Abstract
Sodium benzoate (NaB) is a versatile food preservative that has also found some applications in the treatment of medical disorders. However, till date, its possible widespread effects on the body are not well studied. We examined the likely effect of diet-added NaB on weight/food intake, haematological parameters, neurobehaviour, antioxidant status, lipid profile and anti-inflammatory/apoptotic markers in mice. Animals were assigned randomly into 4 groups of 10 mice each. Groups included normal control (fed rodent chow) and three groups fed NaB at 125 (0.0125%), 250 (0.025% and 500 (0.05%) mg/kg of feed added to diet, respectively, for eight weeks. Body weight and food intake were assessed. At the end of the experimental period animals were euthanized, blood was then taken for the assessment of haematological, biochemical and inflammatory/apoptotic markers. At the lowest concentration, NaB diet increased body weight and food intake. Decrease in haematological cell counts and total antioxidant capacity were observed, whereas serum malondialdehyde levels and superoxide dismutase activity were increased across the three concentrations. Serum tumour necrosis factor-alpha and interleukin-10 decreased, whereas caspase-3 levels showed no significant difference. Lipid profile and biochemical indices of kidney and liver function were also affected by NaB diet. In conclusion, our findings suggest that NaB may be harmful if regulations regarding its limit of consumption are mistakenly or deliberately ignored. Therefore, it is advisable that regulations on quantities to be added to food be enforced.
Keywords: apoptosis, food preservative, anaemia, leucopenia, inflammation, oxidative stress
Graphical Abstract
Graphical Abstract.
Showing the effect of sodium benzoate on body weight, food intake, haematological, lipid profile, lipid peroxidation, antioxidant status, inflammatory and apoptotic markers in mice.
Introduction
Food preservatives prevent or delay the onset of enzymatic, chemical or microbiological changes that occur in food. They fend off visible spoilage of food by bacteria, moulds and yeasts, as well as prevent the formation of toxins [1, 2]. Food preservatives are believed to play crucial roles in ensuring the overall safety of foods and food supply. Nevertheless, there have been suggestions that these food preservatives could also possess potential genotoxic and mutagenic effects [2–4]. In humans, food preservatives are considered safe (especially when used within recommended levels of intake). However, in the last few decades or more, there has been increasing concerns regarding the possible adverse effects of some food additives. These effects could either be acute or occur as a result of long-term use. The acute effects usually include headaches, lethargy, alteration in mental status and immune system hyperactivity, whereas long-term use has been associated with increased risk of developing cancers. That being said, widely used food additives include monosodium glutamate, colouring agents like tartrazine (E102), Quinoline yellow (E104) and food preservatives like benzoic acid (E210) and sodium benzoate (E211).
Sodium benzoate (NaB) is a food preservative, which is a stable, water-soluble sodium salt of benzene carboxylic acid (benzoic acid) with fungistatic and bacteriostatic effects [5, 6]. It has found use in the preservation of a number of foods and food products including flour, salad dressings, jams, carbonated drinks, fruit fillings and juices, and even cosmetics [6, 7]. Also, NaB has found medical use in the management of a number of conditions including acute hyperammonaemia in persons with disorders of the urea cycle [8], hepatic encephalopathy, multiple sclerosis [6, 7] and post-dural puncture headaches [9]. In the last few years, there have also been suggestions that NaB administered at 1 g/day in addition to standard drugs is beneficial in the management of schizophrenia [10]. Although NaB has GRAS (generally regarded as safe) status in many countries [11], acceptable limits of dietary intake of 0–5 mg/kg body weight has been set by the FAO/WHO expert committee on food additives [2, 4, 7]. In Nigeria, there are reports that the National Food and Drug Administration agency (NAFDAC) in accordance with Codex Alimentarius Commission [12] guidelines has set the recommended limit of NaB in foods and drinks at 250 mg/kg [13].
Despite assurance by the regulatory bodies that NaB is safe in humans, its use has continued to be controversial, and there have been reports that excessive intake of NaB could have adverse effects. The consumption of beverages and/or carbonated drinks containing the preservative NaB has been linked with the development of behavioural deficits including memory loss, motor impairment and anxiety [7, 14]. Yetuk et al. [2] following an in vitro study in erythrocytes reported evidence of oxidative stress with NaB. Also, within the last decade or more, there have also been reports that the combination of NaB (or other benzoates) and ascorbic acid (a food antioxidant) especially in drinks was associated with the formation of benzene. This was confirmed in a number of drinks tested by the US Food and Drug Administration in 2006, with the observation of benzene levels in some drinks being above the 5 ppb maximum levels of contaminant set by the Environmental Protection Agency for drinkable water [15]. Although there have been reports from in vitro studies demonstrating the favourable effects of NaB especially when added to drinking water, there is a dearth of in vivo preclinical studies assessing the toxicological profile of NaB when added directly to dry food, especially at the concentrations recommended by the Codex Alimentarius Commission [12]. Hence, this study investigated the effect of NaB added directly to dry feed on body weight, food intake, haematological parameters, lipid profile, oxidative stress parameters and biochemical indices of kidney and liver function in mice. We tested the hypothesis that diet-added NaB is associated with deleterious effects in mice.
Materials and Methods
Materials
Included materials are food-grade NaB (sourced from the Open market in Osogbo, Osun State, Nigeria), assay kits for total cholesterol (TC), low-density lipoproteins (LDLs), triglycerides (TGs), high-density lipoproteins (HDLs), alanine aminotransaminase (ALT), urea, aspartate aminotrasnaminase (AST), creatinine (Randox Laboratory Ltd, UK), tumour necrosis factor-α (TNF-α), interleukin (IL)-10 (ENZO Life Sciences, USA), and caspase-3 (BioVision Inc. USA).
Animals
Swiss mice, weighing 20–25 g were obtained from Empire farms in Osun state, Nigeria and used for this study. Animals were kept in cages (plastic) located in temperature-controlled quarters (23–26 degree Celsius) with 12 hours dark–light cycle. Animals had access to food and water ad libitum. Experiments complied strictly with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines as well as approved protocols of the Ladoke Akintola University of Technology and the provisions of the European Council Directive (EU2010/63) for the use and care of laboratory animals.
Feed
Animals were fed commercially available rodent diet [(standard diet (SD)] sourced from Top Feeds Ltd, Ibadan Nigeria). NaB was incorporated into standard rodent diet at 125 (0.0125%), 250 (0.025%) and 500 (0.050%) mg/kg of feed. The concentrations used were chosen with reference to the recommended limit of NaB in foods and drinks [13].
Experimental methodology
Forty male mice (aged 5–6 months) and weighing between 25–30 g were assigned at random into 4 groups of 10 mice each. The groups included: control fed SD, and three concentrations of NaB incorporated into SD at 125 (0.0125%), 250 (0.025% and 500 (0.05%) mg/kg of feed. NaB or SD was administered for 8 weeks, and body weight was measured weekly. At the end of the experimental period, animals were euthanized by cervical dislocation as previously described [16]. Blood was drawn from the cardiac chamber and used for the estimation of haematological markers, biochemical markers of renal and hepatic injury, lipid peroxidation using malondialdehyde (MDA) levels, lipid profile, superoxide dismutase (SOD), total antioxidant capacity (TAC), TNF-α, IL-10 and caspase-3 levels.
Determination of body weight and food intake
Weekly body weight and daily food intake measurements were carried out using an electronic weighing balance as described [16–19].
Haematological tests
Blood samples collected were used to analyse red cell count (RCC), haemoglobin (Hb), haematocrit, white cell count (WCC), mean platelet volume and plateletcrit (PCT) using the Haematology Autoanalyser Systems (Sysmex Haematology Systems®, Model XE-2100, Sysmex Incorporation, USA).
Liver and kidney biochemistry
Serum levels of aspartate and alanine aminotransaminase were used to measure liver function, whereas the levels of creatinine and urea in the serum were used to measure kidney function.
Determination of aspartate and alanine aminotransferase activity
Serum aspartate aminotransferase (AST) activity was determined as described in an earlier study [20–22]. Serum alanine aminotransferase (ALT) activity was as described earlier [23–25].
Determination of serum urea and creatinine levels
Serum creatinine was measured by a method based on the modified kinetic Jaffe reaction [26]. Creatinine concentration was calculated and expressed as mg/dl. Serum urea was determined by a method based on modified Urease–Berthelot method [27].
Lipid profile
TC, HDL-C, TGs and LDL-C in the serum were analysed using commercially available kits following the manufacturer’s instructions.
Lipid peroxidation (MDA)
The lipid peroxidation kit was used to determine MDA levels following the manufacturer’s instructions.
Antioxidant activity
SOD activity was assayed using the method previously described by Nikishimi et al. [28]. SOD activity was expressed as u/ml. TAC measures the amount of free radicals scavenged by the test solution in any biological sample [29–32]. TAC was based on the principle of the Trolox equivalent (TE) antioxidant capacity [33, 34].
Tumour necrosis factor-α and Interleukin-10
TNF-α and IL-10 were measured using enzyme-linked immunosorbent assay techniques with commercially available kits (Enzo Life Sciences Inc. NY, USA) designed to measure the ‘total’ (bound and unbound) amount of the respective cytokines.
Caspase-3 levels
Caspase-3 levels (BioVision, USA) were determined using commercially available assay kits following the manufacturer‘s instructions.
Statistical analysis
Statistical analysis was carried out using Chris Rorden’s ezANOVA for windows. Data obtained were subjected to analysis of variance and post hoc tests (Tukey’s HSD). Results are expressed as mean ± standard error of mean (SEM), P < 0.05 was taken as the accepted level of significant difference from control.
Results
Effect of NaB on body weight and food intake
Figure 1 shows the effect of diet-added NaB on body weight (upper panel) and food consumption (lower panel). Body weight was significantly in [F (3, 36) = 9.97, P < 0.006] increased with NaB at 125 mg/kg of feed compared with mice fed the control diet. Concentration for concentration comparisons revealed a significant decrease in body weight with NaB at 250 and 500 compared with effects observed at 125 mg/kg of feed.
Figure 1.
Effect of food-added NaB on change in body weight (upper panel) and change in food intake (lower panel). Each bar represents mean ± SEM, *P < 0.05 vs. control, number of mice/group = 10
Food consumption was significantly [F (3, 36) = 2450, P < 0.001] increased with NaB at 125 mg/kg of feed in comparison with mice fed the control diet. Concentration for concentration comparisons revealed a significant decrease in food intake with NaB at 250 and 500 compared with effects observed at 125 mg/kg of feed.
Effect of NaB on haematological parameters
Tables 1, 2 and 3 show the effect of diet-added NaB on haematological parameters. Table 1 depicts the effect of NaB on red cell parameters. RCC was significantly [F (3, 36) = 110, P < 0.001] decreased with NaB at 125, 250 and 500 mg/kg of feed in comparison with mice fed the control diet. Concentration for concentration comparisons revealed a significant decrease in RCC with NaB at 250 and 500 compared with effects observed at 125 mg/kg of feed.
Table 1.
Effect of NaB on red cell parameters
| Groups | RCC(x1012/L) | Haematocrit (%) | Hb(g/dl) | MCV (fl/cell) | MCH (pg/cell) | MCHC (g/dl) |
|---|---|---|---|---|---|---|
| Control | 6.10 ± 0.01 | 39.0 ± 0.10 | 95.30 ± 2.50 | 55.12 ± 1.11 | 13.31 ± 0.20 | 232.0 ± 1.50 |
| NaB 125 | 4.16 ± 0.01* | 30.0 ± 0.12* | 93.50 ± 2.40* | 41.17 ± 1.13* | 13.27 ± 0.20 | 233.5 ± 2.20 |
| NaB 250 | 4.06 ± 0.01*,# | 28.0 ± 0.12* | 92.30 ± 2.50* | 40.64 ± 1.22* | 13.54 ± 0.25 | 234.2 ± 3.20 |
| NaB 500 | 4.03 ± 0.02*,# | 27.0 ± 0.19*,# | 90.50 ± 2.30*,# | 34.50 ± 1.12*3 | 13.68 ± 0.24 | 233.3 ± 2.40 |
Values are presented as mean ± SEM, *P < 0.05 vs. control, #P < 0.05 concentration vs. concentration, number of mice/group = 10.
Table 2.
Effect of NaB on white cell parameters
| Groups | WCC(x109/L) | Granulocytes x109 | Mid cells x109 | Lymphocytes x109 |
|---|---|---|---|---|
| Control | 5.10 ± 0.01 | 0.31 ± 0.05 | 0.20 ± 0.06 | 4.22 ± 0.34 |
| NaB 125 | 3.11 ± 0.01* | 0.30 ± 0.06 | 0.17 ± 0.07* | 2.23 ± 0.10* |
| NaB 250 | 2.80 ± 0.03*,# | 0.29 ± 0.06 | 0.16 ± 0.03* | 2.20 ± 0.28* |
| NaB 500 | 2.53 ± 0.02*,# | 0.28 ± 0.10 | 0.15 ± 0.02*,# | 2.09 ± 0.20*,# |
Values are presented as mean ± SEM, *P < 0.05 vs. control, #P < 0.05 concentration vs. concentration, number of mice/group = 10.
Table 3.
Effect of NaB on platelet parameters
| Groups | Platelet count (x109/L) | MPV | PCT (%) | PDW | P-LCR |
|---|---|---|---|---|---|
| Control | 380.0 ± 0.21 | 6.02 ± 0.01 | 29.4 ± 0.11 | 13.06 ± 0.02 | 0.06 ± 0.01 |
| NaB 125 | 370.0 ± 0.21 | 6.04 ± 0.02 | 30.3 ± 0.10 | 13.16 ± 0.01 | 0.07 ± 0.01 |
| NaB 250 | 375.2 ± 0.23 | 6.10 ± 0.01 | 28.5 ± 0.10 | 13.18 ± 0.01 | 0.07 ± 0.01 |
| NaB 500 | 382.0 ± 0.22 | 6.09 ± 0.01 | 30.2 ± 0.10 | 13.15 ± 0.01 | 0.06 ± 0.01 |
Values are presented as mean ± SEM, *P < 0.05 vs. control, number of mice/group = 10.
Haematocrit [F (3, 36) = 13.24, P < 0.001], Hb concentration [F (3, 36) = 16.23, P < 0.001] and mean corpuscular volume (MCV) [F (3, 36) = 5.24, P < 0.001] were significantly decreased with NaB at 125, 250 and 500 mg/kg in comparison with mice fed the control diet. Concentration for concentration comparisons revealed a significant decrease in haematocrit, Hb concentration and MCV with NaB at 500 compared with effects observed at 125 mg/kg of feed.
Mean corpuscular Hb concentration (MCHC) and mean corpuscular Hb (MCH) did not differ significantly in any of the groups fed NaB in comparison with mice fed the control diet. Concentration for concentration comparisons revealed no significant difference in MCHC or MCH with NaB at any of the concentrations.
Table 2 shows the result of total and differential WCC. Total WCC was significantly decreased [F (3, 36) = 10.33, P < 0.001] with NaB at 125, 250 and 500 mg/kg of feed in comparison with mice fed the control diet. Concentration for concentration comparisons revealed a significant decrease in WCC with NaB at 250 and 500 compared with effects observed at 125 mg/kg of feed. Granulocyte count was not significantly different in any of the groups fed NaB compared with control diet. A significant decrease [F (3, 36) = 5.67, P < 0.001] in mid cells (eosinophils, basophils and monocytes) was observed with NaB at 125, 250 and 500 mg/kg in comparison with mice fed the control diet. A significant [F (3, 36) = 12.5, P < 0.001] decrease in lymphocyte count was observed with NaB at 250 mg/kg and an increase at 500 mg/kg of feed in comparison with the control diet. Concentration for concentration comparisons revealed a significant decrease in mid cell and lymphocyte count with NaB at 500 compared with effects observed at 125 mg/kg of feed.
Table 3 shows the result of platelet parameters. Platelet count, platelet volume., platelet distribution width (PDW), PCT and platelet larger cell ratio (P-LCR) did not differ significantly in any of the groups fed NaB in comparison with mice fed the control diet. Concentration for concentration comparisons revealed no significant difference in platelet parameters with NaB at any of the concentrations.
Effect of NaB on MDA levels and antioxidant status
Table 4 shows the effect of diet-added NaB on lipid peroxidation levels and antioxidant status. Serum MDA level was significantly [F (3, 36) = 1260, P < 0.001] increased with NaB at 125, 250 and 500 mg/kg of feed in comparison with mice fed the control diet. Concentration for concentration comparisons revealed a significant decrease in serum MDA levels with NaB at 250 and 500 compared with effects observed at 125 mg/kg of feed.
Table 4.
Oxidative stress parameters and antioxidant status
| Groups | MDA uM | SOD u/ml | TAC (TE mM) | Total Protein (g/dl) |
|---|---|---|---|---|
| Control | 5.13 ± 0.10 | 0.55 ± 0.10 | 10.25 ± 0.08 | 4.72 ± 0.08 |
| NaB 125 | 43.23 ± 0.16* | 1.99 ± 0.02* | 2.43 ± 0.04* | 4.62 ± 0.04 |
| NaB 250 | 14.3 ± 0.94*,# | 1.89 ± 0.01*,# | 4.45 ± 0.05*,# | 4.17 ± 0.03*,# |
| NaB 500 | 7.58 ± 0.04*,# | 0.2 ± 0.01*,# | 8.25 ± 0.05*,# | 4.94 ± 0.05*,# |
Values are presented as mean ± SEM, *P < 0.05 vs. control, #P < 0.05 concentration vs. concentration, number of mice/group = 10.
Serum SOD activity was significantly increased [F (3, 36) = 138, P < 0.001] with NaB at 125 and 250 mg/kg, and was decreased at 500 mg/kg of feed compared with the control diet. Concentration for concentration comparisons revealed a significant decrease in SOD activity with NaB at 250 and 500 compared with effects observed at 125 mg/kg of feed.
Serum TAC was significantly [F (3, 36) = 3973, P < 0.001] decreased with NaB at 125, 250 and 500 mg/kg in comparison with the control diet. Concentration for concentration comparisons revealed a significant increase in TAC with NaB at 250 and 500 compared with effects observed at 125 mg/kg of feed.
Serum total protein levels were significantly decreased [F (3, 36) = 50.5, P < 0.001] with NaB at 250 mg/kg and was increased at 500 mg/kg of feed in comparison with mice fed the control diet. Concentration for concentration comparisons revealed a significant decrease in serum total protein levels with NaB at 250 and 500 compared with effects observed at 125 mg/kg of feed.
Effect of NaB on inflammatory markers and caspase-3 levels
Table 5 shows the effect of diet-added NaB on IL-10, TNF-α and caspase-3 levels. Serum TNF-α levels were significantly decreased [F (3, 36) = 4710, P < 0.001] with NaB at 125 and 250 mg/kg of feed comparison with mice fed the control diet. Concentration for concentration comparisons revealed a significant increase in TNF-α levels with NaB at 250 and 500 compared with effects observed at 125 mg/kg of feed.
Table 5.
TNF-α-α, IL-10 and caspase-3
| Groups | TNF-α ng/L | IL-10 pg/ml | Caspase-3 (ng/ml) |
|---|---|---|---|
| Control | 91.83 ± 0.25 | 30.69 ± 0.09 | 0.67 ± 0.02 |
| NaB 125 | 63.66 ± 0.31* | 26.27 ± 0.07* | 0.68 ± 0.02 |
| NaB 250 | 68.46 ± 0.13*3 | 27.62 ± 0.19* | 0.66 ± 0.04 |
| NaB 500 | 90.29 ± 0.10*,# | 27.17 ± 0.06 * | 0.67 ± 0.04 |
Values are presented as mean ± SEM, *P < 0.05 vs. control, #P < 0.05 concentration vs. concentration, number of mice/group = 10.
Serum IL-10 levels were significantly decreased [F (3, 36) = 736, P < 0.001] with NaB at 125, 250 and 500 mg/kg of feed compared with mice fed the control diet. Caspase-3 levels did not significantly differ at any of the concentrations compared with mice fed the control diet. Concentration for concentration comparisons revealed no significant difference in IL-10 and caspase-3 levels with NaB.
Effect of NaB on lipid profile
Table 6 shows the effect of diet-added NaB on lipid profile. TC levels were significantly decreased [F (3, 36) = 10.5, P < 0.001] with NaB at 125 and 500 mg/kg of feed in comparison with mice fed the control diet. Concentration for concentration comparisons revealed a significant increase in TC levels with NaB at 250 and a decrease at 500 compared with effects observed at 125 mg/kg of feed.
Table 6.
Lipid profile parameters
| Groups | TC (mmol/L) | TG (mmol/L) | LDL (mmol/L) | HDL (mmol/L) |
|---|---|---|---|---|
| Control | 2.86 ± 0.08 | 1.38 ± 0.04 | 1.53 ± 0.05 | 0.80 ± 0.03 |
| NaB 125 | 1.85 ± 0.35* | 1.23 ± 0.02* | 0.42 ± 0.02* | 0.47 ± 0.06* |
| NaB 250 | 2.70 ± 0.02# | 1.54 ± 0.03*,# | 1.42 ± 0.02*,# | 0.58 ± 0.04* |
| NaB 500 | 1.70 ± 0.01*,# | 1.69 ± 0.02*,# | 0.40 ± 0.01* | 0.63 ± 0.03* |
Values are presented as mean ± SEM, *P < 0.05 vs. control, #P < 0.05 concentration vs. concentration, number of mice/group = 10.
TG levels were significantly increased [F (3, 36) = 53.8, P < 0.001] with NaB at 125 mg/kg and increased at 250 and 500 mg/kg of feed in comparison with mice fed the control diet. Concentration for concentration comparisons revealed a significant increase in TG levels with NaB at 250 and 500 compared with effects observed at 125 mg/kg of feed.
LDL levels were significantly decreased [F (3, 36) = 449, P < 0.001] with NaB at 125, 250 and 500 mg/kg of feed in comparison with mice fed the control diet. Concentration for concentration comparisons revealed a significant increase in LDL levels with NaB at 250 compared with effects observed at 125 mg/kg of feed.
HDL levels were significantly decreased [F (3, 36) = 11.7, P < 0.001] with NaB at 125, 250 and 500 mg/kg of feed in comparison with mice fed the control diet. Concentration for concentration comparisons revealed a significant increase in HDL levels with NaB at 250 and 500 compared with effects observed at 125 mg/kg of feed.
Effect of NaB on biochemical markers of hepatic and renal function
Table 7 shows the effect of diet-added NaB on biochemical markers of liver and kidney function is shown in Table 7. Aspartate aminotrasnaminase levels were not significantly different in any of the groups fed NaB compared with mice fed the control diet. Concentration for concentration comparisons revealed a significant decrease in aspartate aminotrasnaminase levels with NaB at 500 compared with effects observed at 125 mg/kg of feed.
Table 7.
Biochemical markers of liver and kidney function
| Groups | AST (IU/L) | ALT (IU/L) | Urea (mmol/L) | Creatinine (mmol/L) |
|---|---|---|---|---|
| Control | 82.46 ± 0.87 | 41.87 ± 0.3 | 2.74 ± 0.04 | 42.50 ± 0.01 |
| NaB 125 | 83.82 ± 0.97 | 63.22 ± 0.39* | 2.69 ± 0.13 | 44.30 ± 1.47 |
| NaB 250 | 80.97 ± 0.91 | 66.53 ± 0.45*,# | 2.90 ± 0.03*,# | 39.80 ± 0.42*,# |
| NaB 500 | 80.42 ± 0.34# | 64.62 ± 0.86* | 2.98 ± 0.03*,# | 40.70 ± 0.70*,# |
Values are presented as mean ± SEM, *P < 0.05 vs. control, #P < 0.05 concentration vs. concentration, number of mice/group = 10.
Alanine aminotransaminase levels were significantly increased [F (3, 36) = 451, P < 0.001] with NaB at 125, 250 and 500 mg/kg of feed in comparison with the control diet. Concentration for concentration comparisons revealed a significant increase in serum alanine aminotransaminase levels with NaB at 250 compared with effects observed at 125 mg/kg of feed.
Urea levels were significantly increased [F (3, 36) = 58.4, P < 0.001] with NaB at 250 and 500 mg/kg of feed in comparison with the control diet. Concentration for concentration comparisons revealed a significant increase in serum urea levels with NaB at 250 and 500 compared with effects observed at 125 mg/kg of feed.
Creatinine levels were significantly decreased [F (3, 36) = 18, P < 0.001] with NaB at 250 and 500 mg/kg of feed in comparison with the control diet. Concentration for concentration comparisons revealed a significant decrease in serum creatinine levels with NaB at 250 and 500 compared with effects observed at 125 mg/kg of feed.
Discussion
In this study, the effect of diet-added NaB on body weight, food consumption, lipid profile, oxidative stress parameters, inflammatory and apoptotic markers were examined in mice. The study was conducted with a view to ascertaining the possible toxic effects (or otherwise) of diet-added NaB in mice. Results showed that compared with the control diet, food-added NaB was associated with (i) an increase in body weight and food intake at 125 mg/kg of feed, (ii) decrease in red cell parameters, white cell, lymphocyte and mid-cell count, (iii) increase in MDA levels, (iv) increase in SOD activity at 125, 250 and a decrease at 500 mg/kg of feed, (v) decrease in TAC and (vi) a decrease in the TNF-α and IL-10 levels.
In this study, feeding mice with food-added NaB for 8 weeks was associated with an increase in body weight at the lowest concentration and a visual decrease in weight at 250 and 500 mg/kg of feed. An increase in food intake was also observed at 125 mg/kg. A few studies have examined the effect of NaB on body weight [20–22]. The result of this study at higher concentration supports the results of the study by Griffith [20] that reported that the administration of NaB diet at 1.5, 2.0 or 2.5% (concentrations significantly higher than those used in this study) did not significantly alter body weight (compared with controls). Priya et al [21] examined the effects of administering NaB in distilled water daily (at concentrations much lower than those used in this study) for 28 days on body weight of Wistar rats, and observed a time and concentration-dependent decrease in body weight in both male and female rats. However, Saatci et al. [22] observed no significant difference in body weight in dams administered NaB by gavage.
Food intake was increased at 125 mg/kg of feed and showed no significant difference from control at the other concentrations. Dose-for-dose comparisons revealed a significant decrease in body weight and food intake in groups fed higher concentrations of NaB, suggesting that at higher concentrations of NaB, animals consumed less and also gained significantly lesser weight. While there is a dearth of scientific information on the effects of NaB on food intake, there have been reports that NaB reduced feed intake [35]. Saatci et al [22] reported no significant effect of NaB administered by gavage on food intake. Also, a number of the earlier studies did not really suggest what may be exactly responsible for NaB’s effects on food consumption and weight. Overall, our results show that at 125 mg/kg, increase in food intake corresponded with an increase in weight, suggesting that NaB possibly increases the palatability of food at this concentration. There was a decrease in food intake and body weight at higher concentrations, probably because the addition of NaB alters the palatability of food; hence, the observed reduction in food intake (albeit only visual). This finding is supported by a previous report that had demonstrated that chronic exposure to NaB diet in animals was associated with reduced feed intake and reduced growth [35]. The reduction in body weight observed with NaB has also been attributed to the deficiency of blood phospholipids and nutritional factors such as glycine [36, 37]. The results of our study and the other studies show that the effects of NaB on body weight is probably dependent on its concentration, mode of administration, duration of administration and quantity consumed.
Feed-added NaB was also associated with a reduction in all serum cholesterol levels (TC, low and HDLs), but increased TGs. This corroborates the result by Brahmachari et al. [8] and Oghenetekevwe et al. [38] that also reported a decrease in cholesterol levels at similar doses, although the route of administration differed. The decrease in HDL and increase in TGs suggest that NaB may not have the capacity to increase cardiovascular risk factors. However, how this contributes to the overall safety of NaB, and whether this may have a possible clinical application in the management of metabolic disorders may be worthy of further investigation.
A few studies have reported that NaB did not significantly alter serum biochemical parameters [39]. The results of this study showed that ingestion of NaB increased serum alanine aminotransaminase levels, although no significant difference was observed in the aspartate aminotrasnaminase levels. Serum transaminases, including aspartate and alanine aminotransaminase levels, are used for the assessment of liver function, or to determine the presence of liver injury. Both of these transaminases are considered two of the most important tests used to determine the presence of liver injury. However, alanine aminotransaminase is more specific and more-commonly increased in liver injury than aspartate aminotrasnaminase; therefore, the mild increase in alanine aminotransaminase suggests the presence of liver injury, which could also be linked to the oxidative stress that was observed [40–42]. Renal function tests revealed a concentration-dependent increase in urea levels, which partially corroborates the results by Khodaei et al. [39] that reported derangement in biochemical parameters of kidney function. Therefore, our results suggest that NaB may be associated with possible liver and kidney injuries in mice. However, the suggestion that NaB administration may cause kidney injury more than liver injury [39] was not affirmed by the results of the current study.
Oxidative stress is defined as the presence of metabolic and radical substances or so-called reactive (oxygen, nitrogen or chlorine) species [43, 44]. In this study, NaB diet was associated with an increase in lipid peroxidation, SOD activity; and a decrease in TAC and total protein levels. This supports the results of number of in vivo and in vitro studies that have reported the ability of NaB to induce oxidative stress [2, 39, 45]. However, not all previous studies had reported the same trend in the measurement of parameters of oxidative stress following NaB ingestion; for instance, Yetuk et al [2] reported low activity of SOD, contrary to the increased activity observed in our study. This could be attributed to the difference in samples of study (erythrocytes versus serum). The increase in SOD suggests that NaB increases the generation of superoxides that need to be partitioned. A decrease in TAC suggests that the overall balance of antioxidant activity is shifted as a result of the need to overcome the increased oxidative stress that occurs as a result of the ingestion of NaB.
The anti-inflammatory potential of NaB has been reported [8, 46], however, the results of this study point to an overall proinflammatory response. Although a decrease in the levels of TNF-α, a proinflammatory cytokine was observed, levels of IL-10, an anti-inflammatory cytokine were also reduced. IL-10 plays a key role in limiting the host immune response, thereby preventing injury to the host and maintaining normal tissue homeostasis [47]. IL-10 has also been reported to assist in the regulation of the activities of TNF-α, through its actions on TNF-α-converting enzyme [48]. While the interactions of NaB with proinflammatory and anti-inflammatory markers following chronic exposure to NaB require further evaluation, the overall effects of NaB as observed from this study is proinflammatory. The proinflammatory state produced by the administration of NaB could also explain the increase in oxidative stress and reduction in antioxidant status observed at certain concentrations of NaB. However, NaB showed no significant effect on caspase-3 activity,
In this study, the administration of NaB diet was associated with a decrease in red cell parameters; and a decrease in white cell, lymphocyte and mid cell counts. This is consistent with the results of a short-term study (14 days) by Ibekwe et al. [49] that administered NaB by gavage at 0, 30, 60 and 120 mg/kg body weight and reported a dose-dependent decrease in Hb concentration and WCC. Aziz and Zabut [50], and Ahmad et al. [51] had also reported decrease in red blood cell count, Hb concentration and haematocrit following the administration of NaB. However, contrary to the results of our study, they both observed an increase in total white blood cell counts lymphocyte and platelet count [50, 51]. NaB’s effects on haematological parameters could be linked to its ability to induce oxidative stress and inflammation.
Overall, between-group comparisons of the three concentrations revealed a decrease in body weight, food intake, red cell, white cell/mid-cell parameters and lipid peroxidation at the higher concentrations of NaB. Also, increase in the lipid parameters, biomarkers of liver/kidney function and antioxidant status were observed at the higher concentrations, suggesting that the effects of NaB are dependent significantly on quantities consumed.
Conclusions
While food additives generally are important in the maintenance and improvement of our ability to adequately feed an increasing world population, and NaB specifically has found use in clinical scenarios such as the management hyperammonaemia, schizophrenia and depression; issues regarding their safety cannot be ignored; rather, they demand constant re-evaluation. Findings from this study show the widespread effects of NaB in mice, by revealing its effects on several tissues and organs at the concentrations used. It is worthy of note that some of these effects (pancytopenia, proinflammation, oxidative stress and lipid derangements) have far-reaching impact on tissue and organ health, and possible development of chronic diseases. Our findings suggest that NaB may be harmful if regulations regarding its limit of consumption are mistakenly or deliberately ignored. Therefore, it is advisable that regulations on quantities to be added to food be enforced.
Funding
This research did not receive any specific grant from agencies in the public, commercial or not-for-profit sectors.
Conflict of interest statement
None declared.
Contributor Information
Anthony Tope Olofinnade, Department of Pharmacology, Therapeutics and Toxicology, Faculty of Basic Clinical Sciences, College of Medicine, Lagos State University, P.M.B. 21266. 1-5 Oba Akinjobi Way,G.R.A Ikeja, Lagos State, Nigeria; Department of Pharmacology, Ladoke Akintola University of Technology, University Road. P.M.B, 4000, Ogbomoso, Oyo State, Nigeria.
Adejoke Yetunde Onaolapo, Department of Anatomy, Ladoke Akintola University of Technology, University Road. P.M.B, 4000, Ogbomoso, Oyo State, Nigeria.
Olakunle James Onaolapo, Department of Pharmacology, Ladoke Akintola University of Technology, University Road. P.M.B, 4000, Ogbomoso, Oyo State, Nigeria.
Olugbenga Adekunle Olowe, Department of Medical Microbiology and Parasitology, Ladoke Akintola University of Technology, University Road. P.M.B, 4000, Ogbomoso, Oyo State, Nigeria Nigeria.
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