ORIGINAL

 

Radiomodifying effect of organic grape juice supplementation on hematological parameters and organ weight in whole-body X-irradiation in rats

Efecto radiomodificador de la suplementación con mosto de uva de cultivo ecológico sobre parámetros hematológicos y peso de órganos en ratas sometidas a irradiación de cuerpo entero con rayos X

 

 

E. Ramos de Andrade², J. Da Costa Escobar Piccoli², I. B. Mânica da Cruz², J. B. Teixeira Rocha², E. Garzo¹, R. Marina¹, J. L. Mauriz¹, P. González¹ and J. P. Barrio¹

¹Instituto de Biomedicina. Centro de Investigaciones Médicas en Red: Enfermedades Hepáticas y Digestivas (CIBEREHD). Departamento de Ciencias Biomédicas. Univesidad de León. Spain. ²Centro de Ciências Naturais e Exatas, Ciências Biológicas. Programa de Pós-Graduação em Bioquímica Toxicológica. Universidade Federal de Santa Maria. Santa Maria RS. Brazil.

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

Correspondence

 

 

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ABSTRACT

The aim of this study is testing black grape juice as a radiomodifier against whole body X-irradiation using an animal model. Sixteen male Wistar rats were divided into four groups where two were irradiated by X-rays from a 200 kV machine specially designed to biological samples. Animals were fed ad libitum and drank voluntarily 2-10 ml a day of grape juice or placebo (isocaloric glucose and fructose solution) for one week before and two weeks after 6 Gy X-irradiation when they were sacrificed. Results have shown a significant liver weight loss in irradiated placebo group only while grape juice one has presented no losses. Hematological analysis showed typical abnormalities for ionizing radiation exposure, including early leucopenia and anemia. The intake of grape juice induced an increase in granulocyte percent count.

Key words: X-rays. Black grape juice. Radiomodifier. Rats.

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RESUMEN

El propósito de este estudio fue comprobar el efecto radiomodificador del mosto tinto de uva frente a irradiación de cuerpo entero con rayos X usando un modelo animal. Dieciséis ratas macho de raza Wistar fueron irradiadas mediante un aparato de 200 kV diseñado específicamente para muestras biológicas. Los animales fueron alimentados ad libitum y bebieron cada dia voluntariamente entre 2 y 10 ml de mosto de uva o placebo (solución isocalórica de glucosa y fructosa) durante una semana antes y dos semanas después de irradiación con rayos X a una dosis de 6 Gy, momento en que fueron sacrificadas. Los resultados mostraron una pérdida significativa de peso hepático en los animales irradiados tratados con placebo, mientras que los tratados con mosto presentaron valores similares a los controles no irradiados. El análisis hematológico presentó las anomalías típicas de la exposición a radiación ionizante, con disminución leucocitaria temprana y anemia. La ingestión de mosto de uva indujo un aumento del porcentaje de granulocitos.

Palabras clave: Rayos X. Mosto tinto de uva. Radiomodificador. Ratas.

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Introduction

The use of ionizing radiation for a wide range of purposes has been growing up rapidly since the nuclear holocaust. Nuclear and radiological technologies have spread over most of knowledge fields, from Engineering to Health Sciences, even though its uses can be controversial and a bottleneck on radiation protection is a problem to be solved. Exposure to ionizing radiation can be harmful and searching for radiomodifiers (drugs or nutrients) has full epidemiological relevance. The ideal radioprotective agent (radiomodifier) must provide: a) significant protection against radiation effects; b) a general protective effects to all other organs including non-target ones (in case of therapy); c) acceptable route of administration (oral is preferred); d) low toxicity; e) compatibility for use with other drugs.^1,2 Radiomodifiers are substances able to reduce the effect of the ionizing radiation strike, but unable to stop it. Their action is neither like a barrier nor shielding for ionizing radiation, but as scavengers for reactive oxygen species. Clinically relevant radiomodifier substances should have low or none toxicity and synergic action with other drugs. Dealing with these purposes the best option is a functional food or nutraceutical. Unfortunately, there is no compound that manifest all these properties at a time and among more than 300 radiomodifiers developed,² amifostine (WR2721, Ethyol) has proved as most efficient but only authorized for radiotherapy treatment of head and neck cancer.^3,4 Natural compounds in human diet could provide functional antioxidants, such as vitamins, minerals and enzymes acting as radiomodifiers on reducing oxidation damage caused by ionizing radiation exposure. An example is the use of vitamin E for recovering post-irradiation procedures showing good results.^5-7 Radiation exposure might be heterogeneous in terms of dose, dose rate and quality, depending on the type of the radiation source released and the location of the subject on site. Therefore, methods are needed to protect against and treat a wide range of early and later developing radiation-induced injuries. Acute effects of the exposure to ionizing radiation mainly include immune suppression, hematopoietic cell loss, mucosal damage, and potential injury to other sites such as lung, kidney, liver and central nervous system.⁸ Liver plays a particular role in radiosensitivity and as a consequence of its redundant, parallel functional structure, liver is able to deal with high radiation dose as long as only partial irradiation occurs; otherwise, whole organ irradiation leads to hepatocyte failure and RILD (Radiation Induced Liver Disease) such as hepatitis might be installed.⁹

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. The hematopoietic syndrome is significantly important to partial-body or whole-body ionizing radiation exposures exceeding 1Gy.¹⁰ Irradiation of bone marrow stem and progenitor cells at increasing doses results in exponential cellular death.¹⁰ Mitotically active hematopoietic progenitors have a limited capacity to divide after a whole-body radiation dose greater than 2 or 3 Gy.¹¹ After exposure, a hematologic crisis might lead to: a) predisposition to infection, b) bleeding, and c) poor wound healing, among others. A predictable decline in lymphocytes occurs after irradiation, and in fact, there is a 50% decline in absolute lymphocyte count within the first 24 hours after exposure, followed by a further, more severe decline within 48 hours.¹² The predictability of the rate of lymphocytic depletion count, which characterizes a potentially lethal exposure, has led to the development of a model using lymphocyte depletion kinetics (Andrew's curve) as a biodosimetric tool.^12-14 The rate of decline of the absolute lymphocyte count over the initial 12 hours and for a week after exposure is a function of cumulative dose¹⁵ and the lymphocyte depletion kinetics predicts dose assessment for a photon-equivalent dose range within 1 and 10 Gy, range in which most of the radiobiological effects take place. For an optimal screening and a good support for clinical decision-making process a complete blood cell count with leukocyte differential should be obtained immediately after exposure, 3 times per day for the next 2 to 3 days, and then twice per day for the following 3 to 6 days. However, it is recommended at least 3 complete blood counts with differential within the initial 4 days after exposure to calculate a slope for lymphocyte decline for estimating the exposure dose. The onset of other cytopenias varies depending on both dose and dose rate and granulocyte counts may transiently increase before decreasing in exposures less than 5 Gy in humans.¹⁶ This behavior, termed an abortive rise, is a transient increase in the absolute number of cells in any compartment of a nearly depleted hematopoietic cell renewal system, and may indicate a survivable exposure.¹⁶

The probability of occurrence of those effects can be minimized or altered by the radiomodifier action. Several evidences suggest that grape juice and seeds can provide protection levels against exposure to ionizing radiation.¹⁷ Radiobiological effects of ionizing radiation are a brand new issue of Science and have been taking noticeable advances over the last 60 years.¹⁸ However, most of the effort made has been facing a hard pathway, bordering other fields such as Biochemistry, Molecular Biology and Medicine, which turns Radiobiology out a complex field to be explored.

The present work is aimed to test black grape juice as a radiomodifier against whole body X-irradiation using rats as an animal model in order to assess the possible changes in bodily and hematological parameters.

 

Materials and Methods

Animals

Sixteen male Wistar rats weighing 200-250 g (Harlan, Barcelona, Spain), housed at the animal house of University of León (Spain), were included in the study. 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.

Whole Body Irradiation

Animals were divided into four groups: (MN) nonirradiated, grape juice supplemented; (GN) non-irradiated, placebo (isocaloric glucose plus fructose) supplemented; (MR) irradiated, grape juice supplemented, and (GR) irradiated, placebo (isocaloric glucose/fructose solution) supplemented. In order to immobilize the animals, anesthesia was induced by intraperitoneal administration of pentobarbital 0.6% in saline (10 ml/kg 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 on a plexiglas board, so that four animals would be irradiated at a time and exposed to a single dose of 6 Gy of whole body X-irradiation from an X-ray machine (200 kV) MAXISHOT 200 (YXLON, Copenhagen, Denmark), at a radiation dose rate of 0.40 Gy/min, with a source-skin distance (SSD) of 50 cm.

Food and Drink

Animals were fed according to a standard rat chow diet, having free access to ad libitum water and food. After one week adaptation to individual cages, they were allowed to ingest a maximum of 10 ml of test compound (grape juice) or placebo, depending on their assigned group. Environmental conditions were controlled (12-hour photoperiod and 20 ± 2 ºC) throughout the experimental period.

Grape juice and placebo composition

Ecologically-produced (organic) black grape juice was obtained from the city of Garibaldi (Rio Grande do Sul, Brazil), in the main grape-growing region of the state. 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 determined as follows: Resveratrol 3.95 ± 0.01, Quercetin 8.95 ± 0.09, Rutin 3.75 ± 0.03, Gallic acid 81.07 ± 2.03, Caffeic acid 30.28 ± 2.00, Flavonoids 0.249 ± 0.002.¹⁹ 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).

Blood samples collection

Blood samples were collected at 6, 24, 48 hours and 16 days following X-ray exposure using heparinized capillaries by puncturing the retro-orbital plexus after prior mild anesthesia with isofluoran.

 

Results and discussion

As it can be seen in table I, leukocyte count decreased with significant differences for all samples considering MR and GR groups in relation to controls. Lymphocyte counts fell down dramatically, but they showed a relative recovery for MR group, not seen in GR. This fall in lymphocyte counts may be interrupted by an abortive rise¹⁶ followed by final recovery due to the release of damaged blood cells. Monocyte count dropped significantly in comparison to controls only for 24 and 48 hours. Granulocytes decreased, for all sampling times and groups. Erythrocyte count (table III) showed normal values for the first 48 hours and a significant decrease at 16 days after irradiation, indicative that an anemia was installed. Levels of hemoglobin decreased significantly at 48 hours for MR group, and at 16 days in MR and GR, respectively to controls. Although there is absence of statistical significance on hemoglobin levels for MR and GR groups compared each other, at 16 days there is a tendency on keeping higher values for MR group, with higher variability found on GR group. This tendency could be related to the glucose and fructose absorption decrease in small intestine in irradiated rats, with increase in CO₂ production in peripheral blood leading to an increase of hemoglobin concentration.^{20, 21}

 

 

 

Changes in hemoglobin and erythrocyte count are also to be expected after significant damage to bone marrow subsequent to X-irradiation, as shown in a number of studies.^22,23 At this respect, hematocrit values were statistically distinguishable for groups MR and GR with respect to controls only at 16 days. Platelet counts have shown statistical significances for MR and GR only for 16 days respectively to controls. Two animals from GR group showed nasal bleeding possibly related to platelet depletion, in contrast to MR group where no bleeding was noticed over the experimental period. Relative counts (%) for lymphocytes (table II), were significantly decreased for MR and GR respectively to controls for 6, 24 and 48 hours but not for 16 days. However, percent counts for monocytes and granulocytes increased at 6, 24 and 48 hours relative to controls. When comparing the X-irradiated groups at and 48 hours post-irradiation, total leukocyte count was higher for GR than MR group (table I). However, the much higher variability appearing in the data from GR group indicates that some caution has to be taken when discarding possible radioprotective effects of grape juice. Percent lymphocyte count was higher on GR than MR, but percent granulocyte count was higher on MR than GR group, which suggest a real possibility of recovering from damaged bone marrow, in agreement with the proposed radioprotective effect of grape juice supplementation over bone marrow. All in all, these results point to massive bone marrow damage as a consequence of whole-body acute X-irradiation, and further experiments should be carried out at different times post-irradiation with direct bone marrow sampling²³ to clarify the time course of these radiomodifying effects.

The evaluation of the body weight changes before and after X-irradiation (fig. 1) showed significant differences from the irradiated grape juice group (MR) in comparison to the placebo one, even though both presented significant body weight loss. Non-irradiated groups showed the same growth pattern for both juice and placebo ones. The irradiated grape juice group showed no significant deviation when compared to the non-irradiated one. On the other hand, a significant deviation was found in placebo group after irradiation,where a remarkable weight loss took place. The average body weight in the grape juice group was closer to the non-irradiated group than placebo, indicative that grape juice seems to protect against X-irradiation²⁴ over total body weight loss.

Significant differences from non-irradiated groups (table IV) were found for liver, spleen and hepatosomatic index, but not for heart and kidneys, suggestive of a higher radioresistance of these latter tissues, at least on the time window explored here. No liver weight differences were found for MR, MN and GN groups. However, GR showed considerable liver weight loss in comparison to MR and all other groups. Previous reports have shown increased liver weight 6h after X-irradiation in rats²⁵ and abnormalities due to small intestine X-radiation exposure, leading to changes on glycogen levels and liver function.^26,27 The present work shows that there was significant liver weight loss after 16 days for GR group, but not for MR group, which is suggestive of a radioprotective action of black grape juice supplementation. The composition of the ecologically-grown black grape juice shows a high content of bioactive phenolic compounds such as resveratrol, quercetin and rutin,¹⁹ and it is tempting to link the radiomodifying actions of black grape juice to these chemicals. Flavonoids are known to have important antioxidant and anti-inflammatory activities.^28,29 Resveratrol has been since long studied not only as a potential antioxidant stimulating agent³⁰ and radiomodifier, ^24,31 but also because of its anti-mutagen action, its role in mediating anti-inflammatory effects, anti-carcinogenic action by the inhibition of cyclooxygenase and hydroperoxidase activities.³² Resveratrol has also been shown to influence the apoptotic effects of cytokines, chemotherapeutic agents, and ionizing radiation.³³ Pharmacokinetic studies of resveratrol activity revealed that its main target organs are liver and kidney,³³ where its conversion into a sulfated form and a glucuronide conjugate takes place. Nevertheless, quercetin and rutin are per se antioxidant agents³⁴ and so might be potentially co-responsible for the radiomodifying effect of black grape juice we are starting to see from this study.

 

 

It must also be commented out the voluntary nature of black grape juice intake by rats. There was a high individual variability, but all animals from MR and MN groups drank at least 2 ml from a maximum allowable of 10 ml. The placebo solution, on the contrary, was more palatable, with a minimum of 8 out of 10 ml being drank daily. The choice of ceiling for supplement intake was made based on previous considerations of the potential effect of black grape juice on ex vivo studies with human volunteers.³⁵ Food intake was severely decreased by 63±4% the first day after irradiation, but it increased thereafter, to be totally resumed 5 days post-irradiation, without differences due to grape juice supplementation (data not shown). Water intake increased by 49 ± 5% in all groups, X-irradiated and controls, on the irradiation day, but resumed to normal the next day onwards. This effect was attributed to the anesthesia procedure all animals suffered.

 

Conclusions

The authors studied the radiomodifier effect of organic grape juice through changes of physiological and hematological parameters in whole body X-irradiated rats. Most of the results are in agreement to the scientific background concerning to non-irradiated groups response and add new, albeit non conclusive, observations. Ecologically-grown grape juice seems to have a radiomodifier effect over selected hematological parameters on whole body X-irradiated rats. The remarkable result was the maintenance of a normal liver weight for MR group, in comparison to GR group, which had become 25% smaller. There is a background support for the idea that glucose and fructose intake is able to induce liver weight gain.²⁷ In spite of this liver weight gain, there are many experimental data supporting the reduction on glucose and fructose absorption by small intestine, which could imply to reduce the amount of sugar reaching to liver, leading to a consequent reduction on its conversion to fat. Non-conclusive effects were found for haematological parameters in relation to the radiomodifying effect of black grape juice intake, albeit percent granulocyte count showed significant increase at 48 hours post-irradiation in MR group. The anemia and leucopenia observed at 16 days post-irradiation are suggestive of significant damage to bone marrow tissue. More detailed experiments must be carried out in order to improve our understanding about the radiomodifying effect of ecological grape juice over blood cells and liver function.

 

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Correspondence:
Juan Pablo Barrio Lera.
E-mail: jpbarl@unileon.es

Recibido: 5-II-2009.
Aceptado: 6-III-2009.