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Effects of coffee consumption in chronic hepatitis C: A randomized controlled trial
Digestive and Liver Disease 45 (2013) 499–504
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Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld
Liver, Pancreas and Biliary Tract
Effects of coffee consumption in chronic hepatitis C: A randomized controlled trial Romilda Cardin a, Marika Piciocchi a, Diego Martines a, Laura Scribano a, Marino Petracco b, Fabio Farinati a,∗ a b
Department of Surgical, Oncological and Gastroenterological Sciences, Section of Gastroenterology, Padua University, Italy Illycaffè SpA, Trieste, Italy
a r t i c l e
i n f o
Article history: Received 16 April 2012 Accepted 30 October 2012 Available online 11 December 2012 Keywords: Apoptosis Coffee Oxidative DNA damage Telomere
a b s t r a c t Background: Coffee is associated with a reduced risk of hepatocellular carcinoma in patients with chronic C hepatitis. This prospective trial was aimed at assessing the mechanisms underlying coffee-related protective effects. Methods: Forty patients with chronic hepatitis C were randomized into two groups: the first consumed 4 cups of coffee/day for 30 days, while the second remained coffee “abstinent”. At day 30, the groups were switched over for a second month. Results: At baseline, aspartate aminotransferase and alanine aminotransferase were lower in patients drinking 3–5 (Group B) than 0–2 cups/day (Group A) (56 ± 6 vs 74 ± 11/60 ± 3 vs 73 ± 7 U/L p = 0.05/p = 0.04, respectively). HCV-RNA levels were significantly higher in Group B [(6.2 ± 1.5) × 105 vs (3.9 ± 1.0) × 105 UI/mL, p = 0.05]. During coffee intake, 8-hydroxydeoxyguanosine and collagen levels were significantly lower than during abstinence (15 ± 3 vs 44 ± 16 8hydroxydeoxyguanosine/105 deoxyguanosine, p = 0.05 and 56 ± 9 vs 86 ± 21 ng/mL, p = 0.04). Telomere length was significantly higher in patients during coffee intake (0.68 ± 0.06 vs 0.48 ± 0.04 Arbitrary Units, p = 0.006). Telomere length and 8-hydroxydeoxyguanosine were inversely correlated. Conclusion: In chronic hepatitis C coffee consumption induces a reduction in oxidative damage, correlated with increased telomere length and apoptosis, with lower collagen synthesis, factors that probably mediate the protection exerted by coffee with respect to disease progression. © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction The debate about the possible risks and the potential beneficial effects of coffee consumption is open, but sound epidemiological evidence has accumulated regarding a protective role of coffee drinking towards cirrhosis development and/or progression to hepatocellular carcinoma (HCC). In particular in Hepatitis C Virus (HCV) related liver damage [1,2] coffee consumption has been linked to lower disease activity and a reduced risk of mortality for liver cirrhosis [3,4]. With respect to HCC, several studies have reported a 2-to-4 folds reduction in the risk of developing liver cancer [5–8] with coffee consumption, particularly in patients with HCV infection, even though the possibility of potential confounding has not been completely ruled out. A relatively recent meta-analysis confirmed a 2 times lower relative risk of HCC in
∗ Corresponding author at: Dipartimento di Scienze Chirurgiche, Oncologiche e Gastroenterologiche, Policlinico Universitario, Via Giustiniani 2, 35128 Padova, Italy. Tel.: +39 049 8211305; fax: +39 049 8760820. E-mail address: [email protected] (F. Farinati).
the presence of coffee consumption [9,10] and only the lack of randomized prospective studies on the topic prevents from considering the protective effect of coffee with respect to HCC as fully ascertained. The mechanisms underlying this beneficial effect of coffee consumption are still, at least in part, unclear [11] but, coffee antioxidant activity is the more frequently advocated. Whether this anti-oxidant activity is mediated by caffeine itself [12], by one of its metabolic derivatives [13] by other compounds contained in coffee, such as the diterpenes kahweol or cafestol [14], is again still open to debate. Over the last 15 years, the attention of our research group has been focused on the relationship between HCV infection and liver oxidative damage [15]. Chronic hepatitis C is characterized by increased reactive oxygen species (ROS) production [16,17], with depletion in anti-oxidants and accumulation of oxidative DNA damage [18], in turn correlated with modulation of growth factors, oncogene activation [19,20] and deranged proliferation/apoptosis ratio [21–23]. Overall, oxidative DNA damage represents a significant and independent risk factor for HCC in chronic HCV hepatitis [24].
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Cell DNA can be damaged by ROS, particularly in its telomeric segments. Telomeres are indeed particularly rich in guanine residues and, under ROS attack, 8-hydroxydeoxyguanosine (8OHdG) formation [25] is an event more common in telomeric DNA. During the early phases of carcinogenesis, DNA damage leads to telomere shortening and chromosome instability. In the later phases of tumour progression, a reactivation of telomerase activity leads to telomere elongation, cell immortalization and, consequently, a higher risk of neoplastic transformation [26,27]. This randomized study was planned to overcome the almost absolute lack of in vivo studies on the effect of coffee exposure in patients with chronic HCV-related hepatitis. The research protocol, characterized by a cross-over design, was aimed at assessing the mechanisms underlying coffee-related protective effect, in particular considering the extent of oxidative DNA damage in relation to liver inflammation, telomere length (TL), fibrosis, apoptosis, angiogenesis and viral load. 2. Patients and methods 2.1. Patients During a 9-month period consecutive patients with HCV-related chronic liver disease referred to the Gastroenterology Unit of Padua outpatient clinic were prospectively recruited for the study. In all patients anti-HCV antibodies were assayed by a thirdgeneration immunoenzymatic test (Ortho Diagnostic Systems, Raritan, NJ) and by confirmatory recombinant immunoblotting assay, Chiron Corp., Emeryville, CA. A standardized genotyping assay (Inno-Lipa HCV III, Innogenetics, Gent, Belgium) was used. Only patients fulfilling the following inclusion criteria were recruited: biopsy-proven (within the previous 24 months) HCVrelated chronic hepatitis or cirrhosis. A clinical diagnosis of cirrhosis was based on the following criteria: International Normalized Ratio (INR) higher than 1.15, White Blood Cell (WBC) lower than 4.40 × 109 /L and platelets (PLTs) below 150 × 109 /L (at least 2/3) and ultrasound (US) examination showing findings compatible with cirrhosis [coarse liver structure, irregular profiles, dilated portal tract, reduced flow velocity (at least 2/4)]. Anti-HCV and HCV-RNA positivity with aspartate aminotransferase/alanine aminotransferase (AST/ALT) at least 1.5 times the upper limit of normal. Ongoing interferon treatment was an exclusion criteria, nonresponders or relapsers to previous treatment were accepted. The study was approved by the Hospital Ethical Committee (Prot. 1755 P, Azienda Ospedaliera di Padova), registered (ClinicalTrials.gov Identifier: NCT01572103) and all participating patients provided written informed consent. From each patient we collected demographical data, information on coffee and smoking habits, alcohol, fruit, vegetable, anti-inflammatory drugs consumption, energy and nutrients intake. All data were assessed by using a quantity-frequency questionnaire designed for use in the present study. Subjects were asked to maintain their alimentary and preferred habits, while other caffeine-containing beverages were forbidden. At baseline, patients were categorized into two groups in relation to their routine coffee intake: Group A, low coffee intake (0–2 cups/day) and Group B, high coffee intake (3–5 cups/day). This cut-off was chosen because in most papers an effect of coffee consumption is observed for over 2 cups/day [3,28].
randomized using a computer-generated list (StatsDirect statistic program) into two groups, with either 4 coffee cups/day intake for 1 month or abstinence for the same time period. Obviously, blinding was not possible and no wash-out period was prescribed, since baseline sampling was examined in relation to the patients’ routine coffee consumption. After 30 days, patients were re-tested, and then exchanged “treatment”: those who were abstinent in the first month were switched to the coffee arm and vice versa, then all patients were reassessed after 30 days. Each patient was therefore tested three times, at baseline, when entering the study, after coffee and following abstinence, each patient being his own control in the two phases. For the sake of simplicity all data obtained following coffee consumption were compared with the data obtained during abstinence, irrespective of the time sequence of exposure/abstinence. As the primary endpoint of the study we identified the changes in 8-OHdG levels and as secondary endpoints any change in the other parameters considered. Considering (on the basis of published data [9]) as expected a coffee effect in 40% of exposed subjects, with 1 control for case, with 5% alpha error and with 80% power, the sample size required was 20 cases and 20 controls, corresponding to the patients’ number in this study. Additionally, given the cross over design of the study in which each patient is investigated twice, during exposure and during abstinence, the actual number of patients investigated is 40 per arm. The patients were given a form in which they registered the compliance with respect to coffee intake, with contacts to report side effects. To reduce the risk of bias due to inter-individual changes in coffee preparation, the patients were provided with Italian-style coffee machine of the stove-top type (MokaTM Bialetti) and the required amount of coffee for 4 coffee cups/day (8 g for each coffee). The coffee brand was a 100% Coffee arabica product from the shelf. We obtained coffee analyses by a contract lab (Eurofins analytic GmbH – Hamburg, Germany). The brewing recipe is of 5.5 g of coffee powder in 55 mL and the brew’s content is as follows: caffeine (High Performance Liquid Chromatography–Diode-Array Detection, HPLC-DAD) 895 mg/L (49 mg/cup), chlorogenic acids (HPLC) 786 mg/L (43 mg/cup), cafestol (Reverse Phase (RP)-HPLC) 45 mg/L (2.5 mg/cup), kahweol (RP-HPLC) 32 mg/L (1.8 mg/cup), vitamin B3 (HPLC with Postcolumn Fluorescence Derivatization (FLD)) 88 mg/L (4.8 mg/cup). The patients were instructed not to drink any differently prepared coffee and to report any deviation.
2.3. Methods Blood samples were collected and stored at −20 ◦ C for no longer than 3 weeks. In this study the following parameters were evaluated: AST/ALT, gamma-glutamyl-transpeptidase (␥GT) and alkaline phosphatase (ALP) levels; viral load (HCV-RNA titre), markers of oxidative damage: 8-OHdG, nitric oxide (NO), Advanced Oxidation Protein Products (AOPP); marker of genomic stability: TL; markers of cell death: Cytokeratin18 Aspartic acid 396 neo epitope (CK18Asp396)/Cytokeratin18 (CK18); markers of liver fibrosis and angiogenesis: Procollagen Type III (PIIINP) and Vascular Endothelial Growth Factor (VEGF).
2.2. Study design
2.4. HCV-RNA determination
Fig. 1 gives a schematic overview of the cross-over design of the randomized trial. The patients were assessed at baseline and then
HCV-RNA in serum was assessed by amplification with Polymerase Chain Reaction (PCR) of the 5 untranslated region (5 -UTR)
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Fig. 1. Flow chart of randomized trial. Each patient was tested at baseline, after 30 days of coffee exposure (4 cups/day), and after 30 days of coffee abstinence. Each patient was his/her control. For simplicity results of coffee exposure and abstinence were considered overall, irrespective of the order in which they were scheduled.
of HCV, the most conserved region of the virus. The amplification was performed by ‘nested PCR’. 2.5. 8-OHdG determination This assay was carried out as previously described [20] in 3 steps: (i) genomic DNA extraction using a Wizard Genomic DNA Purification Kit (Promega Italia, Milano); (ii) nuclease P1 and ALP hydrolysis of DNA; (iii) 8-OHdG determination using an HPLC equipped with an electrochemical detector (HPLC-ED) (ESA Coulochem II 5200 A, Bedford, MA). The 8-OHdG levels were expressed as the number of 8-OHdG adduct/105 deoxyguanosine (dG).
5 -TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTATel-2: 3 ) and the other for 36B4, used as the single copy gene (36B4u: 5 -CAGCAAGTGGGAAGGTGTAATCC-3 , 36b4d: 5 CCCATTCTATCATCAACGGGTACAA-3 ). Each 25 L PCR reaction included 40 ng of DNA, SYBR Green Master Mix (Applied Biosystems, Foster City, CA) and primers at final concentrations 270 nM Tel-1 and 900 nM Tel-2 or 300 nM 36B4u and 500 nM 36B4d, respectively. PCR amplification was performed in a ABI PRISM 7900 (Applied Biosystem). In each plate, a standard curve was produced ranging from 4 to 80 ng of human reference DNA (Applied Biosystem). All samples were assayed in triplicate to test the reproducibility, with a negative control in each.
2.6. NO determination Serum NO levels were assayed spectrophotometrically by measuring the accumulation of NO stable degradation products, nitrate and nitrite (Oxford Biomedical Research, USA). NO levels were expressed as mol/L. 2.7. AOPP determination Plasma AOPP levels, expressed as mol/L of chloramines-T equivalents, were measured by a spectrophotometric method performed by Witko-Sarsat [29].
2.9. Cell death determination The M30-Apoptosense enzyme-linked immunosorbent assay (ELISA) (Peviva, USA) is a one-step in vitro immunoassay for the quantitative determination of the apoptosis-associated CK18Asp396 neo-epitope in plasma. The M30-Apoptosense ELISA was used in combination with M65 ELISA (Peviva, USA), for the quantitative determination of total soluble CK-18 released from dead cells (necrotic and apoptotic). The concentration of the antigens was expressed as U/L.
2.8. TL analysis by quantitative PCR
2.10. PIIINP and VEGF determination
Genomic DNA extracted was also used to measure TL by using Cawthon’s method [30]. Two 96-well plates were prepared for each experiment, one containing telomere primers (Tel5 -GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT-3 , 1:
Plasma PIIINP levels were determined using ELISA kit (USCN, China). PIIINP levels were expressed as ng/mL. Serum VEGF was determined using an ELISA kit (Bender MedSystems, Austria), data being expressed in ng/L.
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Table 1 Characteristics of the study group. N (%) Number of patients Male sex Mean age (years) ± SD Mean BMI ± SD Ishak Histological grading (mean ± SD) Ishak Histological staging (mean ± SD) Genotype 1 Naive Previous antiviral treatment Routine coffee intake Low (0–2 cups of coffee/day) High (3–5 cups of coffee/day) Dietary habits Food supplements Vitamins supplements Low fruit intake (0–2 portions/day) High fruit intake (3–5 portions/day) Vegetable intake (0–2 portions/day) Current smokers Current alcohol drinkers
37 29 (78%) 58 ± 11 26 ± 5 2 ± 1.6 2 ± 0.8 26 (70%) 14 (38%) 23 (62%) 17 (46%) 20 (54%) 7 (19%) 8 (22%) 27 (73%) 10 (27%) 37 (100%) 8 (22%) 3 (8%)
Food supplements: enriched cereals, protein and mineral salts nutritional supplements. Vitamins supplements: vitamin D, antioxidants as ascorbic acid (vitamin C) and vitamin E, vitamin A, vitamin B.
2.11. Statistical analysis Data were analyzed using paired t-test, one-way ANOVA, Student’s t-test for unpaired data, linear regression and Fisher’s exact test (SPSS, STATSDIRECT) with statistical significance set at p < 0.05 (2-tailed). Given the cross-over design of the study, all comparison regarding coffee exposure and abstinence were carried out within patients and not within groups, using, as said, a paired data approach. 3. Results Forty patients were enrolled, though 37 patients were analyzed because three participants discontinued the study for reasons unrelated to the study protocol. The characteristics of the participants completing the study are listed in Table 1. 3.1. Results at baseline At baseline the patients were categorized into two groups in relation to their routine coffee intake: Group A, low coffee intake (0–2 cups/day) and Group B, high coffee intake (3–5 cups/day). At baseline mean AST, ALP and ␥GT levels were lower in Group B than in Group A (p = 0.05, p = 0.04 and p = 0.08, respectively). HCV-RNA quantitative mean levels were higher in Group B than in Group A (p = 0.05). No difference was observed with respect to the remaining considered parameters (Table 2). 3.2. Results after coffee intake and abstinence in all patients The patients’ compliance was excellent (95%), with only two non-compliers, one assuming 6 cups of coffee and one 2, the latter reporting tachycardia. With the above exceptions, no patient, including teetotalers, reported side-effects due to coffee consumption/abstinence. The significant results obtained are summarized in Table 3. Higher AST levels were detected in patients administered with coffee than in abstainers (69 ± 7 vs 63 ± 7 U/L, p = 0.005 by a paired t-test). Levels of gGT were significantly lower during coffee
Table 2 Liver function tests, HCV-RNA and 8-OHdG determination at baseline and in relation to routine coffee consumption in 37 patients. Group A: 0–2 coffee cups/day; Group B: 3–5 coffee cups/day.
Number of patients AST (U/L) ALT (U/L) ALP (U/L) ␥GT (U/L) HCV-RNA (UI/mL) 8-OHdG (no. add/105 dG)
Group A
Group B
p-Value
17 74 ± 11 39 ± 7 73 ± 7 68 ± 13 (3.9 ± 1.0) × 105 46 ± 23
20 56 ± 6 50 ± 14 60 ± 3 46 ± 8 (6.2 ± 1.5) × 105 27 ± 9
– 0.05 – 0.04 0.08 0.05 –
Data are reported as mean ± SEM and examined by Student’s t test for unpaired data. AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; ␥GT, gamma-glutamyl-transpeptidase; HCV-RNA, hepatitis C viral load; 8-OHdG, 8-hydroxydeoxyguanosine; no. add/105 dG, no. adducts/105 deoxyguanosine.
exposure than without coffee intake (55 ± 8 vs 63 ± 10 U/L p = 0.05 by a paired t-test). 3.2.1. Markers of oxidative damage 8-OHdG levels were significantly lower during coffee intake (15 ± 3 vs 44 ± 16 no. add/105 dG p = 0.05 by a paired t-test), with almost a three-fold decrease. With respect to baseline, the intake of 4 cups of coffee/day leads to a reduction in 8-OHdG levels (Fisher’s exact test, p = 0.02). 3.2.2. HCV-RNA Mean serum HCV-RNA levels were significantly higher during coffee intake than during the abstinence period [(7.9 ± 1.2) × 105 vs (4.8 ± 1.1) × 105 UI/mL, p = 0.002 by a paired t-test]. Only one patient had changes greater than one logarithm. When compared to abstainers, patients drinking 4 cups of coffee/day more frequently presented an increase in HCV-RNA titre (OR = 3.5, 95% CI 1.3–9.3, p = 0.01). This increased OR was confirmed also in comparison with the baseline levels (Fisher’s exact test, p = 0.018). The increase in serum HCV-RNA levels was significantly more pronounced in patients with genotype 1 HCV than in others genotypes, when calculated as the [(4 coffee cups) − (0 coffee cups)] in viral load (p = 0.05) (data not shown). No correlation between HCV-RNA levels and AST/ALT levels was observed. 3.2.3. Markers of genomic stability Telomere length (TL) was significantly higher in patients during coffee intake (0.68 ± 0.06 vs 0.48 ± 0.04, Arbitrary Unit, p = 0.006 by a paired t-test). A significant negative correlation was found between TL and 8-OHdG levels when considering the patients during coffee consumption (r = −0.44, p = 0.03). Additionally, TL and 8-OHdG showed a significant concordant (p = 0.007) trend with respect to coffee exposure/abstinence, with 8-OHdG levels dropping and TL increasing under coffee administration. Regarding the apoptosis marker CK-18, a statistically significant difference was observed during coffee intake with respect to abstinence (712 ± 89 vs 649 ± 93 U/L, p = 0.04 by a paired t-test). A significant positive correlation was found between the apoptosis index and HCV-RNA levels, but only in patients assuming coffee (r = 0.35, p = 0.03). 3.2.4. Markers of liver fibrosis and angiogenesis The mean levels of PIIINP, a marker of collagen synthesis and fibrosis accumulation, were significantly lower during coffee intake than during abstinence (56 ± 9 vs 86 ± 21 ng/mL, p = 0.04 by a paired t-test).
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Table 3 Data obtained during the 2 phases of the study, 30 days of coffee consumption and 30 days of coffee abstinence.
Number of patients AST (U/L) ␥GT (U/L) 8-OHdG (no. add/105 dG) HCV-RNA (UI/mL) TL (Arbitrary Unit) CK-18 (U/L) PIIINP (ng/mL)
4 cups of coffee/day for 30 days
No coffee for 30 days
p-Value
37 69 ± 7 55 ± 8 15 ± 3 (7.9 ± 1.2) × 105 0.68 ± 0.06 712 ± 89 56 ± 9
37 63 ± 7 63 ± 10 44 ± 16 (4.8 ± 1.1) × 105 0.48 ± 0.04 649 ± 93 86 ± 21
0.005 0.05 0.05 0.002 0.006 0.04 0.04
Only significant results are reported in the table, data are reported as mean ± SEM and examined by paired t-test. AST, aspartate aminotransferase; ␥GT: gamma-glutamyltranspeptidase; 8-OHdG, 8-hydroxydeoxyguanosine; no. add/105 dG, no. adducts/105 deoxyguanosine; HCV-RNA, hepatitis C viral load; TL, telomere length; CK-18, Cytokeratin18; PIIINP, Procollagen Type III.
All the remaining parameters investigated (AOPP, NO, CK18/CK18Asp396) demonstrated no significant change in relation to coffee intake/abstinence, while the circulating VEGF showed a trend towards lower levels when the patients were exposed to coffee (p = 0.1) (data not shown). According to the hypothesis for the sample size calculation, a change was observed in 88% of the patients with respect to the primary end-point, 8-OHdG levels, in 89% with respect to telomere length, 81% with respect to apoptosis (CK-18) and 70% with respect to collagen synthesis (PIIINP).
4. Discussion The present study, designed to investigate the mechanisms underlying the effect of coffee consumption in patients with chronic hepatitis C, allowed identification of some interesting effects of coffee, both as a routine habit and as a programmed, acute, exposure. To reduce the risk of bias the patients were provided with the same type of coffee, same brewing machines and instructed to report any deviation from the protocol, not to change their routine alimentary and voluptuary habits and finally to report any consumption of drugs and anti-oxidants. The compliance to the study protocol was actually very high and only two patients slightly deviated. A longer exposure/abstinence might have induced deeper changes but with probably a lower compliance by the patients, mostly used to drinking coffee frequently. In this study, patients with a higher routine consumption of coffee showed significantly lower AST and ALP serum levels and with a higher HCV-RNA titre. The effects of coffee consumption on the two parameters were then assessed during the two test periods: HCVRNA levels significantly increased during coffee consumption while lower ␥GT and slightly higher AST levels were observed during acute coffee consumption. No clear explanation is available for coffee selectively modifying AST levels after both acute and chronic administration. The respective roles of AST and ALT levels in patients with chronic liver damage are still debatable but for the role of AST/ALT ratio in cirrhotics [31]. A recent study [32] identified AST as an independent predictor of inflammation in HCV-related hepatitis in patients with mildly elevated aminotransferase levels. Also the patients we investigated showed relatively low levels of transaminases and it may well be that in this subgroup of HCV patients, AST more clearly reflects the extent of liver damage and, consequently, the effects of coffee consumption. An acute exposure to coffee might well induce different changes from a chronic one. A different and differently mediated effect of chronic and acute caffeine exposure has been demonstrated for instance for cerebral haemodynamics [33] where acute caffeine administration results in A1 and A2A adenosine receptors activation, while chronic exposure seem to affect only A2A.
Indeed, in this study opposite results were obtained with respect to AST after chronic and acute coffee exposure, but the changes observed were quite limited, and irrelevant from the clinical point of view. The intriguing correlation between coffee exposure and higher HCV-RNA titres was instead confirmed both in chronic and acute coffee exposure. No data have been previously reported on this novel finding that requires some clinical considerations: the increase is almost never higher than one logarithm and is not therefore clinically significant [34] and, in contrast with what happens in HBV-related damage, in HCV infection viral load is not a prognostic factor [35]. Finally HCV-RNA titre does not correlate, in this study, with transaminase levels. Coffee exposure significantly increased apoptosis and, given the correlation between viral load and apoptosis, the increase in viral load following coffee exposure might well represent a consequence of liver cell death, with release of viral RNA into the blood from apoptotic hepatocytes. Previously published data confirm a role of coffee in cell apoptosis [14,36] but additional mechanisms leading to the modulation of viral replication rate cannot be excluded. During coffee consumption a significant reduction of oxidative DNA damage was observed confirming our working hypothesis, i.e. that the protection exerted by coffee with respect to HCC might be mediated by a reduced accumulation of oxidized bases in cell DNA, and consequently, of DNA mutations. This reduction was observed in 88% of the investigated patients, well above the target of 40% hypothesized to define the sample size. The relevance of oxidative damage in carcinogenesis [37] and, in particular in patients with HCV infection [15,38], is well known. That coffee consumption is associated with lower oxidative DNA damage was recently reported also by Miˇsík [39] in healthy volunteers. We here demonstrated in vivo, in patients with HCV infection, that coffee exposure significantly limits the extent of oxidative DNA damage in circulating leukocytes, but 8-OHdG levels from leukocytes and liver tissues, in patients with chronic HCV-mediated damage, strictly correlate [16]. Oxidative DNA damage in telomeric sequences leads to telomere shortening, an event linked to chromosomal instability [27]. One month coffee exposure induced, in this study, a 40% increase in telomere length in 89% of the patients, a finding that strongly confirms coffee effects on oxidative DNA damage and indicates that coffee consumption leads to a stabilization of chromosomal DNA, thus protecting from neoplastic evolution. Finally, we observed during coffee exposure a significant reduction in the levels of pro-collagen III, a serum marker of fibrosis [40], involving 70% of the patients. This reduction indicates a drop in collagen deposition in the liver that may, at least in part, justify the slower progression to cirrhosis associated with long term coffee consumption, an effect recently confirmed also in nonalcoholic steatohepatitis [41]. This reduced collagen deposition may be due to caffeine activity in antagonizing adenosine receptors, since adenosine and its receptors have been found to promote liver
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fibrosis [42], or to paraxanthine, a caffeine metabolite, suppressing the expression of the pro-fibrogenic connective tissue growth factor (CTGF) [13]. In summary, this study demonstrates that coffee consumption, in patients with chronic hepatitis C, reduces oxidative DNA damage, increases apoptosis, leads to telomere elongation and DNA stabilization and finally reduces pro-collagen III deposition. Such a set of factors may well play a major role in reducing the risk of disease progression and of evolution to HCC. Since a number of studies demonstrated that caffeine can modulate both innate and adaptive immunity [43], and given that, for viral clearance, both immune responses are required, the data obtained prompt additional investigations on the effect of coffee on the human immune-response in patients with HCV-related liver damage. Secondly, the respective effects of coffee, of the diterpenes kahweol and cafestol, strong antioxidant, and of furan, a coffee volatile aroma, potentially carcinogenic compound, should be tested in established experimental models of liver damage, again in relation to oxidative damage. Source of support The study was funded, in part, by the Institute of Scientific Information on Coffee (ISIC), which is a scientific consortium of major European Coffee Companies. ISIC had absolutely no role in designing the study, in the collection, analysis and interpretation of data, in either writing or submitting the report. Conflict of interest statement The authors declared no personal conflict of interest and no personal financial relationship with the organization “Institute of Scientific Information on Coffee” (ISIC) partially funding the study. Dr. M. Petracco is an employee of Illy, a coffee factory in Italy. The role of Dr. M. Petracco was to supervise the analyses on the coffee used in the experiments, that was regularly bought. Dr. Petracco also helped in designing the study and evaluating the results obtained. Illy S.p.a had absolutely no role in the study, nor it participated in financing it. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.dld.2012.10.021. References [1] Costentin CE, Roudot-Thoraval F, Zafrani ES, et al. Association of caffeine intake and histological features of chronic hepatitis C. Journal of Hepatology 2011;54:1123–9. [2] Muriel P, Arauz J. Coffee and liver diseases. Fitoterapia 2010;81:297–305. [3] Tverdal A, Skurtveit S. Coffee intake and mortality from liver cirrhosis. Annals of Epidemiology 2003;13:419–23. [4] Stroffolini T, Cotticelli G, Medda E, et al. Interaction of alcohol intake and cofactors on the risk of cirrhosis. Liver International 2010;30:867–70. [5] Gelatti U, Covolo L, Franceschini M, et al. Coffee consumption reduces the risk of hepatocellular carcinoma independently of its aetiology: a case–control study. Journal of Hepatology 2005;42:528–34. [6] Gallus S, Bertuzzi M, Tavani A, et al. Does coffee protect against hepatocellular carcinoma? British Journal of Cancer 2002;87:956–9. [7] Kurozawa Y, Ogimoto I, Shibata A, et al. Coffee and risk of death from hepatocellular carcinoma in a large cohort study in Japan. British Journal of Cancer 2005;93:607–10. [8] Inoue M, Yoshimi I, Sobue T, et al. Influence of coffee drinking on subsequent risk of hepatocellular carcinoma: a prospective study in Japan. Journal of the National Cancer Institute 2005;97:293–300. [9] Bravi F, Bosetti C, Tavani A, et al. Coffee drinking and hepatocellularcarcinoma: an update. Hepatology 2009;50:1317–8. [10] Morgan TR. Chemoprevention of hepatocellular carcinoma in chronic hepatitis C. Recent Results in Cancer Research 2011;188:85–99. [11] Masterton GS, Hayes PC. Coffee and the liver: a potential treatment for liver disease? European Journal of Gastroenterology and Hepatology 2010;22:1277–83.
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[22] Farinati F, Cardin R, Bortolami M, et al. Up and down regulation of apoptosis in hepatitis C virus-related liver damage. Journal of Hepatology 2004;41:883–5. [23] Kotsafti A, Farinati F, Cardin R, et al. Bax inhibitor-1 down-regulation in the progression of chronic liver diseases. BMC Gastroenterology 2010;10:35. [24] Chuma M, Hige S, Nakanishi M, et al. 8-Hydroxy-2 -deoxy-guanosine is a risk factor for development of hepatocellular carcinoma in patients with chronic hepatitis C virus infection. Journal of Gastroenterology and Hepatology 2008;23:1431–6. [25] Oikawa S, Kawanishi S. Site-specific DNA damage at GGG sequence by oxidative stress may accelerate telomere shortening. FEBS Letters 1999;453:365–8. [26] Artandi SE, DePinho RA. Telomeres and telomerase in cancer. Carcinogenesis 2010;31:9–18. [27] Rudolph KL, Hartmann D, Opitz OG. Telomere dysfunction and DNA damage checkpoints in diseases and cancer of the gastrointestinal tract. Gastroenterology 2009;137:754–62. [28] Zani C, Donato F, Chiesa M, et al. Alcohol and coffee drinking and smoking habit among subjects with HCV infection. Digestive and Liver Disease 2009;41:599–604. [29] Witko-Sarsat V, Friedlander M, Capeillère-Blandin C, et al. Advanced oxidation protein products as a novel marker of oxidative stress in uremia. Kidney International 1996;49:1304–13. [30] Cawthon RM. Telomere measurement by quantitative PCR. Nucleic Acids Research 2002;30:e47. [31] Fontana RJ, Lok AS. Noninvasive monitoring of patients with chronic hepatitis C. Hepatology 2002;36:S57–64. [32] Cheong JY, Kim DJ, Hwang SG, et al. Serum markers for necroinflammatory activity in patients with chronic viral hepatitis and normal or mildly elevated aminotransferase levels. Liver International 2011;31:1352–8. [33] Pellegrino DA, Xu HL, Vetri F. Caffeine and the control of cerebral hemodynamics. Journal of Alzheimer’s Disease 2010;20:S51–62. [34] Deltenre P, Corouge M, Canva V, et al. Usefulness of viral kinetics for early prediction of a sustained virological response in HCV-1 non-responders re-treated with pegylated interferon and ribavirin. Journal of Hepatology 2011;55:989–95. [35] Massard J, Ratziu V, Thabut D, et al. Natural history and predictors of disease severity in chronic hepatitis C. Journal of Hepatology 2006;44:S19–24. [36] Choi MJ, Park EJ, Oh JH, et al. Cafestol, a coffee-specific diterpene, induces apoptosis in renal carcinoma Caki cells through down-regulation of antiapoptotic proteins and Akt phosphorylation. Chemico-Biological Interactions 2011;190:102–8. [37] Kryston TB, Georgiev AB, Pissis P, et al. Role of oxidative stress and DNA damage in human carcinogenesis. Mutation Research 2011;711:193–201. [38] Moriya K, Nakagawa K, Santa T, et al. Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis. Cancer Research 2001;61:4365–70. [39] Miˇsík M, Hoelzl C, Wagner KH, et al. 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Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld
Liver, Pancreas and Biliary Tract
Effects of coffee consumption in chronic hepatitis C: A randomized controlled trial Romilda Cardin a, Marika Piciocchi a, Diego Martines a, Laura Scribano a, Marino Petracco b, Fabio Farinati a,∗ a b
Department of Surgical, Oncological and Gastroenterological Sciences, Section of Gastroenterology, Padua University, Italy Illycaffè SpA, Trieste, Italy
a r t i c l e
i n f o
Article history: Received 16 April 2012 Accepted 30 October 2012 Available online 11 December 2012 Keywords: Apoptosis Coffee Oxidative DNA damage Telomere
a b s t r a c t Background: Coffee is associated with a reduced risk of hepatocellular carcinoma in patients with chronic C hepatitis. This prospective trial was aimed at assessing the mechanisms underlying coffee-related protective effects. Methods: Forty patients with chronic hepatitis C were randomized into two groups: the first consumed 4 cups of coffee/day for 30 days, while the second remained coffee “abstinent”. At day 30, the groups were switched over for a second month. Results: At baseline, aspartate aminotransferase and alanine aminotransferase were lower in patients drinking 3–5 (Group B) than 0–2 cups/day (Group A) (56 ± 6 vs 74 ± 11/60 ± 3 vs 73 ± 7 U/L p = 0.05/p = 0.04, respectively). HCV-RNA levels were significantly higher in Group B [(6.2 ± 1.5) × 105 vs (3.9 ± 1.0) × 105 UI/mL, p = 0.05]. During coffee intake, 8-hydroxydeoxyguanosine and collagen levels were significantly lower than during abstinence (15 ± 3 vs 44 ± 16 8hydroxydeoxyguanosine/105 deoxyguanosine, p = 0.05 and 56 ± 9 vs 86 ± 21 ng/mL, p = 0.04). Telomere length was significantly higher in patients during coffee intake (0.68 ± 0.06 vs 0.48 ± 0.04 Arbitrary Units, p = 0.006). Telomere length and 8-hydroxydeoxyguanosine were inversely correlated. Conclusion: In chronic hepatitis C coffee consumption induces a reduction in oxidative damage, correlated with increased telomere length and apoptosis, with lower collagen synthesis, factors that probably mediate the protection exerted by coffee with respect to disease progression. © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction The debate about the possible risks and the potential beneficial effects of coffee consumption is open, but sound epidemiological evidence has accumulated regarding a protective role of coffee drinking towards cirrhosis development and/or progression to hepatocellular carcinoma (HCC). In particular in Hepatitis C Virus (HCV) related liver damage [1,2] coffee consumption has been linked to lower disease activity and a reduced risk of mortality for liver cirrhosis [3,4]. With respect to HCC, several studies have reported a 2-to-4 folds reduction in the risk of developing liver cancer [5–8] with coffee consumption, particularly in patients with HCV infection, even though the possibility of potential confounding has not been completely ruled out. A relatively recent meta-analysis confirmed a 2 times lower relative risk of HCC in
∗ Corresponding author at: Dipartimento di Scienze Chirurgiche, Oncologiche e Gastroenterologiche, Policlinico Universitario, Via Giustiniani 2, 35128 Padova, Italy. Tel.: +39 049 8211305; fax: +39 049 8760820. E-mail address: [email protected] (F. Farinati).
the presence of coffee consumption [9,10] and only the lack of randomized prospective studies on the topic prevents from considering the protective effect of coffee with respect to HCC as fully ascertained. The mechanisms underlying this beneficial effect of coffee consumption are still, at least in part, unclear [11] but, coffee antioxidant activity is the more frequently advocated. Whether this anti-oxidant activity is mediated by caffeine itself [12], by one of its metabolic derivatives [13] by other compounds contained in coffee, such as the diterpenes kahweol or cafestol [14], is again still open to debate. Over the last 15 years, the attention of our research group has been focused on the relationship between HCV infection and liver oxidative damage [15]. Chronic hepatitis C is characterized by increased reactive oxygen species (ROS) production [16,17], with depletion in anti-oxidants and accumulation of oxidative DNA damage [18], in turn correlated with modulation of growth factors, oncogene activation [19,20] and deranged proliferation/apoptosis ratio [21–23]. Overall, oxidative DNA damage represents a significant and independent risk factor for HCC in chronic HCV hepatitis [24].
1590-8658/$36.00 © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dld.2012.10.021
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Cell DNA can be damaged by ROS, particularly in its telomeric segments. Telomeres are indeed particularly rich in guanine residues and, under ROS attack, 8-hydroxydeoxyguanosine (8OHdG) formation [25] is an event more common in telomeric DNA. During the early phases of carcinogenesis, DNA damage leads to telomere shortening and chromosome instability. In the later phases of tumour progression, a reactivation of telomerase activity leads to telomere elongation, cell immortalization and, consequently, a higher risk of neoplastic transformation [26,27]. This randomized study was planned to overcome the almost absolute lack of in vivo studies on the effect of coffee exposure in patients with chronic HCV-related hepatitis. The research protocol, characterized by a cross-over design, was aimed at assessing the mechanisms underlying coffee-related protective effect, in particular considering the extent of oxidative DNA damage in relation to liver inflammation, telomere length (TL), fibrosis, apoptosis, angiogenesis and viral load. 2. Patients and methods 2.1. Patients During a 9-month period consecutive patients with HCV-related chronic liver disease referred to the Gastroenterology Unit of Padua outpatient clinic were prospectively recruited for the study. In all patients anti-HCV antibodies were assayed by a thirdgeneration immunoenzymatic test (Ortho Diagnostic Systems, Raritan, NJ) and by confirmatory recombinant immunoblotting assay, Chiron Corp., Emeryville, CA. A standardized genotyping assay (Inno-Lipa HCV III, Innogenetics, Gent, Belgium) was used. Only patients fulfilling the following inclusion criteria were recruited: biopsy-proven (within the previous 24 months) HCVrelated chronic hepatitis or cirrhosis. A clinical diagnosis of cirrhosis was based on the following criteria: International Normalized Ratio (INR) higher than 1.15, White Blood Cell (WBC) lower than 4.40 × 109 /L and platelets (PLTs) below 150 × 109 /L (at least 2/3) and ultrasound (US) examination showing findings compatible with cirrhosis [coarse liver structure, irregular profiles, dilated portal tract, reduced flow velocity (at least 2/4)]. Anti-HCV and HCV-RNA positivity with aspartate aminotransferase/alanine aminotransferase (AST/ALT) at least 1.5 times the upper limit of normal. Ongoing interferon treatment was an exclusion criteria, nonresponders or relapsers to previous treatment were accepted. The study was approved by the Hospital Ethical Committee (Prot. 1755 P, Azienda Ospedaliera di Padova), registered (ClinicalTrials.gov Identifier: NCT01572103) and all participating patients provided written informed consent. From each patient we collected demographical data, information on coffee and smoking habits, alcohol, fruit, vegetable, anti-inflammatory drugs consumption, energy and nutrients intake. All data were assessed by using a quantity-frequency questionnaire designed for use in the present study. Subjects were asked to maintain their alimentary and preferred habits, while other caffeine-containing beverages were forbidden. At baseline, patients were categorized into two groups in relation to their routine coffee intake: Group A, low coffee intake (0–2 cups/day) and Group B, high coffee intake (3–5 cups/day). This cut-off was chosen because in most papers an effect of coffee consumption is observed for over 2 cups/day [3,28].
randomized using a computer-generated list (StatsDirect statistic program) into two groups, with either 4 coffee cups/day intake for 1 month or abstinence for the same time period. Obviously, blinding was not possible and no wash-out period was prescribed, since baseline sampling was examined in relation to the patients’ routine coffee consumption. After 30 days, patients were re-tested, and then exchanged “treatment”: those who were abstinent in the first month were switched to the coffee arm and vice versa, then all patients were reassessed after 30 days. Each patient was therefore tested three times, at baseline, when entering the study, after coffee and following abstinence, each patient being his own control in the two phases. For the sake of simplicity all data obtained following coffee consumption were compared with the data obtained during abstinence, irrespective of the time sequence of exposure/abstinence. As the primary endpoint of the study we identified the changes in 8-OHdG levels and as secondary endpoints any change in the other parameters considered. Considering (on the basis of published data [9]) as expected a coffee effect in 40% of exposed subjects, with 1 control for case, with 5% alpha error and with 80% power, the sample size required was 20 cases and 20 controls, corresponding to the patients’ number in this study. Additionally, given the cross over design of the study in which each patient is investigated twice, during exposure and during abstinence, the actual number of patients investigated is 40 per arm. The patients were given a form in which they registered the compliance with respect to coffee intake, with contacts to report side effects. To reduce the risk of bias due to inter-individual changes in coffee preparation, the patients were provided with Italian-style coffee machine of the stove-top type (MokaTM Bialetti) and the required amount of coffee for 4 coffee cups/day (8 g for each coffee). The coffee brand was a 100% Coffee arabica product from the shelf. We obtained coffee analyses by a contract lab (Eurofins analytic GmbH – Hamburg, Germany). The brewing recipe is of 5.5 g of coffee powder in 55 mL and the brew’s content is as follows: caffeine (High Performance Liquid Chromatography–Diode-Array Detection, HPLC-DAD) 895 mg/L (49 mg/cup), chlorogenic acids (HPLC) 786 mg/L (43 mg/cup), cafestol (Reverse Phase (RP)-HPLC) 45 mg/L (2.5 mg/cup), kahweol (RP-HPLC) 32 mg/L (1.8 mg/cup), vitamin B3 (HPLC with Postcolumn Fluorescence Derivatization (FLD)) 88 mg/L (4.8 mg/cup). The patients were instructed not to drink any differently prepared coffee and to report any deviation.
2.3. Methods Blood samples were collected and stored at −20 ◦ C for no longer than 3 weeks. In this study the following parameters were evaluated: AST/ALT, gamma-glutamyl-transpeptidase (␥GT) and alkaline phosphatase (ALP) levels; viral load (HCV-RNA titre), markers of oxidative damage: 8-OHdG, nitric oxide (NO), Advanced Oxidation Protein Products (AOPP); marker of genomic stability: TL; markers of cell death: Cytokeratin18 Aspartic acid 396 neo epitope (CK18Asp396)/Cytokeratin18 (CK18); markers of liver fibrosis and angiogenesis: Procollagen Type III (PIIINP) and Vascular Endothelial Growth Factor (VEGF).
2.2. Study design
2.4. HCV-RNA determination
Fig. 1 gives a schematic overview of the cross-over design of the randomized trial. The patients were assessed at baseline and then
HCV-RNA in serum was assessed by amplification with Polymerase Chain Reaction (PCR) of the 5 untranslated region (5 -UTR)
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Fig. 1. Flow chart of randomized trial. Each patient was tested at baseline, after 30 days of coffee exposure (4 cups/day), and after 30 days of coffee abstinence. Each patient was his/her control. For simplicity results of coffee exposure and abstinence were considered overall, irrespective of the order in which they were scheduled.
of HCV, the most conserved region of the virus. The amplification was performed by ‘nested PCR’. 2.5. 8-OHdG determination This assay was carried out as previously described [20] in 3 steps: (i) genomic DNA extraction using a Wizard Genomic DNA Purification Kit (Promega Italia, Milano); (ii) nuclease P1 and ALP hydrolysis of DNA; (iii) 8-OHdG determination using an HPLC equipped with an electrochemical detector (HPLC-ED) (ESA Coulochem II 5200 A, Bedford, MA). The 8-OHdG levels were expressed as the number of 8-OHdG adduct/105 deoxyguanosine (dG).
5 -TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTATel-2: 3 ) and the other for 36B4, used as the single copy gene (36B4u: 5 -CAGCAAGTGGGAAGGTGTAATCC-3 , 36b4d: 5 CCCATTCTATCATCAACGGGTACAA-3 ). Each 25 L PCR reaction included 40 ng of DNA, SYBR Green Master Mix (Applied Biosystems, Foster City, CA) and primers at final concentrations 270 nM Tel-1 and 900 nM Tel-2 or 300 nM 36B4u and 500 nM 36B4d, respectively. PCR amplification was performed in a ABI PRISM 7900 (Applied Biosystem). In each plate, a standard curve was produced ranging from 4 to 80 ng of human reference DNA (Applied Biosystem). All samples were assayed in triplicate to test the reproducibility, with a negative control in each.
2.6. NO determination Serum NO levels were assayed spectrophotometrically by measuring the accumulation of NO stable degradation products, nitrate and nitrite (Oxford Biomedical Research, USA). NO levels were expressed as mol/L. 2.7. AOPP determination Plasma AOPP levels, expressed as mol/L of chloramines-T equivalents, were measured by a spectrophotometric method performed by Witko-Sarsat [29].
2.9. Cell death determination The M30-Apoptosense enzyme-linked immunosorbent assay (ELISA) (Peviva, USA) is a one-step in vitro immunoassay for the quantitative determination of the apoptosis-associated CK18Asp396 neo-epitope in plasma. The M30-Apoptosense ELISA was used in combination with M65 ELISA (Peviva, USA), for the quantitative determination of total soluble CK-18 released from dead cells (necrotic and apoptotic). The concentration of the antigens was expressed as U/L.
2.8. TL analysis by quantitative PCR
2.10. PIIINP and VEGF determination
Genomic DNA extracted was also used to measure TL by using Cawthon’s method [30]. Two 96-well plates were prepared for each experiment, one containing telomere primers (Tel5 -GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT-3 , 1:
Plasma PIIINP levels were determined using ELISA kit (USCN, China). PIIINP levels were expressed as ng/mL. Serum VEGF was determined using an ELISA kit (Bender MedSystems, Austria), data being expressed in ng/L.
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Table 1 Characteristics of the study group. N (%) Number of patients Male sex Mean age (years) ± SD Mean BMI ± SD Ishak Histological grading (mean ± SD) Ishak Histological staging (mean ± SD) Genotype 1 Naive Previous antiviral treatment Routine coffee intake Low (0–2 cups of coffee/day) High (3–5 cups of coffee/day) Dietary habits Food supplements Vitamins supplements Low fruit intake (0–2 portions/day) High fruit intake (3–5 portions/day) Vegetable intake (0–2 portions/day) Current smokers Current alcohol drinkers
37 29 (78%) 58 ± 11 26 ± 5 2 ± 1.6 2 ± 0.8 26 (70%) 14 (38%) 23 (62%) 17 (46%) 20 (54%) 7 (19%) 8 (22%) 27 (73%) 10 (27%) 37 (100%) 8 (22%) 3 (8%)
Food supplements: enriched cereals, protein and mineral salts nutritional supplements. Vitamins supplements: vitamin D, antioxidants as ascorbic acid (vitamin C) and vitamin E, vitamin A, vitamin B.
2.11. Statistical analysis Data were analyzed using paired t-test, one-way ANOVA, Student’s t-test for unpaired data, linear regression and Fisher’s exact test (SPSS, STATSDIRECT) with statistical significance set at p < 0.05 (2-tailed). Given the cross-over design of the study, all comparison regarding coffee exposure and abstinence were carried out within patients and not within groups, using, as said, a paired data approach. 3. Results Forty patients were enrolled, though 37 patients were analyzed because three participants discontinued the study for reasons unrelated to the study protocol. The characteristics of the participants completing the study are listed in Table 1. 3.1. Results at baseline At baseline the patients were categorized into two groups in relation to their routine coffee intake: Group A, low coffee intake (0–2 cups/day) and Group B, high coffee intake (3–5 cups/day). At baseline mean AST, ALP and ␥GT levels were lower in Group B than in Group A (p = 0.05, p = 0.04 and p = 0.08, respectively). HCV-RNA quantitative mean levels were higher in Group B than in Group A (p = 0.05). No difference was observed with respect to the remaining considered parameters (Table 2). 3.2. Results after coffee intake and abstinence in all patients The patients’ compliance was excellent (95%), with only two non-compliers, one assuming 6 cups of coffee and one 2, the latter reporting tachycardia. With the above exceptions, no patient, including teetotalers, reported side-effects due to coffee consumption/abstinence. The significant results obtained are summarized in Table 3. Higher AST levels were detected in patients administered with coffee than in abstainers (69 ± 7 vs 63 ± 7 U/L, p = 0.005 by a paired t-test). Levels of gGT were significantly lower during coffee
Table 2 Liver function tests, HCV-RNA and 8-OHdG determination at baseline and in relation to routine coffee consumption in 37 patients. Group A: 0–2 coffee cups/day; Group B: 3–5 coffee cups/day.
Number of patients AST (U/L) ALT (U/L) ALP (U/L) ␥GT (U/L) HCV-RNA (UI/mL) 8-OHdG (no. add/105 dG)
Group A
Group B
p-Value
17 74 ± 11 39 ± 7 73 ± 7 68 ± 13 (3.9 ± 1.0) × 105 46 ± 23
20 56 ± 6 50 ± 14 60 ± 3 46 ± 8 (6.2 ± 1.5) × 105 27 ± 9
– 0.05 – 0.04 0.08 0.05 –
Data are reported as mean ± SEM and examined by Student’s t test for unpaired data. AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; ␥GT, gamma-glutamyl-transpeptidase; HCV-RNA, hepatitis C viral load; 8-OHdG, 8-hydroxydeoxyguanosine; no. add/105 dG, no. adducts/105 deoxyguanosine.
exposure than without coffee intake (55 ± 8 vs 63 ± 10 U/L p = 0.05 by a paired t-test). 3.2.1. Markers of oxidative damage 8-OHdG levels were significantly lower during coffee intake (15 ± 3 vs 44 ± 16 no. add/105 dG p = 0.05 by a paired t-test), with almost a three-fold decrease. With respect to baseline, the intake of 4 cups of coffee/day leads to a reduction in 8-OHdG levels (Fisher’s exact test, p = 0.02). 3.2.2. HCV-RNA Mean serum HCV-RNA levels were significantly higher during coffee intake than during the abstinence period [(7.9 ± 1.2) × 105 vs (4.8 ± 1.1) × 105 UI/mL, p = 0.002 by a paired t-test]. Only one patient had changes greater than one logarithm. When compared to abstainers, patients drinking 4 cups of coffee/day more frequently presented an increase in HCV-RNA titre (OR = 3.5, 95% CI 1.3–9.3, p = 0.01). This increased OR was confirmed also in comparison with the baseline levels (Fisher’s exact test, p = 0.018). The increase in serum HCV-RNA levels was significantly more pronounced in patients with genotype 1 HCV than in others genotypes, when calculated as the [(4 coffee cups) − (0 coffee cups)] in viral load (p = 0.05) (data not shown). No correlation between HCV-RNA levels and AST/ALT levels was observed. 3.2.3. Markers of genomic stability Telomere length (TL) was significantly higher in patients during coffee intake (0.68 ± 0.06 vs 0.48 ± 0.04, Arbitrary Unit, p = 0.006 by a paired t-test). A significant negative correlation was found between TL and 8-OHdG levels when considering the patients during coffee consumption (r = −0.44, p = 0.03). Additionally, TL and 8-OHdG showed a significant concordant (p = 0.007) trend with respect to coffee exposure/abstinence, with 8-OHdG levels dropping and TL increasing under coffee administration. Regarding the apoptosis marker CK-18, a statistically significant difference was observed during coffee intake with respect to abstinence (712 ± 89 vs 649 ± 93 U/L, p = 0.04 by a paired t-test). A significant positive correlation was found between the apoptosis index and HCV-RNA levels, but only in patients assuming coffee (r = 0.35, p = 0.03). 3.2.4. Markers of liver fibrosis and angiogenesis The mean levels of PIIINP, a marker of collagen synthesis and fibrosis accumulation, were significantly lower during coffee intake than during abstinence (56 ± 9 vs 86 ± 21 ng/mL, p = 0.04 by a paired t-test).
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Table 3 Data obtained during the 2 phases of the study, 30 days of coffee consumption and 30 days of coffee abstinence.
Number of patients AST (U/L) ␥GT (U/L) 8-OHdG (no. add/105 dG) HCV-RNA (UI/mL) TL (Arbitrary Unit) CK-18 (U/L) PIIINP (ng/mL)
4 cups of coffee/day for 30 days
No coffee for 30 days
p-Value
37 69 ± 7 55 ± 8 15 ± 3 (7.9 ± 1.2) × 105 0.68 ± 0.06 712 ± 89 56 ± 9
37 63 ± 7 63 ± 10 44 ± 16 (4.8 ± 1.1) × 105 0.48 ± 0.04 649 ± 93 86 ± 21
0.005 0.05 0.05 0.002 0.006 0.04 0.04
Only significant results are reported in the table, data are reported as mean ± SEM and examined by paired t-test. AST, aspartate aminotransferase; ␥GT: gamma-glutamyltranspeptidase; 8-OHdG, 8-hydroxydeoxyguanosine; no. add/105 dG, no. adducts/105 deoxyguanosine; HCV-RNA, hepatitis C viral load; TL, telomere length; CK-18, Cytokeratin18; PIIINP, Procollagen Type III.
All the remaining parameters investigated (AOPP, NO, CK18/CK18Asp396) demonstrated no significant change in relation to coffee intake/abstinence, while the circulating VEGF showed a trend towards lower levels when the patients were exposed to coffee (p = 0.1) (data not shown). According to the hypothesis for the sample size calculation, a change was observed in 88% of the patients with respect to the primary end-point, 8-OHdG levels, in 89% with respect to telomere length, 81% with respect to apoptosis (CK-18) and 70% with respect to collagen synthesis (PIIINP).
4. Discussion The present study, designed to investigate the mechanisms underlying the effect of coffee consumption in patients with chronic hepatitis C, allowed identification of some interesting effects of coffee, both as a routine habit and as a programmed, acute, exposure. To reduce the risk of bias the patients were provided with the same type of coffee, same brewing machines and instructed to report any deviation from the protocol, not to change their routine alimentary and voluptuary habits and finally to report any consumption of drugs and anti-oxidants. The compliance to the study protocol was actually very high and only two patients slightly deviated. A longer exposure/abstinence might have induced deeper changes but with probably a lower compliance by the patients, mostly used to drinking coffee frequently. In this study, patients with a higher routine consumption of coffee showed significantly lower AST and ALP serum levels and with a higher HCV-RNA titre. The effects of coffee consumption on the two parameters were then assessed during the two test periods: HCVRNA levels significantly increased during coffee consumption while lower ␥GT and slightly higher AST levels were observed during acute coffee consumption. No clear explanation is available for coffee selectively modifying AST levels after both acute and chronic administration. The respective roles of AST and ALT levels in patients with chronic liver damage are still debatable but for the role of AST/ALT ratio in cirrhotics [31]. A recent study [32] identified AST as an independent predictor of inflammation in HCV-related hepatitis in patients with mildly elevated aminotransferase levels. Also the patients we investigated showed relatively low levels of transaminases and it may well be that in this subgroup of HCV patients, AST more clearly reflects the extent of liver damage and, consequently, the effects of coffee consumption. An acute exposure to coffee might well induce different changes from a chronic one. A different and differently mediated effect of chronic and acute caffeine exposure has been demonstrated for instance for cerebral haemodynamics [33] where acute caffeine administration results in A1 and A2A adenosine receptors activation, while chronic exposure seem to affect only A2A.
Indeed, in this study opposite results were obtained with respect to AST after chronic and acute coffee exposure, but the changes observed were quite limited, and irrelevant from the clinical point of view. The intriguing correlation between coffee exposure and higher HCV-RNA titres was instead confirmed both in chronic and acute coffee exposure. No data have been previously reported on this novel finding that requires some clinical considerations: the increase is almost never higher than one logarithm and is not therefore clinically significant [34] and, in contrast with what happens in HBV-related damage, in HCV infection viral load is not a prognostic factor [35]. Finally HCV-RNA titre does not correlate, in this study, with transaminase levels. Coffee exposure significantly increased apoptosis and, given the correlation between viral load and apoptosis, the increase in viral load following coffee exposure might well represent a consequence of liver cell death, with release of viral RNA into the blood from apoptotic hepatocytes. Previously published data confirm a role of coffee in cell apoptosis [14,36] but additional mechanisms leading to the modulation of viral replication rate cannot be excluded. During coffee consumption a significant reduction of oxidative DNA damage was observed confirming our working hypothesis, i.e. that the protection exerted by coffee with respect to HCC might be mediated by a reduced accumulation of oxidized bases in cell DNA, and consequently, of DNA mutations. This reduction was observed in 88% of the investigated patients, well above the target of 40% hypothesized to define the sample size. The relevance of oxidative damage in carcinogenesis [37] and, in particular in patients with HCV infection [15,38], is well known. That coffee consumption is associated with lower oxidative DNA damage was recently reported also by Miˇsík [39] in healthy volunteers. We here demonstrated in vivo, in patients with HCV infection, that coffee exposure significantly limits the extent of oxidative DNA damage in circulating leukocytes, but 8-OHdG levels from leukocytes and liver tissues, in patients with chronic HCV-mediated damage, strictly correlate [16]. Oxidative DNA damage in telomeric sequences leads to telomere shortening, an event linked to chromosomal instability [27]. One month coffee exposure induced, in this study, a 40% increase in telomere length in 89% of the patients, a finding that strongly confirms coffee effects on oxidative DNA damage and indicates that coffee consumption leads to a stabilization of chromosomal DNA, thus protecting from neoplastic evolution. Finally, we observed during coffee exposure a significant reduction in the levels of pro-collagen III, a serum marker of fibrosis [40], involving 70% of the patients. This reduction indicates a drop in collagen deposition in the liver that may, at least in part, justify the slower progression to cirrhosis associated with long term coffee consumption, an effect recently confirmed also in nonalcoholic steatohepatitis [41]. This reduced collagen deposition may be due to caffeine activity in antagonizing adenosine receptors, since adenosine and its receptors have been found to promote liver
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fibrosis [42], or to paraxanthine, a caffeine metabolite, suppressing the expression of the pro-fibrogenic connective tissue growth factor (CTGF) [13]. In summary, this study demonstrates that coffee consumption, in patients with chronic hepatitis C, reduces oxidative DNA damage, increases apoptosis, leads to telomere elongation and DNA stabilization and finally reduces pro-collagen III deposition. Such a set of factors may well play a major role in reducing the risk of disease progression and of evolution to HCC. Since a number of studies demonstrated that caffeine can modulate both innate and adaptive immunity [43], and given that, for viral clearance, both immune responses are required, the data obtained prompt additional investigations on the effect of coffee on the human immune-response in patients with HCV-related liver damage. Secondly, the respective effects of coffee, of the diterpenes kahweol and cafestol, strong antioxidant, and of furan, a coffee volatile aroma, potentially carcinogenic compound, should be tested in established experimental models of liver damage, again in relation to oxidative damage. Source of support The study was funded, in part, by the Institute of Scientific Information on Coffee (ISIC), which is a scientific consortium of major European Coffee Companies. ISIC had absolutely no role in designing the study, in the collection, analysis and interpretation of data, in either writing or submitting the report. Conflict of interest statement The authors declared no personal conflict of interest and no personal financial relationship with the organization “Institute of Scientific Information on Coffee” (ISIC) partially funding the study. Dr. M. Petracco is an employee of Illy, a coffee factory in Italy. The role of Dr. M. Petracco was to supervise the analyses on the coffee used in the experiments, that was regularly bought. Dr. Petracco also helped in designing the study and evaluating the results obtained. Illy S.p.a had absolutely no role in the study, nor it participated in financing it. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.dld.2012.10.021. References [1] Costentin CE, Roudot-Thoraval F, Zafrani ES, et al. Association of caffeine intake and histological features of chronic hepatitis C. Journal of Hepatology 2011;54:1123–9. [2] Muriel P, Arauz J. Coffee and liver diseases. Fitoterapia 2010;81:297–305. [3] Tverdal A, Skurtveit S. Coffee intake and mortality from liver cirrhosis. Annals of Epidemiology 2003;13:419–23. [4] Stroffolini T, Cotticelli G, Medda E, et al. Interaction of alcohol intake and cofactors on the risk of cirrhosis. Liver International 2010;30:867–70. [5] Gelatti U, Covolo L, Franceschini M, et al. Coffee consumption reduces the risk of hepatocellular carcinoma independently of its aetiology: a case–control study. Journal of Hepatology 2005;42:528–34. [6] Gallus S, Bertuzzi M, Tavani A, et al. Does coffee protect against hepatocellular carcinoma? British Journal of Cancer 2002;87:956–9. [7] Kurozawa Y, Ogimoto I, Shibata A, et al. Coffee and risk of death from hepatocellular carcinoma in a large cohort study in Japan. British Journal of Cancer 2005;93:607–10. [8] Inoue M, Yoshimi I, Sobue T, et al. Influence of coffee drinking on subsequent risk of hepatocellular carcinoma: a prospective study in Japan. Journal of the National Cancer Institute 2005;97:293–300. [9] Bravi F, Bosetti C, Tavani A, et al. Coffee drinking and hepatocellularcarcinoma: an update. Hepatology 2009;50:1317–8. [10] Morgan TR. Chemoprevention of hepatocellular carcinoma in chronic hepatitis C. Recent Results in Cancer Research 2011;188:85–99. [11] Masterton GS, Hayes PC. Coffee and the liver: a potential treatment for liver disease? European Journal of Gastroenterology and Hepatology 2010;22:1277–83.
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