Nutritional supplementation with arginine protects radiation-induced effects . An experimental study 1

PURPOSE: To investigate the protective effect of L-arginine on the prostate (nonneoplasic) of rats with radiation-induced injury. METHODS: Twenty-nine Wistar rats, male adult, allocated into three groups: Control group (C) was not exposed to irradiation (n=10); Radiated group (R) had undergone pelvic irradiation (n=10); Supplemented and radiated group (R+S) had undergone pelvic irradiation plus L-arginine supplementation (n=9). The animals were observed for signs of toxicity. After euthanization, the prostate was dissected under magnification and stained by hematoxylin and eosin to study acinar structures and stained with Picrosirius red for collagen analysis. RESULTS: After radiation exposure, all animals presented diarrhea, but supplementation with L-arginine reduced this effect. The weight gain in the R+S group was significantly higher than in the C and R groups. In the R+S group the collagen density and the prostate acinar area was similar to the R and C groups. Epithelial height was significantly reduced in group R compared with group C (p<0.0001). When comparing the group R+S with R, a statistical difference was observed to be present (p<0.0001). CONCLUSIONS: Pelvic radiation promotes systemic effects and some structural modifications in the ventral prostate of rats. These modifications can be prevented by oral supplementation with L-arginine.


Introduction
In recent years, many cytoprotective agents have been developed to protect normal cells from the toxic effects of anticancer chemotherapy and radiotherapy.From a theoretical standpoint, the ideal cytoprotective agent would be capable of maintaining the relative intensity of chemotherapy or a radiotherapy dose, protecting a wider range of organs and preserving antitumor activity, having lower intrinsic toxicity and protecting against free radicals 1 .
On this basis one can hypothesize that L-arginine, by possessing its own mechanisms for inactivation of free radicals produced by irradiation, would be a potentially radioprotective substance.Although the body itself produces L-arginine under certain conditions, such as in infection and in cases of stress, supplementation of these amino acids is essential.L-arginine is a semi-essential amino acid produced in the body, but in insufficient quantity for all needs.In the urea cycle, arginine is used in the synthesis of creatine and ornithine, enabling the synthesis of polyamines 2 .
The radioprotective potential of L-arginine relates to the synthesis of nitric oxide, arising from the oxidation of one of its guanidine nitrogens 3 .Nitric oxide is a potent mediator, with diverse roles in regulating cellular functions and signaling pathways 4 .
The aim of the study was to investigate the protective effect of L-arginine on the prostate (nonneoplasic) of rats with radiation-induced injury.

Methods
All applicable international, national, and institutional guidelines for the care and use of animals were followed.All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.This research was approved by the Institutional Animal Bioethics Committee of the Biological Sciences Center, Universidade Estadual do Rio de Janeiro (protocol number: CEA/224/2008).
Twenty-nine Wistar rats, male adult (90 days old) were kept in a room with controlled temperature (25±1°C), artificial dark-light cycle (lights on from 7:00 am to 7:00 pm) and fed standard rat chow and water ad libitum.
The rats were randomly allocated into three groups: Control group (C) was maintained under standard conditions and was not exposed to irradiation (n=10).Radiated group (R) had undergone pelvic irradiation (n=10) on the eighth day of the experiment.The supplemented and radiated group (R+S) had undergone pelvic irradiation plus L-arginine supplementation (n=9).This group (R+S) was also exposed to radiation on the eighth day of the experiment and was supplemented with L-arginine (Sigma-Aldrich®) from the beginning of the experiment (day 0) until death.L-arginne was administered by gavage in a dose of 0.65 g/Kg of body weight, diluted in distilled water [5][6][7][8] .
The animals in the R and R+S groups had each received a single dose of pelvic radiation 9 .
During all experiment stages, the animals were observed for signs of toxicity such as inappetence, weight loss, piloerection, hyper or hypo activity.
All animals were euthanized by an overdose of sodium thiopental on the 22nd day (14 days after radiation exposure).

Stereological analysis
The prostates were dissected with aid of a stereoscopic microscope, and ventral lobes fixed in 4% buffered formaldehyde.
The specimens were processed for paraffin embedding and sections of 5µm thickness were obtained.Samples were stained by hematoxylin and eosin to study acinar structures and Picrosirius red for collagen analysis.
Micrographs were captured by a digital camera (DP70 -Olympus ® ) coupled to a light microscope (BX51 -Olympus ® ).All analysis was performed on random fields using ImageJ ® software (National Institute of Health, USA).
After calibration, the area of the prostatic acini and epithelium height were measured with "freehand selection" and "straight line selection" tools respectively.The acini area was magnified x200; epithelium height analysis was performed using x1000 magnification.
For collagen analysis, a 100 points grid was superimposed over the images, and the point counting method [10][11] was used to determine objectively collagen surface density, expressed as a percentage.The analysis was performed using x400 magnification.

Statistical analysis
For parametric values, both analysis of variance (ANOVA) followed by the Student t test were used.For nonparametric data, both the Kruskal-Wallis test followed by the Mann-Whitney test were used.The GraphPad Prism 5.0 software was used for statistical analysis.The significance level for rejecting the null hypothesis was 5% (p≤ 0.05).

Results
After radiation exposure, all animals presented diarrhea.Supplementation with L-arginine considerably reduced this effect in approximately 40% of the animals (p=0.0329).Over 50% the animals in the radiated group had nosebleeds.In the supplemented group, 20% also had nosebleeds.No other toxicity sign was observed.
The weight gain in the R+S group (27.65g) was significantly higher than in the C (20.83) and R (3.11) groups (p=0.0000 and 0.0060, respectively).
Epithelial height was significantly reduced in group R compared to group C (p<0.0001).When comparing the group R+S with R, statistical difference was observed (p<0.0001).
The collagen density between the C and R+S groups showed no statistical difference (p=0.1237).Collagen was less in group R+S when compared with group R (p=0.8674), but the values did not differ significantly (Figures 1 and 2).The prostate acinar area of group R was similar to that of the C group (p=0.9715).The R+S group had a decrease when compared with the C (p=0.5648) and R groups (p=0.3470),but there was no statistically significant difference.
All data are summarized in Table 1.

Discussion
Pelvic radiation is well recognized as a cause of major side effects contributing to morbidity of oncologic patients.
One of the consequences of the radiation-matter interaction on cell structures is the production of a reactive oxygen species and oxidative damage.The immediate effects of radiation can be easily observed in tissues with great proliferative capability, such as the epithelium, leading to vascular injuries, hypoxia, and cell death 12 .
In recent years, L-arginine supplementation or restriction has been proposed and trialed in for a number of disorders, including vascular diseases and asthma, mitochondrial encephalopathy lactic acidosis and stroke-like episodes (MELAS), glutaric aciduria type I and disorders of creatine metabolism, both in production and transportation into the central nervous system 13 .
Supplementation with L-arginine was shown to have systemic protective effects, since it reduced radiation-induced diarrhea in approximately 40% of the animals, maintained body weight and increased weight gain.In another study, the authors also reported cases of diarrhea with oral supplementation of L-arginine 13 .
Possibly the systemic protective effects of oral supplementation with L-arginine are related to their ability to act in various systems such as cardiovascular, endocrine-metabolic and kidney, promoting, for example, stimulation of insulin secretion, growth hormone and reduction of angiotensin-converting enzyme activity.These beneficial effects have been associated with improvement in endothelial function with increased production of endothelium-derived relaxing factors 14 .
With specific reference to the prostate, radiation promoted a significant change in the acinar epithelium height, decreasing it by approximately 36% when compared to the control group and 41% for the supplemented group.This modification corroborates with what was previously pointed 12 .
Studies in rats showed that L-arginine protected vesical modifications, alterations in penile structures after pelvic radiation 15 , acting against oxidative damage [16][17][18] .However, there is a lack of information regarding L-arginine in preserving and maintaining the integrity of the prostate after pelvic radiotherapy.
The present study establishes through quantitative methods the benefits of oral supplementation with L-arginine for protecting the prostate from radiation.
Supplementation with L-arginine regenerated the acinar epithelium, as confirmed by the statistical difference between this group and the irradiated group, as well as between it and the control.According to Cukier 16 , L-arginine is the major oxidative fuel for the epithelial cells, consisting of an oxidizable amino acid precursor, a nitric oxide and polyamines, as well as being associated with the metabolism of urea.
Oxidative stress is detrimental to animals and the arginine may function as a potential substance against oxidative stress, through the enhancement of antioxidant capacity and inhibition of the expression of inflammatory cytokines (IL-6 and TNF-a mRNA) 17 .
The effects of L-arginine supplementation on human physiology appear to be multicausal and dose-related.Doses of 3-8 g/day appear to be safe and not to cause acute pharmacological effects in humans 18 .In this study, the L-arginne was administered by gavage in a dose of 0.65 g/Kg of body weight.
According to histomorphometric data, radiation significantly affected the rat prostate.After supplementation with L-arginine, there was a significant increase in the height of the epithelium and a decrease, not statistical, in collagen distribution.L-arginine appears to exert no effect in individuals with low ADMA levels, whereas in those with high levels, L-arginine restores the L-arginine/ADMA ratio to normal and, thereby, normalizes endothelial function 18 .
In another study, when L-arginine was used, the authors concluded that pelvic radiation leads to significant vesical modifications, as in the morphology of blood vessels and in VEGF and FGF expression in the wall of rat bladder.All these changes, however, were prevented by L-arginine treatment.The authors emphasized the potential use of this amino acid as a radioprotective drug 19 .
In addition to the radioprotective effect, L-arginine supplementation has demonstrated protective effects related to reduced dapsone-induced methemoglobin levels.This effect is mediated by nitric oxide formation, since the reduction in methemoglobin levels by L-arginine is blocked by simultaneous administration with L-NAME, a nitric oxide synthase inhibitor 20 .
It is possible that an analysis of the effects of pelvic radiation in prostate rats made over longer periods than that used in this study, 21 days, could result in more drastic changes than those observed in this work.
Also with respect to the radioprotective results of this study, many studies have shown that L-arginine has a protective effect on the radiation-induced injury in several systems (urogenital, skin tissue, immune, cardiac and intestinal mucosa), as can be observed in Supplement 1.

Conclusions
The pelvic radiation promotes systemic effects and some structural modifications in the ventral prostate of rats.These modifications can be prevented by oral supplementation with L-arginine.
The acinar area was similar to the control.Intravenous or dietary (oral) administration of relatively large doses of L-arginine has been shown to result in enhanced nitric oxide formation in individuals with impaired endothelial function at baseline.Recently, it became clear that endogenous levels of asymmetric dimethylarginine (ADMA), a competitive inhibitor of L-arginine metabolism by nitric oxide, might determine an individual's response to L-arginine supplementation.

TABLE 1 -
Morphometric data of ventral prostate from control, radiated and radiated with L-arginine supplementation rats.