In vivo evidence of hepato- and reno-protective effect of garlic oil against sodium nitrite-induced oxidative stress.

Sodium nitrite (NaNO2), a food color fixative and preservative, contributes to carcinogenesis. We investigated the protective role of garlic oil against NaNO2-induced abnormalities in metabolic biochemical parameters and oxidative status in male albino rats. NaNO2 treatment for a period of three months induced a significant increase in serum levels of glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), bilirubin, urea and creatinine as well as hepatic AST and ALT. However, significant decrease was recorded in liver ALP activity, glycogen content, and renal urea and creatinine levels. In parallel, a significant increase in lipid peroxidation, and a decrease in glutathione content and catalase activity were observed in the liver and the kidney. However, garlic oil supplementation showed a remarkable amelioration of these abnormalities. Our data indicate that garlic is a phytoantioxidant with powerful chemopreventive properties against chemically-induced oxidative stress.

Introduction

Natural and synthetic food additives approved by the U.S. Food and Drug Administration are commonly used to maintain or improve safety, the nutrient value, and the taste and texture of food 1. Although many of the 3,000 these additives enhance our food supply, others are the subject of fierce controversy. The discovery that children at the age of nursery consume food containing great amounts of additives prompted the scientific community to oversee this issue. Sodium nitrite (NaNO2) is present in vegetables and is routinely used as a color fixative and preservative for meats and fish 2. The hazardous effect of NaNO2 derives from the reaction of nitrites with amines to produce nitrosamines, and with amides to produce nitrosamides. The toxic effects of nitrates and nitrites are well documented in mammalians, including impairment of reproductive function 3, hepatotoxicity and methaemogobenemia 4, dysregulation of inflammatory responses and tissue injury 5, growth retardation 6, and endocrine disturbance 7. The wide use of nitrates as preservatives in food technology elevates the importance of studying their effects.

Although NaNO2 is generally accepted as a weak carcinogen 8, 9, Wistar rats exposed to 0.3% NaNO2 in their drinking water for at least one year developed squamous papillomas of the forestomach 10. Using F344 rat in a multiorgan carcinogenesis model, 0.3% NaNO2 given in the drinking water for 28 weeks increased the incidence of forestomach neoplasm in the post-initiation period 11. Thus, it cannot be precluded that NaNO2 has very weak carcinogenic potential, particularly in the squamous epithelium of the forestomach. Moreover, in combination with other chemicals, NaNO2 has also been shown to form carcinogens or to enhance carcinogenesis. For instance, highly carcinogenic N-nitroso-compounds are produced when nitrite reacts with secondary amines and N-alkyl amides under acidic conditions in vitro 8, 12 and in vivo 13. Other studies have demonstrated that treatment with NaNO2 in combination with phenolic compounds 11, 14 or ascorbic acid 15 strongly enhanced forestomach carcinogenesis in a rat two-stage carcinogenesis model.

Recent trends in controlling and treating diseases tend to favor natural antioxidant compounds rather than synthetic ones 16. The human diet, which contains large number of natural compounds, is essential in protecting the body against the development of diseases, and the garlic Allium sativum is one of the well known plant with remarkable anti-carcinogenic properties is 16, 17. Garlic, is a commonly worldwide used food, and its medical properties have been well recognized since the ancient times. Garlic is known for its antibacterial 18, anticarcinogenic 19, hypolipidemic 20, hypoglycemic 21, antifungal 22, and anti-atherosclerotic 23 properties, and an antioxidant against free radicals 24, 25.

Here, we investigated the role of garlic oil in preventing NaNO2-induced abnormalities in the biochemical parameters associated with the oxidative stress in male albino rats.

Materials and methods

Chemicals

NaNO2 (Sigma Aldrich, St Louis, MO) was applied as a freshly prepared solution and given by gavages at a dose of 80 mg/kg body weight as previously described 26. Garlic oil was purchased from El-Captain Company (Cairo, Egypt). Garlic oil was given by gavages at a dose of 5ml/kg as described 27.

Animals

Male Albino Rats (Rattus rattus) weighing about 100-120 g were used in this study. The animals were kept under good ventilation and received a balanced diet and water ad libitum throughout the experimental period. Rats were divided into four main groups (n= 6) as follow: 1) Control group received, standard diet without any treatment; 2) Garlic oil-treated group, received standard diet, which was supplemented orally with garlic oil at a dose of 5 ml/kg body weight for a period of 3 months; 3) NaNO2-treated group, received standard diet supplemented orally with sodium nitrite at dose of 80 mg/kg body weight for a period of 3 months; and 4) NaNO2+garlic oil-treated group, received standard diet and were supplemented orally with similar doses of NaNO2 and garlic oil as group 3 for similar period of 3 months.

At the end of the experimental period, overnight fasted animals were sacrificed by cervical dislocation, and blood samples were collected in centrifuge tubes. Serum was separated from coagulant blood by centrifugation at 860g for 20 min, and then quickly frozen at -20°C for biochemical analysis. Small pieces of liver and kidney tissues were separated, weighed, homogenized in ice cold water and stored at -20°C for subsequent measurements.

Biochemical analysis

Serum glucose level was determined using the Biomerieux reagent kits 28. Liver glycogen content was determined according to the method described by Van-Handle 29. Serum alanine aminotransfrase (ALT) and aspartate aminotransfrease (AST) activities were determined according to the method described by Reitman and Frankel 30, whereas alkaline phoasphatase (ALP) activity was estimated by Belfield method 31. Total protein, bilirubin, urea and creatinine levels were determined using Diamond Diagnostic Kit as previously reported 32-33. The product of lipid peroxidation, thiobarbituric acid reactive substances (TBARS), was determined as previously described 34. Glutathione (GSH) content was estimated by the method of Prins and Loose 35, and the activity of catalase was determined by the method of Aebi 36.

Statistical analysis

The data was analyzed using the Statistical Package for Social Science program (S.P.S.S. 11). For comparison between different experimental rat groups, one way analysis of variance (ANOVA) was used followed by Tukey's test. The results were expressed as means + SE and % of change, and P < 0.05 was considered to be statistically significant.

Results

A number of biochemical parameters were examined in the serum collected from each group and the results are summarized in table 1. While rats fed on standard diet supplemented with garlic oil did not show any significant changes in the majority of the parameters examined, a significant increase in serum levels of glucose, bilirubin, urea and creatinine as well as the activity of the AST, ALT and ALP enzymes were observed in rats treated with NaNO2 for a period of three months. The total protein content significantly decreased in serum (Table 1). However, supplementation of NaNO2- intoxicated rats with garlic oil ameliorated the nitrite adverse effects as evidenced by a significant increase of serum total protein content, and a decrease of serum glucose, bilirubin, urea and creatinine levels, as well as the activity of AST, ALT and ALP enzymes (Table 1). The same parameters were examined in liver and kidney tissues, and the data are shown in table 2. In the NaNO2-treated rats, a statistically significant inhibition of hepatic glycogen and total protein contents, and the activity of the enzymes ALT and AST as well as the levels of renal urea, creatinine and total protein content (Table 2). The activity of hepatic ALP was significantly increased in the NaNO2-treated rats (Table 2). However, administration of garlic oil to the NaNO2-intoxicated rats significantly restored these parameters in the liver and kidney organs (Table 2).

Table 1

Serum biochemical parameters in different rat groups. Results are presented as means ± SE (n=5) and % of change. AST: aspartate aminotransfrease ; ALT: alanine aminotransfrase; ALP: alkaline phoasphatase

a: Compared to control group; b: Compared to NaNO2 treated rats group

* % of change compared with control group.

** % of change compared with NaNO2 treated rats group.

Table 2

Hepatic and renal biochemical parameters in different rat groups. Results are presented as means ± SE (n=5) and % of change. AST: aspartate aminotransfrease ; ALT: alanine aminotransfrase; ALP: alkaline phoasphatase

a: Compared to control group; b: Compared to NaNO2 treated rats group

* % of change compared with control group.

** % of change compared with NaNO2 treated rats group.

Further, we assessed oxidative stress parameters and antioxidant activity in the liver and the kidney and the results are summarized in table 3. The data indicate that TBARS concentration increased significantly, while GSH content, as well as catalase activity were decreased in both organs of NaNO2-intoxicated rats (Table 3). However, combination of garlic oil with NaNO2 reduced TBARS concentration and restored the levels of GSH as well as the activity of catalase (Table 3).

Table 3

Hepatic and renal oxidative stress and antioxidant parameters in different rat groups. Results are presented as means ± SE (n=5) and % of change. TBARS: Thiobarbituric acid reactive substance; GSH: Glutathione; CAT: Catalase; MDA: malondialdehyde

a: Compared to control group; b: Compared to NaNO2 treated rats group

* % of change compared with control group.

** % of change compared with NaNO2 treated rats group.

Discussion

The NaNO2 and other additives may react with amines of the foods in the stomach and produce nitrosamines and free radicals. Such products may increase lipid peroxidation, which can be harmful to different organs including liver and kidney 37. On the other hand, these free radicals, known to cause oxidative stress, can be prevented or reduced by dietary natural antioxidants through their capacity to scavenge these products 38. The present study was undertaken to determine whether garlic oil can prevent and/or reduce NaNO2-induced oxidative stress by examining different biochemical parameters of oxidative damage in the serum, the liver and the kidney of male rats.

Our results clearly showed that there was a significant increase in serum glucose concentration and a decrease in liver glycogen content of NaNO2-treated rats. The findings suggest nitrite-stimulation of gluconeogenesis 39, and glucose shift from tissue to blood or an impairment of glucose mobilization. Furthermore, nitroso-compounds can alter the antioxidant system causing disturbance in the metabolic processes leading to hyperglycemia 40. However, serum glucose and liver glycogen levels were ameliorated upon garlic oil supplementation. The hypoglycemic effect of garlic oil and its organo-sulfur compounds might be due to their ability to enhance insulin secretion 41, 42.

Our results also indicate an inhibitory effect NaNO2 on the biosynthesis of protein, which was restored by garlic oil supplementation. These data suggest a stimulation of the thyroid and the adrenal glands by NaNO2 which can lead to a blockade in protein synthesis, fast breakdown, increased rate of free amino acids, and decreased protein turnover 43. In addition, nitrite interactions results into nitric oxide release, which can inhibit total protein synthesis 44. However, the increase in bilirubin concentration as well as the activity of AST, ALT and ALP enzymes in the serum of NaNO2-treated rats could be attributed to the toxic effect of nitroso-compounds, formed in the acidic environment of the stomach, in causing severe hepatic necrosis 45. These abnormalities were prevented by supplementation of garlic oil, perhaps due to its role in stabilizing the cell membrane and protect the liver from free radical-mediated liver cell toxicity 46.

In response to NaNO2 treatment, urea and creatinine were increased in the serum but decreased in the kidney, suggesting an impairment of kidney functions. These effects could also be attributed to the changes in the threshold of tubular re-absorption, renal blood flow and glomerular filtration rate 47. Garlic oil showed a clear improvement in kidney functions, perhaps due to the antioxidant properties of garlic in scavenging free radicals leading to reduced levels of nitric oxide and lipid peroxidation. Moreover, NaNO2-inhibited glutathione content and catalase enzyme activity in the liver and the kidney may be attributed to the observed induction of lipid peroxidation 48. However, garlic improved the antioxidant mechanism due to the ability of Diallyl disulfide and Diallyl trisulfide present in garlic oil in modulating the oxidative stress and detoxifying enzyme system 49, 50, 51.

In conclusion, from the results achieved it can be concluded that the administration of garlic has an extremely beneficial role in overcoming the occurred adverse effects of chronic ingestion of sodium nitrite, which is probably through its excellent antioxidant properties and highly nutritional values.