDAB. (B) The levels of 8-oxo-Gua in the DNA of mouse livers. The 8-oxo-Gua value is expressed as the number of 8-oxo-dG per 105 deoxyguanosine. \*1: *P* < 0.0005 *vs*. group B, *P* < 0.05 vs. group E, *P* < 0.01 vs. group F; \*2: *P* < 0.0001 vs. group C, *P* < 0.005 vs. group D, *P* < 0.01 vs. group E, *P* < 0.05 vs. group F; \*3: *P* < 0.05 vs. group E, *P* < 0.005 vs. group F; \*4: *P* < 0.05 vs. group F. (C) 8-Oxo-Gua nicking activity in the mouse livers. The activity was calculated as the ratio of the excised fragment intensity to the total substrate (unexcised substrate intensity plus excised fragment intensity). \*: *P* < 0.005 vs. group A. (D) Western blotting of mOGG1 protein in the mouse livers. Mouse livers were removed and homogenized to produce crude extracts. The extracts were electrophoresed and blotted onto a PVDF membrane. A western blot analysis was performed using an anti-mouse OGG1 antibody. The arrowhead indicates fragmented mOGG1. mOGG1: mouse OGG1. This figure was published in reference [37], Copyright Elsevier (license#: 2633951132321).

**A** 

 **B C** 

**Repair activity**

**ABCDEF**

**\***

**\***

**Group**

**D**  Fig. 2. (A) The experimental protocol. A: control [ND / water] for 10 months; B: [ND / water] for the first 2 months and [0.06% 3'-MeDAB / water] for the last 8 months; C: [ND / alcohol] for 10 months; D: [ND / alcohol] for the first 2 months and [0.06% 3'-MeDAB / alcohol] for the last 8 months; E: [ND / alcohol] for the first 2 months, [0.06% 3'-MeDAB / alcohol] for the next 4 months, and [0.06% 3'-MeDAB / water] for the last 4 months; F: [ND / water] for the first 2 months, [3'-MeDAB / water] for the next 4 months, and [3'-MeDAB / alcohol] for the last 4 months. ND: normal diet, DAB: 3'-MeDAB. (B) The levels of 8-oxo-Gua in the DNA of mouse livers. The 8-oxo-Gua value is expressed as the number of 8-oxo-dG per 105 deoxyguanosine. \*1: *P* < 0.0005 *vs*. group B, *P* < 0.05 vs. group E, *P* < 0.01 vs. group F; \*2: *P* < 0.0001 vs. group C, *P* < 0.005 vs. group D, *P* < 0.01 vs. group E, *P* < 0.05 vs. group F; \*3: *P* < 0.05 vs. group E, *P* < 0.005 vs. group F; \*4: *P* < 0.05 vs. group F. (C) 8-Oxo-Gua nicking activity in the mouse livers. The activity was calculated as the ratio of the excised fragment intensity to the total substrate (unexcised substrate intensity plus excised fragment intensity). \*: *P* < 0.005 vs. group A. (D) Western blotting of mOGG1 protein in the mouse livers. Mouse livers were removed and homogenized to produce crude extracts. The extracts were electrophoresed and blotted onto a

**ABCDEF**

**\*4**

**\*3**

**Group**

**0 0.2 0.4 0.6 0.8 1**

**\*1**

**\*2**

**8-oxo-Gua**

PVDF membrane. A western blot analysis was performed using an anti-mouse OGG1 antibody. The arrowhead indicates fragmented mOGG1. mOGG1: mouse OGG1. This figure

was published in reference [37], Copyright Elsevier (license#: 2633951132321).

To address this issue, we focused on the effects of instant coffee consumption on the generation of 8-oxo-Gua. In our previous work, we analyzed the 8-oxo-Gua level and the 8 oxo-Gua repair system in the livers of mice fed with / without a 0.1% instant coffee solution. In addition, we employed an autoclaved diet (low vitamin diet; LV diet) to maintain half of the mice under low vitamin conditions. We found that instant coffee consumption does not alter the 8-oxo-Gua generation level and *OGG1* mRNA expression, although it prevents lowvitamin diet-induced 8-oxo-Gua production [53] (Fig. 3). Our study suggested that daily instant coffee consumption may provide more beneficial effects than detrimental effects. However, we have not examined other types of DNA damage. To clarify the role of coffee consumption on human health, further studies should be performed.
