Hydroxyhydroquinone Interferes With the Chlorogenic Acid-induced Restoration of Endothelial Function in Spontaneously Hypertensive Rats

American Journal of Hypertension, Jan 2008

Coffee is a rich source of antioxidative polyphenols, but epidemiological studies and interventional trials have failed to demonstrate any clear beneficial effects of coffee consumption on hypertension. The interaction between hydroxyhydroquinone (HHQ) and 5-caffeoylquinic acid (CQA) was examined, in an attempt to understand the controversial effects of coffee on hypertension.

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Hydroxyhydroquinone Interferes With the Chlorogenic Acid-induced Restoration of Endothelial Function in Spontaneously Hypertensive Rats

AMERICAN JOURNAL OF HYPERTENSION | Hydroxyhydroquinone Interferes With the Chlorogenic Acid-induced Restoration of Endothelial Function in Spontaneously Hypertensive Rats Atsushi Suzuki 0 Akihiko Fujii 0 Hiroko Jokura 0 Ichiro Tokimitsu 0 Tadashi Hase 0 Ikuo Saito 0 Biological Science Laboratories, Kao Corporation , Tochigi, japan BACKGROUND Coffee is a rich source of antioxidative polyphenols, but epidemiological studies and interventional trials have failed to demonstrate any clear beneficial effects of coffee consumption on hypertension. The interaction between hydroxyhydroquinone (HHQ) and 5-caffeoylquinic acid (CQa ) was examined, in an attempt to understand the controversial effects of coffee on hypertension. METHODS Chemicals. CQA was purchased from the Cayman Chemical (Ann Arbor, MI). HHQ was purchased from Wako Pure Chemical Industries (Osaka, Japan). Phenylephrine,acetylcholine (ACh), and sodium nitroprusside were purchased from Sigma composition (in mmol/l): NaCl 110.8, KCl 5.9, NaHCO 3 Chemical (St. Louis, MO). The total NO assay kit was pu-r 25.0, mgSO 4 1.07, CaCl2 2.49, NaHPO4 2.33, and glucose 11.51. chased from Assay Designs (Ann Arbor, MI), and used for The tissues were maintained at 37°C under a 1-g tension and the quantitative determination of nitrite/nitrate (NOx) in equilibrated for 1 h before initiating the experimental protocols. biological fluids.13 Urinary H2O2 concentrations were deter- Vascular reactivity was measured in aortic rings with the funcmined using a quantitative H2O2 assay kit (OXIS International, tional endothelium precontracted with submaximal concentraPortland, OR).14 tions of phenylephrine (10−6 mol/l). Endothelium-dependent or endothelium-independent relaxation was evaluated from conAnimals. Male SHR (SHR/Izm; 14 weeks old) and normo- centration response curves to ACh (10−9–10−6 mol/l) or sodium tensive Wistar Kyoto (WKY) rats (WKY/Izm; 14 weeks old), nitroprusside (10−9–10−6 mol/l), respectively. Relaxation was purchased from SLC (Shizuoka, Japan), were used in the calculated as the percent of precontractile vascular tone. The experiments. All rats were maintained at 25 ± 1°C, 55 ± 10% responses of tissues were recorded using isometric transducers humidity, and 12-h on/off light cycle (lights on 7:00 am.–7:00 (Kishimoto Medical Instruments, Kyoto, Japan) and recorders pm). The rats had free access to the diets described below and (Sekonic, Tokyo, Japan). to drinking water. All animal experiments were conducted with the approval of the Ethics Review Committee for Animal Experimentation of the Kao Corporation. Immunohistochemical staining of nitrotyrosine. The immunohistochemical detection of tyrosine nitration in thoracic aortas from the same rats mentioned above wasperformed as described Study design. A powdered CE-2 diet (CLEA Japan, Tokyo, previously.17 Aortas were fixed in 10% buffered formalin and Japan) was used as the control diet. The components of the 8-µm sections were prepared from paraffin-embedded tissues. CE-2 diet were protein (24.9%), fats (4.6%), ash (6.7%), fiber After deparaffinization, endogenous peroxidase was quenched (3.7%), soluble non-nitrogenous compounds (51.4%), and by incubation with 3% H2O2 for 10 min. Nonspecific adsorpwater (8.6%). The CE-2 diet was combined with 0.005% HHQ tion wasminimized by incubating the sections in 2% normal (~3 mg/kg/day) and/or 0.5% CQA (~300mg/kg/day) and was goat serum in phosphate-buffered saline for 20min. The secused as the test diet. We have previously reported that a 6-week tions were incubated with a primary anti-nitrotyrosine antibody treatment with a green coffee bean extract (chlorogenic acids, (Biomol Research Laboratories, Plymouth meeting, PA) for 200 mg/kg/day) clearly inhibited the progression of hyperten- 30 min at room temperature. The sections were then incubated sion in SHR;15 therefore, we used a higher dose (300mg/kg/day) with the secondary antibody for 30min, and specific labeling and a longer treatment period (8 week) in the present study. was detected with horseradish peroxidase/streptavidin. The amount of chlorogenic acids in coffee varies from 0.2 to 3.8 g/kg and that of HHQ varies from 6 to 20mg/kg; 11 thus, Statistical analysis. All values are expressed as the mean ± the ratio of HHQ/chlorogenic acids in coffee is ~1/100. Based s.e.m. Statistical analyses were performed using StatView (SAS on this ratio, rats were fed HHQ at a level of 3mg/kg of body Institute, Cary, NC). Data were initially analyzed using an analweight/day. Four groups each of WKY rats and SHR (n = 5 or ysis of variance for each group. When a significanFt -value (P< 6 per group, 14 weeks old at the initiation of the experiment) 0.05) was obtained, a Dunnett’s test was performed as a post-hoc were given the control diet or the CQA and/or HHQ diets for analysis. Vasorelaxant responses are reported as the percentan 8-week period. Food intake, body weight, systolic blood age of phenylephrine-induced precontraction. Comparisons of pressure (SBP), and heart rate at the tail artery were measured dose–response curves were evaluated by means of a two-way at 2-week intervals in conscious rats. At the eighth week of analysis of variance for repeated measures. the experiment, daily urine was collected using a metabolic cage (Natsume Seisakusho, Tokyo, Japan) and urinary nitrite/ RESULTS nitrate or hydrogen peroxide concentrations were determined. Blood pressure, body weight, heart weight, and heart rate In the same rats, blood and thoracic aortas were collected from In WKY rats, the baseline SBP (14 weeks old) was 130± 2 mm Hg. pentobarbital-anesthetized rats at the end of the 8-week expe-r In SHR, the baseline SBP (14 weeks old) was 186± 4 mm Hg. imental period. Plasma concentrations of a metabolite of CQA After 8 weeks, the SBP in the HHQ or HHQ+CQA groups was (caffeic acid) were determined by ion-trap liquid chromatog- not different from the control group for either strain (Figure 1). raphy/tandem mass spectrometry. The SBP was significantly lower in SHR that were on the CQA diet than for SHR on the control diet. This effect was signifiVascular reactivity. This experiment was performed according to cantly inhibited by the concomitant administration of HHQ. In a previously described method16 in the rats mentioned above. contrast, in the WKY rats, no significant difference in results was At the end of the 8-week experimental period, rats were anes- found between the control diet and the CQA diet . There were thetized with an intraperitoneal injection of sodium pentobar- no significant differences in body weight (370–389g in WKY; bital. The descending thoracic aorta was excised from the pen- 343–355 g in SHR), heart weight (2.7–2.9g/kg body weight tobarbital-anesthetized rats, freed of fat and connectivteissue, in WKY; 4.0–4.2 g/kg body weight in SHR), and heart rate cut into rings ~2–3 mm in length, and placed in gassed (95% (353–378 beats/min in WKY; 350–379 beats/min in SHR) O2 and 5% CO2) Krebs–Henseleit solution with the following among the control, HHQ, CQA, and HHQ+CQA diet groups 24 Downloaded from https://academic.oup.com/ajh/article-abstract/21/1/23/137155 by guest on 13 June 2018 ja NUa Ry 2008 | VOLUME 21 NUMBER 1 | AMERICAN JOURNAL OF HYPERTENSION WKY Control CQA after 8 weeks for either the WKY rats or the SHR. No significant differences were found in daily food intake (20–21 g) among the four diet groups in either WKY rats or SHR. Vascular reactivity No significant differences were found between the control, HHQ, and HHQ+CQA diet groups in ACh-induced endothelium- endothelium-independent vasodilation was found in either dependent vasodilation measured in WKY and SHR aorta prepa- strain (Figure 2c,d). rations that were excised after 8 weeks (Figure 2ab, ). In contrast, in SHR aortas, ACh-induced endothelium-dependent vasodila- Urinary NOx and H2O2 and plasma metabolite of CQA tion was greater in the CQA diet group compared to the con- No significant differences were found in urinary volume trol diet group. No difference in sodium nitroprusside–induced among the groups for either strain. In the control, HHQ, and Control HHQ CQA HHQ+CQA * ** *** a ) n i teo60 r p g m / lo40 m n ( e t a itr 20 n iitt/r e N 0 Control HHQ CQA HHQ+CQA * ** *** b ) e n i itn 200 a e r c l/go 150 m µ ( ide 100 x o r e np 50 e g o r ydH 0 WKY SHR WKY SHR HHQ+CQA groups, no significant differences were found in of NOx and a decrease in the urinary excretion of H2O2, urinary NOx or H2O2 excretion for either strain (Figure3a,b). enhanced ACh-induced endothelium-dependent vasodilation, In the CQA diet SHR group, urinary NOx excretion was sig- and decreased immunohistochemical staining for nitrotyrosine nificantly increased compared to the SHR control diet group, in the aorta. Under the conditions of this study, urinary NOx but no significant difference was found among the WKY groups excretion was lower in SHR than in WKY. Both high and low (Figure 3a). The urinary excretion of H2O2 in the control diet urinary NOx excretion in SHR have been reported.18,19 These group was significantly increased in SHR compared to WKY contradictory results might be due to differences either in SHR (Figure 3b). The ingestion of CQA for 8 weeks, led to a signifi- breeders or age. In humans, there is a significant negative correcant decrease of H2O2 levels in the urine when compared to the lation between blood pressure and plasma NOx concentration2.0 control diet and the effect of CQA was significantly inhibited by Because CQA ingestion increased the urinary NOx excretion in HHQ in SHR (Figure 3b). The plasma concentration of caffeic SHR in this study, it is likely that CQA intake might improve NO acid, a metabolite of CQA, in the HHQ+CQA treated group, as bioavailability only in SHR. However, the biological site of action determined by ion-trap liquid chromatography/tandem mass for CQA on NO regulation, including NOS activity or protein spectrometry was equivalent to that in the CQA treated group expression level in the endothelium, remains to be elucidated. (33 ± 29 and 20 ± 7 nmol/l) in SHR. Urinary H2O2 is used as a direct marker of oxidative stress in the human body.10 Lacy reported an association between high Immunohistochemical staining of nitrotyrosine plasma H2O2 concentration and high blood pressure in essenNitrotyrosine was detected using immunohistochemistry stain- tial hypertensives.21 According to Rodriguez-Itube et al., the ing techniques (Figure 4). Nitrotyrosine staining was negative plasma H2O2 concentration is significantly higher in SHR than when the non-specific IgG antibody was used in both the WKY in WKY.22 In our study, the urinary H2O2 concentration was aortas (Figure 4a) and in the SHR aortas (Figure4b). In the higher in SHR than in WKY, suggesting that oxidative stress is control, HHQ, and HHQ+CQA diet SHR groups, clear stain- increased in SHR. The significant CQA-induced decrease in the ing was observed in the intima (Figure4e,f,h). In contrast to urinary H2O2 concentration in SHR, suggests that CQA reduced this, no clear staining was detected in the CQA diet SHR group oxidative stress in genetic hypertension. NAD(P)H oxidase is (Figure 4g) or in the control, HHQ (Figure 4c,d), CQA, and thought to be the main source of O2− in blood vessels and in the HHQ+CQA diet WKY groups (data not shown). kidney in SHR, as well as other experimental models of hypertension.1 In the previous study, the findings demonstrate that the DISCUSSION aortic NAD(P)H oxidase activity was significantly decreased in Based on the content ratio of HHQ and chlorogenic acid in the CQA diet group compared to the control diet group in SHR, coffee, the interaction of HHQ with CQA on hypertension but not in WKY, suggesting that CQA reduces ROS production and vascular reactivity was investigated in normotensive and from NAD(P)H oxidase in the vasculature of SHR2.3 We predict hypertensive rats. HHQ clearly inhibited the CQA-induced that the reduced production of ROS from vascular NADPH oxiimprovement in blood pressure and NO bioavailability in SHR. dase reflects a decrease in urinary ROS in the CQA diet group These findings explain, at least in part, the basis for the con- in SHR; however, the involvement of other ROS sources in this flicting action of coffee drinking on hypertension and vascular study remains unclear. Because the protein expression or activity function. of vascular NAD(P)H oxidase is not known in this study, further ROS appears to play a critical role in the pathogenesis of investigation should be done in WKY rats and SHR. vascular damage1, but the effect of an exogenous generator of ROS levels were increased in the blood vessels of SHR comROS on vascular function is largely unknown. Coffee drinking pared to normotensive WKY rats.1 Because CQA reduces ROS increases urinary H2O2 levels in males, and HHQ, which is pro- production from vascular cells only in the case of SHR,23 CQA duced during the roasting of green coffee beans, is considered to might protect NO activity against ROS attack and improve be the causative substance1.0 Accordingly, we hypothesized that endothelial function mainly in SHR, but not in WKY rats, and the administration of HHQ might impair vascular function via lead to blood pressure-lowering actions in hypertensive rats. the breakdown of NO. Contrary to our expectations, an 8-week In the high concentrations of ACh used in the present study, HHQ (3 mg/kg/day) treatment had no effect on blood pressure the loss of an augmentation of ACh-induced endotheliumor endothelium-dependent and -independent vasodilation in dependent relaxation in CQA-treated aortic rings from SHR normotensive and hypertensive rats in this study. No change was was observed. It has been reported that high concentrations of observed in urinary H2O2 excretion and nitrotyrosine staining ACh could evoke endothelium-dependent contractions in the in aortas due to the HHQ treatment; however, the possibility aorta of SHR but not in that of WKY2.4 Our data suggest that exists that this dose of HHQ might not have been sufficient to CQA might not affect ACh-induced contraction in SHR aorevoke oxidative stress in rats. Further studies with higher doses tas. In addition, further study is needed to determine whether of HHQ or a longer experimental period might be necessary to CQA ingestion influences vascular reactivity in resistant small investigate the influence of the exogenous ingestion of HHQ on arteries, which are major determinants of blood pressure, in blood pressure or vascular reactivity. hypertensive animals. In the present study, the antihypertensive effect of CQA in Coffee contains a number of pharmacologically active compoSHR was accompanied by an increase in the urinaryexcretion nents including caffeine (1,3,7-trimethylxanthine). Since caffeine 26 Downloaded from https://academic.oup.com/ajh/article-abstract/21/1/23/137155 by guest on 13 June 2018 ja NUa Ry 2008 | VOLUME 21 NUMBER 1 | AMERICAN JOURNAL OF HYPERTENSION is an inducer of hypertension, it has been extensively studied, but a recent study demonstrated that caffeine intake has little effect on blood pressure in regular coffee drinkers.8 In sharp contrast, green coffee bean extracts reduce blood pressure in human hypertensive subjects, in spite of the fact that its caffeine content is equivalent to that of roasted coffee2.5,26 These findings led us to hypothesize that a coffee component, other than caffeine, inhibits the effects of antihypertensive substances in coffee such as chlorogenic acids. Because HHQ is known as a source of ROS in coffee,10 we hypothesized that HHQ inhibits the hypotensive effect of chlorogenic acids. It is noteworthy that HHQ in this dose inhibited the CQA-induced improvement in blood pressure, the urinary excretion of NOx and H O2, and endothelial 2 function in spite of having little effect by itself. The mechanisms underlying the inhibiting action of HHQ are proposed to be either a chemical interaction with CQA, an inhibition of CQA absorption in the gastrointestinal tract, or an impairment in the bioavailability of NO by HHQ-derived ROS production. In this study, the plasma concentration of a metabolite of CQA (ca-f feic acid) in the HHQ+CQA treated group was equal to that in the CQA treated group, thereby excluding a scenario in which HHQ chemically interacts with CQA or decreases the absorption of CQA in the gastrointestinal tract. Recently, Covleet al. reported that H O2 produces oxidative stress in endothelial cells 2 by increasing intracellular O2− levels through NOS and NADPH oxidase in porcine aortic endothelial cells2.7 HHQ-derived H O 2 2 might interfere with the CQA-induced enhancement of bioavailability of NO through the production of O2−, even though the action of HHQ-derived ROS by itself appeared to be weak in this study. Experiments designed to investigate the detailed mechanisms of the interaction between these molecules are currently in progress. In summary, HHQ alone had little effect on blood pressure and vascular function at the dose used in the present study in WKY rats and SHR, but it clearly abolished the CQA-induced improvement in hypertension and endothelial dysfunction in SHR. Consequently, the controversial action of coffee drinking on blood pressure and vascular function might be partially due to the presence of HHQ. a cknowledgments: We thank Mr S. 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Suzuki, Atsushi, Fujii, Akihiko, Jokura, Hiroko, Tokimitsu, Ichiro, Hase, Tadashi, Saito, Ikuo. Hydroxyhydroquinone Interferes With the Chlorogenic Acid-induced Restoration of Endothelial Function in Spontaneously Hypertensive Rats, American Journal of Hypertension, 2008, 23-27, DOI: 10.1038/ajh.2007.3