Effect Of Black Grape Juice Intake On Liver Lipoperoxidation And Body Weight Loss In Whole Body X-Irradiated Rats

J Med CBR Def | Volume 7, 2009 Submitted 12 May 2009 | Accepted 26 June 2009 | Revised 3 August 2009 | Published 8 August 2009

Effect Of Black Grape Juice Intake On Liver Lipoperoxidation And Body Weight Loss In Whole

Body X-Irradiated Rats Edson R Andrade2*, Jacqueline CE Piccoli1, Ivana BM Cruz1,2, Joao BT Rocha2, Veronica VR Andrade2, Paquita González1, Liliane F Bauermann1,2, Juan P Barrio1,2. 1 Institute of Biomedicine, Biomedical Sciences Department, University of León (Spain). 2 Toxicological Biochemistry Post-graduation Program, Federal University of Santa Maria (Brazil). * Corresponding Author: Campus Universitario, 24071 León (España) Tel : +34 987 291000 ext. 5658 | Fax: +34 987 291267 | Email: [email protected]

Abstract

Grape products such as black grape juice (BGJ) are rich in phenolic compounds and are effective in modifying the effects of radiation either as a radioprotector or damage mitigator. In this study, the effect of BGJ on lipid peroxidation has been analyzed by testing a possible radiomodifiying activity of BGJ against radiation-induced damage in rat, focused on body weight and liver lipid peroxidation. Twenty male Wistar rats were divided into four groups, two of them being irradiated by X-rays at 200 kVp. The animals were fed ad libitum and drank voluntarily 2-10 mL per day BGJ or placebo (isocaloric glucose plus fructose solution) for one week before and two weeks after 6 Gy X-irradiation, when they were sacrificed. Lipid peroxidation estimation on liver was made using the Thiobarbituric Acid Reactive Substance (TBARS) Assay, and showed a higher concentration of lipid peroxidation product, malondialdehyde (MDA) for X-irradiated group given placebo when compared to BGJ group. An analysis of BGJ supplementation effects showed that weight loss was compensated about 60%, while MDA hepatic levels were nearly 45% less in BGJ group. Further experiments are being designed for confirming the ability of BGJ as a radiomodifier food supplement with possible application to military operations in radioactive scenarios and radiotherapy.

Introduction


The development of radiomodifiers (drugs or nutrients) to protect against exposures to ionizing radiation (IR) is especially to public health in the case of accidents or attacks using various forms of radiation. An ideal radiomodifier should provide: (a) significant protection against radiation effects; (b) general protective effects to non-target organs (in case of therapy); (c) acceptable route of administration; (d) low toxicity; (e) compatibility for use with other drugs [Maisin 1998; Hosseinimehr et al. 2007]. The radiomodifying action is not one of a barrier or IR-shielding, but acting mainly as scavengers for reactive oxygen species. If a naturally occurring food substance can be used, the difficulties and costs in developing, manufacturing and distributing pharmaceutical for protection in the event of an exposure can be avoided. Natural compounds in human diet could provide functional antioxidants, such as vitamins, minerals and other bioactive substances acting on reducing oxidation damage caused by IR exposure [Hosseinimehr 2007]. Acute effects of the IR exposure mainly include immune suppression, hematopoietic cell loss, mucosal damage, and potential injury to other sites such as lung, kidney, liver and central nervous system [Gold et al. 1988; Ende 2004]. Liver plays a particular role in radiosensitivity and has the capability to deal with high radiation doses as long as only partial irradiation occurs; otherwise, whole organ irradiation leads to hepatocyte failure and Radiation-Induced Liver Disease (RILD) such as hepatitis might be installed [Khozouz et al. 2008]. Long-term effects include dysfunction, fibrosis, and cancer in a wide range of organs and tissues. Blood counts can help to manage the decision-making process in clinical decisions [Goans et al. 1997].

The composition of Black Grape Juice (BGJ) shows a high content of bioactive phenolic compounds such as resveratrol, quercetin and rutin [Machado 2009] and it is tempting to link the radiomodifying actions of BGJ to these chemicals. Flavonoids are good exogenous antioxidants against free-radical initiated lipid peroxidation and recent studies have suggested that flavonoids exhibit desirable biological activities, including anti-allergenic, anti-viral and anti-inflammatory capacity [Seyoum et al. 2006]. Most ingested flavonoids are extensively degraded to various phenolic acids, some of which still possess a radical-scavenging ability [Chaudhuri et al. 2007].

Absorbed flavonoids and their metabolites may display an in vivo antioxidant activity as demonstrated by increased plasma antioxidant status, the sparing effect on vitamin E on the preservation of erythrocyte membrane polyunsaturated fatty acids [Pietta 2000]. Flavonoids, such as resveratrol, have also been shown to influence the apoptotic effects of cytokines, chemotherapeutic agents, and gamma radiation [Aggarwal et al. 2004]. Nevertheless, quercetin and rutin are per se antioxidant agents [Choquenet et al. 2008] and so might be potentially co-responsible for the possible radiomodifying effects of BGJ. The present work is focused on exploring the potential of BGJ as a radiomodifier, with emphasis on lipid peroxidation measurements and liver and body weight effects.

J Med CBR Def | Volume 7, 2009 Submitted 12 May 2009 | Accepted 26 June 2009 | Revised 3 August 2009 | Published 8 August 2009 Materials And Methods Radiation damage was measured by changes in body weight (overall damage), changes in lipid peroxidation (indicative free radical damage) and by the amount of lipid peroxidation.

Animals, Feed and Whole-Body X-Irradiation

Twenty male Wistar rats weighing 200–250g (Harlan, Barcelona, Spain) were included in the study, being housed at the animal house of University of León (Spain) under controlled environment conditions. The experimental protocol used was approved by the University of León Ethical Committee, and adhered to the European Community Guiding Principles for the Care and Use of Animals. They were divided into four groups, each group including five animals: (JN) non-irradiated, BGJ-supplemented; (PN) non-irradiated, placebo (isocaloric equimolar glucose plus fructose solution) supplemented; (JR) irradiated, grape juice supplemented, and (PR) irradiated, placebo (isocaloric equimolar glucose plus fructose solution) supplemented. In order to immobilize the animals during the procedure, anesthesia was induced by intraperitoneal administration of 0.6% penthobarbital in saline (10 mL/kg of body weight), at noon, 15 minutes before irradiation, ensuring the loss of palpebral and plantar reflex activity and spontaneous respiration throughout the procedure. The animals were placed in decubitus pronus and four animals were irradiated at a time and exposed to a single dose of 6 Gy (90% of LD50 in rats, equivalent to 3 Gy in human beings) of whole body X-irradiation from an X-ray machine (200 kV) MAXISHOT 200 (YXLON, Copenhagen, Denmark), at a radiation dose rate of 0.4 Gy/min, with a source-skin distance (SSD) of 50 cm. All animals were quickly weighed twice per week to avoid additional stress. After irradiation, animals were monitored during 16 days until sacrificed.

Animals were fed according to a standard rat chow diet, having free access to water and food. After one week adaptation to individual cages, and 5 days before irradiation they were allowed to ingest a maximum of 10 mL of test compound (grape juice) or placebo, depending on their assigned group. The animals continued drinking BJG or placebo during all the study time. Organic BGJ was obtained from the city of Garibaldi (Brazil), in the main grape-growing region of the state. Black grapes were cultivated in 2007 and the juice was prepared the same year. The concentration (mg/L) of phenolic compounds in the grape juice was previously determined [Machado 2009] as: (a) Resveratrol 3.95±0.01, (b) Quercetin 8.95±0.09, (c) Rutin 3.75±0.03, (d) Gallic acid 81.07±2.03 and (e) Caffeic acid 30.28±2.00. Placebo solution was made using an equimolar mixture of glucose and fructose to be isocaloric with the sugar composition in the grape juice (95 g/L). Earlier work by Park et al. [Park 1993] reported that the best antioxidant system response was 14 days after gamma irradiation in mice. The time of animal sacrifice in other literature varies, minutes to hours, based on specifics of the investigations [B. Lakshmi 2005; Enginar,

J Med CBR Def | Volume 7, 2009 Submitted 12 May 2009 | Accepted 26 June 2009 | Revised 3 August 2009 | Published 8 August 2009 Cemek et al. 2007] till weeks [Cetin, Kaynar et al. 2008].

The choice of ceiling for BGJ supplement intake was made based on previous considerations of the potential effect of BGJ on preliminary studies with healthy human volunteers (data not shown and not published), who were given 300 mL BGJ per 70 kg average body weight corresponding proportionally to 10 mL for rats weighed within 200–250g. The human volunteers were given BGJ, and 20-mL blood samples were taken following the schedule: 30 min before BJG intake, and 30 min, 1 hour and 2 hours after BJG intake. The samples were gamma-irradiated using a radiotherapy unit Theratron 780C located at the radiotherapy unit at Federal University of Santa Maria, Brazil, University Hospital. Thiobarbituric acid reactive substances (TBARS) assays, an assay for oxidative stress, in the blood samples appeared to be lower for those volunteers who drank the BGJ.

Measurements of Lipid Peroxidation in the Liver Biochemical measurements of lipid peroxidation were made using the

TBARS assay, which is used to quantify the colorimetric reaction of the lipid peroxidation product malondialdehyde (MDA) with thiobarbituric acid (TBA). The reaction produces a colored compound which absorbs maximally at λ=532 nm. One gram of liver in 9 mL of potassium phosphate 0.1 M, pH 7.4 was homogenated using a Polytron mixer (Kinematica AG, Switzerland). After heating at 90°C for reacting with TBA, tubes were cooled and centrifuged at 2000×g. The organic layer (supernatant) was collected and the absorbance was read at 532 nm using a spectrophotometer. Thiobarbituric acid-reactive species (TBARS) production was determined as described by Wills [Wills 1987].

Quantification of Rat Liver Protein The amounts of lipid peroxidation were normalized to the amount of liver protein by measuring the MDA per mg of liver protein. The quantification of the protein was performed following Bradford method [Bradford 1976], where the maximum absorbance for the acidic solution of Coomassie Brilliant Blue due to its interaction to Bovine serum albumin (BSA) protein, occurs at 595 nm. The homogenate was obtained by adding 0.5 g of liver in 4.5 mL of sodium potassium buffer (0.1M, pH 7.4), before centrifugation at 12000 rpm for 25 min at 4°C. Standard and sample curves were assessed as following: (a) H2O MilliQ was added to each used position in a 96-position plate; (b) a standard solution of 0.5 mg of protein/mL BSA (-20°C), for a final volume of 160 uL; (c) for sample curve, 1 uL of homogenate in 159 uL of H2O MilliQ was piped for quantifying proteins content in homogenate. All filled positions in a plate received 40 uL of Coomassie Brilliant Blue solution Bradford (ref. 500-0006 Bio-Rad), followed by 10 min in dark place, at room temperature before reading the absorbance at 595 nm.

J Med CBR Def | Volume 7, 2009 Submitted 12 May 2009 | Accepted 26 June 2009 | Revised 3 August 2009 | Published 8 August 2009 Results

Previous work established the correlation between body weight loss and overall damage due to irradiation [Andrade et al. 2009]. The relative body weight losses were calculated and the ratios to the placebo control group were used as average protection values of the radiomodifying effect of BGJ, presented in figure 1 and Table 1. The animals that had been irradiated and given BGJ lost less body weight compared to placebo. The smaller body weight loss can be interpreted as a measure of protection. Thus the radiomodifying protection attributed to BGJ intake was 60%, over the experimental period.

Lipid peroxidation has been established as a measure of free radical activity and cellular damage in the body. We measured the lipid peroxidation by measuring the MDA per mg of liver protein estimated by the Bradford method in each animal. The greater the damage, the more MDA should be formed. In our model, this means the highest MDA per mg of liver protein should be in the animals irradiated and given placebo, not juice [Figure 2]. MDA levels were higher for the PR group, in agreement with those reported by us, where animals in the PR group showed a decrease in liver weight around 25%, suggestive of liver damage. Table 1 summarizes the average body weight measurements taken at 2, 5, 8 and 11th day after irradiation, average differences and MDA formation per milligram of liver protein for each group and treatment (PN, PR, JN, JR).

Statistical significance for MDA formation as a product of lipid peroxidation in liver was assessed by using the Student-Newman-Keuls Multiple Comparisons Test was performed by GraphPad® Prism 5 software. Values are expressed as means ± s.e.m. A value of p<0.05 was considered statistically significant for MDA levels from PR in comparison with JR group. No statistical differences were found for PN vs JR, PN vs JN, and JN vs JR.

Discussion And Conclusions

By observing the changes in two parameters, body weight and lipid peroxidation, in whole-body X-irradiated rats we are able to measure a radiomodifier effect provided by BGJ. The results are in agreement the radiation protection role of antioxidants. The radiomodifier effect of BGJ is apparent; the body weight loss was 60% less in X-irradiated animals receiving BGJ than in those receiving placebo. Protection levels estimated by liver PR/JR MDA formation ratios were found to be nearly 45%, indicating that BGJ offers protection for lipid peroxidation in liver. The radiomodifying effect of BGJ could be related to its flavonoid content, given the antioxidant properties of these compounds [Dani et al. 2008].

High MDA levels can be indicative of oxidative damage on mitochondrial and hepatocyte cell membrane. It is possible that oxidative damage caused by X-irradiation could promote cell death processes due to membrane damage such

J Med CBR Def | Volume 7, 2009 Submitted 12 May 2009 | Accepted 26 June 2009 | Revised 3 August 2009 | Published 8 August 2009 as radiation-induced apoptosis by ceramide pathway [Carini et al. 1992; Verheij et al. 1998]. Actually, there is a mixture of different products formed during lipid peroxidation, including toxic aldehydes such as MDA, that are strongly bound to proteins, modifying their function and leading to a degree of damage that can be assessed based on the MDA level. Although the TBARS assay is not specific enough to distinguishing all those products formed in lipid peroxidation process, it is capable to estimate the oxidative damage on cell membrane due to its high sensitivity.

Overall, changes in MDA levels can be a signature of changes in membrane physiology, and BGJ seems to protect against this physiological unbalance. Significant differences in lipid peroxidation for PR and JR groups [Figure 2] infer that BGJ flavonoid content, and its action as scavengers for hydroxyl radical, might be the reason for the protection offered to the membrane site, and reduction on MDA formation/detection for JR group. Moreover, quercetin (the most abundant flavonoid in BGJ) was recently reported as a protective agent against oxidative damage in rat primary hepatocytes [Liu et al. 2009], even though quercetin was not studied under oxidative damage conditions induced by radiation.

BGJ flavonoids might be responsible for the protection of rat liver cells by scavenging radical activity including both superoxide and hydroxyl radicals. Recent reports [Miccadei et al. 2008; Londhe et al. 2009] support the idea that flavonoids effectively prevent lipid peroxidation and protein oxidation in rat liver mitochondria. However those conclusions are drawn from studies of quercetin isolated from the flavonoid content, not in a natural mixture as it is presented in BGJ and discussed in the present study.

The lower relative body weight loss in the JR group in comparison with PR (see figure 1) might indicate that grape juice seems to protect against acute whole body X-irradiation, an effect possibly linked, once again, to the higher concentration of quercetin (30 uM) present on it. It is important to note that although quercetin is the most abundant flavonoid present in BGJ, it is possible that the mixture of all of them could provide a more efficient overall radiomodifier effect. Moreover, effects of different harvest conditions and changes in environmental conditions could alter the proportions of the main chemicals responsible for the radiomodifying effects.

The overall protection offered by BGJ can be measured as the ratio between body weight loss difference (placebo minus BGJ) and body weight loss for placebo. (Figure 1 and Table 1.) This protection for body weight loss is a very important result since body weight loss is one of the most important symptoms of acute radiation syndrome. Recent work shows the importance of developing a rapid method for helping people involved in a radiation accident, especially when the radiological nature of the accident is not immediately recognized [Bertho and Roy 2009]. Further detailed experiments are being carried out in order to improve our understanding about the radiomodifying effect of BGJ on pathophysiology and liver function.

We conclude that overall BGJ appears to act as a radiomodifier protection agent against the effects of whole body X-irradiation. This protection might permit

J Med CBR Def | Volume 7, 2009 Submitted 12 May 2009 | Accepted 26 June 2009 | Revised 3 August 2009 | Published 8 August 2009 the use of BGJ as a food and not as a pharmaceutical, which will facilitate its production, distribution, and administration in the event of a radiological disaster or dispersal.

Acknowledgements This work has been supported by a study-leave grant from CAPES (Brazil) and Ministerio de Educación y Ciencia (Spain) (Brazil-Spain cooperation project PHB2007-0071-PC). Additional support from Department of Biomedical Sciences (University of León, Spain) is also acknowledged.


Figures

Figure 1: Effect of Black Grape Juice Treatment on Body Weight. The average differences of body weight loss for Juice and Placebo irradiated groups relative to their respective control groups. The average weight loss is greater for the placebo treated rats. Each group had 5 rats (n = 5) on the sacrifice day except PR group, which had 4 (n = 4) because one rat died on day 16 after irradiation at YXLON MAXSHOT 200 X-rays machine at irradiation facility (University of León - Spain). Values are expressed as means ±sem.


Figure 2: Measurement of Lipid Peroxidation. The MDA formation, a measure of lipid peroxidation, in rat livers was estimated by TBARS assay measurements, according to [Wills 1987]. The higher the MDA formation, the greater the lipid peroxidation and the greater the cellular damage. Student-Newman-Keuls Multiple Comparisons Test was performed by GraphPad® Prism 5 software, * means significant for JR and PR, # means significant for PR and PN. There is no significance for JR in comparison to JN and PN.


Tables

Group Animal Average body

weight MDA/mg protein Group Animal

Average body weight

MDA/mg protein

1 296.87 0.878 11 270.17 2.056 2 298.27 0.474 12 264.23 1.442 3 296.33 0.517 13 289.89 0.946 4 289.09 0.642 14 297.77 death

JN 5 295.80 0.629 PR 15 280.51 1.489 6 305.17 1.106 16 304.94 0.476 7 285.06 0.955 17 301.20 0.698 8 291.39 0.640 18 327.76 0.334 9 276.21 0.695 19 286.80 0.747

JR 10 290.46 0.846 PN 20 306.18 0.569 Table 1. The Change in Body Weights due to Radiation. The changes in body weights after irradiation are presented, along with the MDA/ mg of protein, the measure of liver peroxidation. The greater the body weight loss, the greater the extent of radiation damage. The higher the MDA/mg protein, the greater the amount of lipid peroxidation. The PN, non irradiated placebo group, the PR, irradiated placebo group, the JN, non-irradiated group give BGJ, and the JR, irradiated group given BGJ.


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Effect Of Black Grape Juice Intake On Liver Lipoperoxidation And Body Weight Loss In Whole Body X-Irradiated Rats

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