Dietary melatonin and liver cancer incidence in Japan: From the Takayama study

Abstract

There is some biological plausibility that exogenous melatonin plays a role in preventing liver carcinogenesis. There has been little research on the association between melatonin intake in a normal diet and health outcomes. We evaluated the association between dietary melatonin intake and the incidence of liver cancer in a population-based prospective study in Japan. This study included 30,824 residents of Takayama city who were 35 years of age or older in 1992 and had participated in the Takayama study, Japan. Dietary intake was assessed using a validated food frequency questionnaire at the baseline. Melatonin content in foods was measured by liquid chromatography–tandem mass spectrometry. Cancer incidence was confirmed through regional population-based cancer registries in Gifu. Liver cancer was defined as code C22 according to the International Classification of Diseases and Related Health Problems, 10th Revision. Hazard ratios for liver cancer were estimated for the tertile groups of melatonin intake using a Cox proportional hazards model. During the mean follow-up period of 13.6 years, 189 individuals developed liver cancer. Compared with subjects in the lowest tertile of melatonin intake, those in the middle and highest tertiles had decreased risks of liver cancer, with a significant linear trend after multivariate adjustments (hazard ratios: 0.64 and 0.65, respectively, trend p = 0.023). There was no significant interaction by sex (interaction p = 0.54). This initial finding, which needs to be confirmed by further studies, suggests that consuming melatonin-containing foods might play a role in the prevention of liver cancer.

Abbreviations
• CI
• confidence interval
• FFQ
• food frequency questionnaire
• MET
• metabolic equivalent

INTRODUCTION

Melatonin, a derivative of tryptophan, is a neurohormone initially isolated from the bovine pineal gland¹ that is widely distributed in body tissues including the gastrointestinal tract.^2,3 Melatonin is involved in various physiological functions such as antioxidation, anti-inflammation, and immunomodulation.^4–6 In addition, melatonin has a potential oncostatic effect on a variety of cancer cells and tissues through anti-metastatic, anti-proliferative, and pro-apoptotic mechanisms.^7,8

Melatonin is also synthesized and metabolized in the liver. Melatonin levels are reportedly higher in the bile and liver than in the pineal grand.^3,9 It is suggested that the liver has specific circadian rhythms independent of the brain and other organs.¹⁰ Disruption of the circadian clock is associated with liver steatosis, inflammation, and hepatocarcinogenesis.^6,7 Melatonin protects the biliary epithelium from oxidative damage, exerts cytoprotective effects in liver injury, prevents liver carcinogenesis, and acts against cancer cell proliferation, motility, and invasiveness.^9,11 The experimental administration of melatonin has been shown to suppress hepatitis, non-alcoholic fatty liver disease, liver fibrosis, liver cirrhosis, and liver tumors.^7,11

Exogenous sources of melatonin include supplements, drugs, and foods. Although the therapeutic uses of melatonin are mainly restricted to sleep regulation and re-synchronization of disrupted circadian rhythms, its efficacy alone or with other drugs has been reported in various clinical settings, including liver cancer.^7,12 Dietary melatonin has been identified and quantified in a variety of foods, including plants, nuts, fungi, and animal foods,^13,14 with relatively high levels in vegetables, seeds, and eggs.^15,16 Melatonin levels are much lower in foods than in supplements or drugs, but circulating melatonin levels reportedly increase with the consumption of melatonin-rich foods.^17,18 However, there has been little research on the association between melatonin intake in a normal diet and health outcomes.¹⁹ Previously, we reported that higher total dietary melatonin was associated with a decreased mortality risk.¹⁹ No studies have investigated the association between dietary melatonin and cancer incidence.

Therefore, in the present study, we evaluated the association between dietary melatonin intake and the incidence of liver cancer in a population-based prospective study in Japan. We speculated that dietary melatonin might be associated with a lower risk of liver cancer.

MATERIALS AND METHODS Participants and design

The participants were part of the Takayama study, which was conducted to evaluate the associations between lifestyle factors and death or cancer incidence in Japan. The details of the study have been described previously.²⁰ Among 36,990 non-hospitalized residents in Takayama City who were 35 years of age or older in September 1992, 31,552 (85.3%) were enrolled in the study (Figure S1). A self-administered questionnaire including a food frequency questionnaire (FFQ) was used to obtain information on demographic characteristics, medical history, diet, physical activity, smoking status, and reproductive factors. Follow-up was conducted until the end of March 2008. This study was approved by the institutional review board of the Gifu University Graduate School of Medicine.

Dietary melatonin intake and other exposure variables

The FFQ consisted of questions on the consumption frequency of foods and dishes (169 items) and the usual portion size of meals over the past year.²¹ Dietary intake was computed by multiplying the consumption frequency of each food by the nutrient or food content of the specified portion. Of the items included in the FFQ, 177 foods were selected for melatonin measurement, including white bread, biscuits and snacks, oils, sugars, seasonings such as salt, vinegar, sauces, beer, and wine. No complete database on melatonin content in various foods is available. The use of melatonin supplements was not approved in Japan at the time of the survey. Melatonin content in individual foods was measured by liquid chromatography–tandem mass spectrometry. Details on the measurements are described elsewhere.^22,23 The quantification limit was 0.2 pg/g for some greasy foods and 0.1 pg/g for others. For foods that showed less than the quantification limit, the value of the quantification limit was assigned. Melatonin values in the measured foods ranged from 0.1 to 218.04 pg/g, with relatively high levels in eggs, seeds, and vegetables, similar to previous reports.^15,16 For foods in which melatonin content was not analyzed, the values of similar foods or of a different form of the same food were assigned. For example, the value of “dried soybeans” was assigned to “boiled soybeans” after considering the moisture content. These assignments covered 88.2% of the cumulative total number of foods in the FFQ, and these foods accounted for 98.6% and 99.3% of the total energy intake of men and women, respectively. For the remaining foods, the median melatonin values of the measured foods within the same food group were assigned. The total melatonin intake for each participant was calculated by adding up the estimated melatonin levels of the foods in the FFQ. The intake of other nutrients and food groups was estimated using the Japanese Standard Table of Food Composition, 5th revised and enlarged edition.²⁴ To estimate amino acid content in some foods for which data were not available in the Japanese Standard Table of Food Composition, values published by the United States Department of Agriculture were used.²⁵

In a subsample of the present study, Spearman's correlation coefficient for melatonin intake between the FFQ and 12-day dietary records over 1 year was 0.39 for men and 0.46 for women.¹⁹ In another sample of women,²⁶ the relationship between melatonin intake estimated from the FFQ and the first-void morning urine levels of 6-sulfatoxymelatonin, a major metabolite of melatonin, was evaluated. The correlation coefficient was 0.10 after controlling for age, body mass index, menopausal status, sleep duration at night, asleep/awake status at midnight, duration of daylight on the day before urine collection, and history of hypertension.

Smoking habit and, if an ever-smoker, the number of years of smoking were determined. Smokers were defined as people who had smoked a total of at least 20 packs of cigarettes during their life. Physical activity was assessed by asking about the average time they spent on various intensities of physical activities over the past year. The physical activity score was calculated by multiplying the duration of activity (h/week) by energy expenditure (metabolic equivalent [MET]) for each intensity-level activity and summing up the products (MET·h/week). The details and validity of the physical activity score have been described elsewhere.²⁷

Outcomes and follow-up

After excluding 728 people who were diagnosed with liver cancer before baseline and/or reported a positive history of cancer in the baseline questionnaire, 30,824 participants (14,240 men and 16,584 women) were included in the analysis (Figure S1). Information on death and emigration was obtained from residential registers or family registers. The cause of death was identified from death certificates provided by the Legal Affairs Bureau. Cancer incidence was confirmed mainly through two regional population-based cancer registries in Gifu. Liver cancer was defined as code C22 according to the International Classification of Diseases and Related Health Problems, 10th Revision. The mortality-to-incidence ratio was 0.75. Because 54.0% of patients were identified in the registry only on the death certificate, a backward tracking review of the incidence date based on the description given in the death certificate revealed that 19.1% of patients had an unknown incidence date before the date of death. During the study period, 1580 persons (5.1%) moved out of the study area. Among 251 participants whose date of emigration was unknown (0.8%), their last date of residence in the study area was assigned as their censored date.

Statistical analysis

Melatonin intake was controlled for energy intake by the residual method proposed by Willett.²⁸ The study participants were categorized into tertile groups (T1–T3) according to their energy-adjusted melatonin intake. Follow-up periods were calculated as the time from baseline to the date of cancer diagnosis, date of death, date of moving out of the study area, or the end of follow-up, whichever came first. During the mean follow-up period of 13.6 years, 189 individuals developed liver cancer.

The characteristics of the participants were calculated as the mean (standard deviation) or percentage of each category according to the tertile groups of energy-adjusted melatonin intake.

Hazard ratios and 95% confidence intervals (CIs) for liver cancer were estimated for the tertile groups of melatonin intake using a Cox proportional hazards model. The reference group was set as the lowest tertile of melatonin intake. After potential risk factors for liver cancer were identified through a literature review, the confounders included sex (male, female), age (years, continuous), body mass index (quartiles), education years (≤8, 9–11, 12–14, ≥15 years), history of diabetes (yes, no), physical activity score (MET·h/week, continuous), smoking status (never, former, current smoker who had smoked for ≤30 years, current smoker who had smoked for ≥31 years), alcohol consumption (g/day, continuous), total energy intake (kcal/day, continuous), coffee consumption (none, less than once a day, once a day or more), and menopausal status (premenopausal, postmenopausal). The analysis was repeated with additional adjustments for sleep duration (≤6, 7, 8, ≥9 h), taking into account potential effects on endogenous melatonin. Dummy variables were created for missing data for categorical covariates. Tests for a linear trend were performed using the median values of melatonin intake for each category.

A sensitivity analysis was performed after excluding cases who were diagnosed with liver cancer in the first 2 years because they might have had latent cancer at baseline. In addition, because nonsteroidal anti-inflammatory drugs and beta-blockers could influence melatonin secretion,²⁹ the use of painkillers and antihypertensive medicine was additionally adjusted.

All analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC). p-values were calculated using a two-sided test. A p-value of less than 0.05 was considered statistically significant in all analyses.

RESULTS

The characteristics of the participants are shown according to the tertile groups of energy-adjusted melatonin intake in Table 1. Participants with higher melatonin intake were more likely to be women, to have reported a history of diabetes and a shorter duration of sleep, to have never smoked, and to drink at least one cup of coffee a day. Participants in the lowest tertile of melatonin intake had higher alcohol consumption than those in the other tertiles. Participants in the middle tertile of melatonin intake were less educated, physically less active, and had lower total energy intake than those in the other tertiles. The main food sources of melatonin in this population were vegetables (49%), cereals (34%), eggs (5%), and coffee (4%).

TABLE 1 Characteristics of study participants at baseline in the Takayama study, Japan.

	Intake of energy-adjusted melatonin (ng/day, range)a
	T1 (≤26.7)	T2 (27.7–31.9)	T3 (≥31.9)
n of participants	10,275	10,275	10,274
Female, n (%)	4045 (39.4%)	6122 (59.6%)	6417 (62.5%)
Age (y.)	55.4 (12.6)	56.4 (13.3)	55.6 (12.6)
Body mass index (kg/m2)	22.3 (2.9)	22.1 (2.9)	22.3 (2.9)
History of diabetes (yes), n (%)	445 (4.3%)	422 (4.1%)	513 (5.0%)
Education years, n (%)
≤8 years	2514 (24.8%)	2834 (28.0%)	2486 (24.5%)
9–11 years	3875 (38.2%)	3694 (36.5%)	3751 (37.0%)
12–14 years	2980 (29.4%)	2856 (28.2%)	3052 (30.1%)
≥15 years	771 (7.6%)	731 (7.2%)	840 (8.3%)
Physical activity score (METs·h/week)	23.3 (37.0)	21.2 (33.6)	24.0 (35.4)
Sleep duration, n (%)
≤6 h	2218 (22.6%)	2324 (23.7%)	2480 (25.1%)
7 h	3794 (38.6%)	3833 (39.0%)	3941 (39.9%)
8 h	2755 (28.0%)	2733 (27.8%)	2572 (26.0%)
≥9 h	1057 (10.8%)	934 (9.5%)	884 (9.0%)
Smoking status, n (%)
Never	3799 (39.3%)	5281 (55.7%)	5437 (57.1%)
Former	1949 (20.2%)	1445 (15.2%)	1401 (14.7%)
Current (for <30 years)	2408 (24.9%)	1781 (18.8%)	1781 (18.7%)
Current (for ≥30 years)	1508 (15.6%)	972 (10.3%)	901 (9.5%)
Alcohol consumption (g/d)	38.3 (44.3)	15.5 (25.2)	15.5 (26.3)
Coffee consumption, n (%)
None	2461 (24.0%)	2626 (25.6%)	2410 (23.5%)
Less than once a day	4256 (41.4%)	3979 (38.7%)	3786 (36.9%)
Once a day or more	3558 (34.6%)	3670 (35.7%)	4078 (39.7%)
Dietary intake
Melatonin (ng/d)	25.0 (9.1)	26.0 (8.6)	41.5 (17.4)
Total energy (kcal/d)	2561 (915)	2074 (728)	2404 (860)

Note: Mean (standard deviation) or n (%).

Abbreviations: MET, metabolic equivalent; T1–T3, the tertile groups of energy-adjusted melatonin intake.

^aMelatonin intake adjusted for total energy intake by Willett method.

Compared with participants in the lowest tertile of melatonin intake, those in the middle and highest tertiles had decreased risks of liver cancer, with a significant linear trend after multivariate adjustments (hazard ratios: 0.64 and 0.65, respectively, trend p = 0.025) (Table 2). Additional adjustments for sleep duration did not alter the results substantially (trend p = 0.023). When the analyses were repeated by sex, the hazard ratios of liver cancer for the middle and highest tertiles versus the lowest tertile of melatonin intake were 0.70 (95% CI: 0.45–1.10) and 0.52 (95% CI: 0.32–0.85), respectively, in men (trend p = 0.008), and 0.74 (95% CI: 0.43–1.28) and 0.62 (95% CI: 0.34–1.11), respectively, in women (trend p = 0.11) (Table S1). There was no significant interaction by sex (interaction p = 0.54).

TABLE 2 Hazard ratios (95% confidence interval) for liver cancer according to melatonin intake in the Takayama study, Japan.

[Table omitted. See PDF]

Abbreviations: HR, hazard ratio; T1–T3, the tertile groups of energy-adjusted melatonin intake.

^aMelatonin intake adjusted for total energy intake by Willett method.

^bHazard ratios adjusted for sex (male/female), age (years), body mass index (quartiles), education years (≤8, 9–11, 12–14, ≥15 years), history of diabetes (yes, no), physical activity score (MET-h/week), smoking status (never, former, current smoker who had smoked for 30 years or less, current smoker who had smoked for 31 years or more), alcohol consumption (g/d), intakes of total energy (kcal/d), coffee consumption (none, less than once a day, once a day or more), and menopausal status (premenopausal, postmenopausal) (only for women).

^cHazard ratios adjusted for sleep duration (≤6, 7, 8, ≥9 h) in addition to the variable included in HR1.

Intake of tryptophan, a precursor of melatonin, was not associated with liver cancer; the hazard ratios of liver cancer for the middle and highest tertiles versus the lowest tertile of tryptophan intake were 0.93 (95% CI: 0.63–1.35) and 1.00 (95% CI: 0.69–1.47), respectively (trend p = 0.97).

When we performed sensitivity analysis after excluding 25 cases of liver cancer during the first 2 years of follow-up, the hazard ratios of liver cancer for the middle and highest tertiles versus the lowest tertile of melatonin intake were 0.69 (95% CI: 0.47–1.01) and 0.67 (95% CI: 0.45–0.99), respectively (trend p = 0.052). After additional adjustments for the use of painkillers and antihypertensive medicine, the results were not substantially altered.

DISCUSSION

In this prospective study in Japan, we observed a significant association between increased melatonin intake and a decreased risk of liver cancer, with no significant difference between men and women. This is the first report to suggest that melatonin intake might have a beneficial impact on the risk of developing liver cancer.

Despite the biological plausibility that exogenous melatonin might play a role in preventing liver carcinogenesis and suppressing hepatic tumors,^6,9,11 there is little epidemiological evidence to support this hypothesis.^30–32 In some randomized controlled trials of patients with non-alcoholic fatty liver disease, which could possibly lead to hepatocarcinogenesis in the advanced condition, melatonin administration reduced the levels of pro-inflammatory cytokines and improved the parameters of fat metabolism and the severity of fatty liver.^32–34 In a randomized controlled trial of patients with advanced liver cancer, melatonin given concurrently with transcatheter arterial chemoembolization improved the survival rate.³¹ Therefore, dietary melatonin might also inhibit the transition from fatty liver to steatohepatitis and liver cancer and might delay the progression of liver cancer. However, there have been no previous reports on the association between dietary melatonin or melatonin biomarkers (e.g., urinary 6-sulfatoxymelatonin) and liver cancer. It is unclear whether the observed inverse association between dietary melatonin and the risk of liver cancer might be mediated by circulating melatonin, although positive associations between circulating melatonin levels and the intake of some melatonin-rich foods have been reported.^17,18

The present study found no significant association between tryptophan intake and liver cancer. Endogenous melatonin is produced from tryptophan, an essential amino acid, via the serotonin-melatonin pathway. Although tryptophan administration reportedly increases circulating melatonin levels,³³ the association between dietary tryptophan and circulating melatonin levels is unknown. Since tryptophan has other metabolic pathways, such as the kynurenine and indole pathways, other metabolites might have influenced the observed associations. There are several reports on various cancers including liver cancer and the changes of tryptophan and its metabolites, but no consistent conclusions have been reached.^35,36

The main strengths of our study are its prospective design and the measurement of melatonin using liquid chromatography–tandem mass spectrometry, which is highly recommended for determining the melatonin content of foods. Other strengths include a good participation rate, a long follow-up period, and the consideration of several confounding factors. However, there are several limitations that should be mentioned. The correlation coefficients for melatonin between the FFQ and 12-day dietary records were not high, which might have led to some misclassification of melatonin intake. The observed association between dietary melatonin and liver cancer might have been underestimated because measurement errors due to such misclassifications are likely to have occurred independent of cancer incidence. It is possible that other components in melatonin-containing foods or synergic effects with them are responsible for the positive association between melatonin intake and liver cancer. It might reflect the effects of a healthy diet containing vegetables and cereals (a low-calorie and low-fat diet) rather than melatonin consumption per se. However, additional adjustments for fat intake did not substantially alter the observed associations (data not shown). Diet or lifestyle habits at baseline might have been influenced by preclinical signs or underlying disease. However, the exclusion of liver cancer cases during the first 2 years of follow-up did not change the results significantly. Information regarding melatonin intake and confounders was collected only at baseline, and the changes during follow-up were not evaluated. In addition, our study did not include information on hepatitis virus, nonalcoholic fatty liver disease, and cirrhosis, which are major risk factors for liver cancer. If participants with these diseases had changed their dietary habits, a reverse causal relationship could have occurred. The sample size was limited in the analysis by sex, particularly in women with a small number of cases. Finally, the generalizability of the study results might be limited to the Japanese population.

In conclusion, this prospective study observed that higher melatonin intake was significantly associated with a decreased risk of liver cancer in Japanese individuals. This initial finding, which needs to be confirmed by further studies, suggests that consuming melatonin could reduce the risk of liver cancer.

AUTHOR CONTRIBUTIONS

Keiko Wada: Conceptualization; data curation; formal analysis; investigation; resources; writing – original draft; writing – review and editing. Atsuhiko Hattori: Resources; writing – review and editing. Yusuke Maruyama: Resources; writing – review and editing. Tomoka Mori: Writing – review and editing. Masaaki Sugino: Writing – review and editing. Yuma Nakashima: Writing – review and editing. Michiyo Yamakawa: Writing – review and editing. Masayuki Yamamoto: Resources; writing – review and editing. Akihiro Hori: Resources; writing – review and editing. Mitsuru Seishima: Resources; writing – review and editing. Shinobu Tanabashi: Resources; writing – review and editing. Shogen Matsushita: Resources; writing – review and editing. Chisato Nagata: Conceptualization; investigation; resources; supervision; writing – review and editing.

ACKNOWLEDGEMENTS

None.

FUNDING INFORMATION

This work was supported by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (15K15222).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest. Dr. Chisato Nagata is a current Editorial Board Member of Cancer Science.

ETHICS STATEMENT

-Approval of the research protocol by an Institutional Reviewer Board: This study protocol was approved by the institutional review board of the Gifu University Graduate School of Medicine (26–277).

-Informed Consent: Consent was waived due to the valid reasons for lack of consent at the time of the survey, the difficulty in obtaining consent now, and the public necessity of this research.

-Registry and the Registration No. of the study/trial: N/A.

-Animal Studies: N/A.