HABITUALCAFFEINE CONSUMPTION AND ITS INFLUENCE ON MYOCARDIAL FIBERS, BLOOD PRESSURE AND OXIDATIVE STATUS IN PREGNANT ALBINO WISTAR RATS

David E Ehichioya 1 *, Tolulope O Oyesola 1 , Rosemary C Ogbonna 1 , Olusoji A Oyesola 2 and Kolawale I Ajiboye 1 . 1. Department of Physiology, Benjamin S. Carson (Snr.) School of Medicine, Babcock University, Ilishan, Ogun State, Nigeria. 2. Department of Physiology, Faculty of Basic Medical Sciences, OlabisiOnabanjo University, Ikenne Campus, Ogun State, Nigeria. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 4 (10), 1845-1854 1846 Studies have suggested that regular consumption of caffeine is associated with adverse cardiovascular (CV) effects (LaCroix et al., 1986;Panagiotakos et al., 2003;Happonen et al., 2004). It has been reported to increase heart rate (HR) and blood pressure (BP) by the stimulation of sympathetic nervous system (Arno et al., 2011). Caffeine has a powerful and continual intense constrictor effect, which could increase the risk(s) of CV disease through its effects on BP. It is known to affect BP through adenosine receptor inhibition and an increased release of selected neurotransmitters such as adrenaline and noradrenaline (Amaresh et al., 2011). Tofovic et al., (2002) stated that tolerance was not developed to the hemodynamic effects of caffeine in regular caffeine consumers. Thesignificance of this is that for each time caffeine is ingested, it mildly raisesBP and HRirrespective of how often it is consumed. In contrary, older studies suggested that habitual use of caffeine leads to the development of tolerance to its physiological effects on BP (Ammon et al., 1983;Robertson et al., 1984).
Caffeine has been found to increase the aortic stiffness and aortic pressure in healthy and hypertensive adults (Vlachopoulos et al., 2005;Papaioannou et al., 2005;Mahmud et al., 2001).
Although caffeine has been proven tohave CNS and gastrointestinal benefits (Schardt, 2008), its effects on the heart has been a source of debate (Pelchovitz andGoldberger, 2011). Some researchers claim that cardiac toxicity can be induced by excess caffeine consumption. On the other hand, it has been observed that caffeine possess antioxidant properties which benefits CV function.
With caffeine consumption linked with altered heart physiology, it is quite possible that CVabnormalities could occur as a result of regular caffeine use in humans. This study is therefore aimed at ascertaining the influence of caffeine on BP, myocardial fibres and oxidative status in pregnant condition.

Materials and Method:-
Ethics Statement:-All animal handling and experimental protocols implemented in this study was in conformation with the international principles for laboratory animals as obtained in the Helsinki's Declaration (NIH, 1985). Also ethical clearance was obtained from Babcock University Health Research Ethics Committee (BUREC).

Animals/Experimental Design:-
Twenty four (24)pregnant Wister rats with average weight of 115grams were used in this study. They were obtained and housed in standard plastic cages at room temperature at the animal house, Babcock University, Ilishan, Nigeria. They were exposed to light and dark (12:12) hours and given water and pelletized feed(Caps Feed Ibadan)adlibtum.
Virginal smears were taken to determine estrus phases of these animals. Those discovered to be in proestrus phase were exposed to male rats for mating. The day a vaginal plug was discovered or sperm cells found to be present in smear was designated as pregnancy day zero. Pregnant rats were randomized into four groups and treated via oral gavage for at least four days in a week till end of term. Group Aprovidedwith vehicle (water), group B was treated with 1mg/kg of caffeinedissolved in vehicle, group C was treated with 6mg/kg of caffeine dissolved in vehicle and group D was treated with 15mg/kg of caffeine dissolved in vehicle. These caffeine treatmentsmark a circulating blood levels equal to the consumption of between 1-4 cups of coffee in humans (Fredholm, 1995). Caffeine was obtained from Sigma-Aldrich, St. Louis, MO, USA.
BP, cardiac histology and oxidative status were determined on pregnant rats at term. BP was determined immediately after term. 2 heart tissues per group were used for histology and serum was analyzed for oxidative status.
Blood Pressure (BP) Determination:-BP was determined by using non-invasive CODA tail cuff system and a volume pressure recording sensor (Kent Scientific Cooperation, USA). The animal was place in the rear of the holder and the rear hatch to the holder was secured. The nose cone was in a position to limit the animal from turning around while inside the holder. The animal was allowed about 5minutes to acclimate to the holder, likewise thermoregulate. Cuff placement, attachment of the cuff to the CODA controller and determination of blood pressure was carried out as described by Daugherty et al., (2009).The systolic and diastolic BP were obtainedfrom the CODA spreadsheet after the cycle was completed. Mean Arterial BP (MABP) was determined by using the formula: (SBP + 2*DBP)/3.

Cardiac Histology:-
Hearts of experimental animals were fixed by perfusion of hearts with 4% paraformaldehyde solution (obtained from Anatomy Department, Babcock University) containing 150 mMKCl and 5 mMEDTA. Hearts were embedded in paraffin, sectioned, mounted on slides and analysed as described by Wendler et al., (2009).
Antioxidant study:-Lipid peroxidation was measured by the formation of thiobarbituric acid reactive substances (TBARS) using the methods postulated by Beuge and Aust, (1978). The absorbance was measured at 535 nm.
The level of superoxide dismutase(SOD) activity in plasma Liver and heart tissue homogenates were determined by the method of Misra and Fridovich (1972).The catalase activity (CAT)was measured according to the method of Sinha et al., (1972). The exponential disappearance of H 2 O 2 was at 570nm. Reduced glutathione (GSH) level was determined by the method of Ellman modified by Jollow et al. (1974). The absorbance was measured immediately at 412 nm.The GSH contents were calculated using GSH as standard and expressed as µmol/mg protein.

Statistical analysis:-
Results are presented as means ± SEM and comparison of the means was done using the one way analysis of variance, followed by Student's Newman-Keuls post hoc test, using the GraphPad software. A p-value < 0.05 was considered statistically significant.

Results:-
Antioxidant Analysis:-Malondialdehyde (MDA) analysisin serum and liver tissue revealed that results were significantly lower (p<0.05) in treatment groups compared to vehicle. From the heart tissue MDA analysis, only the group administered 15mg/kg of caffeine was significantly lower(p<0.05) at 3.53±0.22 nmol/mlcompared to vehicle (4.78±0.09nmol/ml) and the group administered 1mg/kg of caffeine (4.55±0.11nmol/ml). SOD and CAT results were significantly higher (p<0.05)across treatment groups for serum, liver and heart tissues when compared to Vehicle.
GSH results for liver and heart tissues were significantly higher (p<0.05) across treatment groups compared to control group.While values forserum (GSH) were 3.94±0.14 µMol/ml for group treated with 1mg/kg of caffeine(not significant compared to vehicle -3.53±0.07µMol/ml), 5.60±0.39 µMol/ml for group treated with 6mg/kg of caffeine (significant higher compared to vehicle), and at 6.62±0.55 µMol/ml for group treated with 15mg/kg of caffeine (significant higher compared to vehicle). Figure I show values of the systolic blood pressure (SBP), diastolic blood pressure (DBP), pulse pressure (PP), mean arterial blood pressure (MABP), heart rate (HR) and rate pressure product (RPP) for vehicle and the treatment groups immediately after treatment period.MABP, SBP and DBP valueswere significantly higher (p<0.05) across the three treatment groups when compared to vehicle.

Blood Pressure (BP) Parameters:-
Histological results:-As observed in plate I, Collagen fibers appeared gold orange in colour. Micrograph showed hypertrophic tissue (arrow) a day after delivery even though tissue had good histoarchitecture and fair nuclei distribution (panel B). In 1848 panels C and D, signs of dystrophy (arrows) were observed and panel D showed some level of histoarchitectural disruption. In plate II, fibres appeared to be more in panels B, C and D compared to panel A. Some degree of hypertrophy was observed in panels C and D (arrows).Results for left ventricular wall thickness across treatment groups revealed that thickness was significantly increased compared to vehicle.

Discussion:-
Caffeine is known to affect several systems of the body. Its use in food and food additives has made it necessary to thoroughly understand its effects on the body system. This study was designed to provide understanding on the effect of sub-chronic caffeine consumption in pregnancy and its effect on some cardiovascular parameters, as well as anti-oxidant status.
After about four weeks of habitual caffeine consumption, BP was significantly raised in the three treatment groups. Increase in BP detected in this studyis an indication of no tolerance to caffeine developed after sub-chronic consumption. This is in line with the finding of Tofovic et al., (2002), who reported no tolerance to the hemodynamic effects of caffeine in regular consumers. The rate of metabolism and clearance of caffeine by the hepatic cytochrome P-450 (CYP) isoenzyme is one major factor that can hinder tolerance to caffeine use.
Increase in BP andHR have been describedto occur as a result of stress. Stress (likely resulting from caffeine intake), mediates high levels of cortisol, adrenaline and nonadrenaline (Echeverri et al., 2010). A continual state of increased stress can chronically raise BP, disturb HRand rhythmicity (Rafetto et al., 2004). Increased BP which was evident in the present study could also be as a result of the inhibition of parasympathetic activity by the antagonism of adenosine receptors (Loomans et al., 2012). Increase in BPwas correlated to the doses administered. This may wellimply that higher concentrations of caffeine may promote incessant and lingering stress condition and/or unswerving inhibition of parasympathetic activity by the antagonism of adenosine receptors thereby endorsing hypertension, which is one of the major causes of heart failure.
Myosin heavy chain synthesis can increase by 35% within hours after a pressure overload occurrence in heart tissues, leading toan increase in the number of sarcomeres in the myocyte and consequently increase in cardiac muscle mass and thickness (Lorell and Carabello, 2000). Our study reveals significant thickness in the LV of rat heart LV wall, substantial accumulation of collagen and reticulin fibres, as well as increase in the number of reticulin fibres across treatment groups. This is a sign of hypertrophy and increase in cardiomyocyte size. Buscariollo et al., (2014) accounted that the increased wall thickness was consistent with cardiac concentric hypertrophy, characterized by reduced chamber volume. High BP has been linked with about a 10-fold increase in the incidence of LV hypertrophy (Levy, 1988).This study demonstrate a connection between high BP, increase in cardiomyocyte size, increased LV hypertrophy and wall thickness. These are indications of cardiomegaly (Shi et al., 2014). Reports have indicated that steady raise in BP can stimulate increased collagen synthesis, in same manner consequential fibrosis and hypertrophy (Gordon et al., 1986;Morgan et al., 1985).

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The increased BP in the present study cannot be correlated to MDA, SOD, GSH nor CAT. The progressive reduction (MDA) and increase (SOD, GSH and CAT) across treatment groups seem more like the influence of caffeine on these antioxidant enzymes is in a dose dependent fashion.Previous studies have specified that caffeine impedes lipid peroxidation especially in the liver tissues of rats (Kamat et al., 2000;Pasaoglu et al., 2011), instigates and augments the activities of CAT and SOD (Mukhopadhyay et al., 2003).
Caffeine may contain antioxidant properties by instigating oxidative enzymes in the prevention of tissue oxidation and damage.It is also evident that it is capable of giving rise to series of physiological and anatomical mechanisms detrimental to cardiovascular health during pregnancy. Studies will be necessary to ascertain if these effects will be manifested in the offspring.