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Effects of commonly used food additives on haematological parameters of Wistar rats

This study was done to investigate the effects of common food additives such as sodium benzoate (SB) and ascorbic acid (AA) on haematological parameters of male Wistar rats. Forty-eight (48) male albino rats with an average weight of 105 g were grouped into twelve (n = 4) of Basal Control and other 11 groups orally administered 1 mg of SB, 10 mg of SB, 10 mg of AA, 0.2 mg of AA + 0.5 mg of SB, 0.2 mg of AA + 1 mg of SB, 0.2 mg of AA + 10 mg of SB, 0.2 mg of SB + 0.1 mg of AA, 0.2 mg of SB + 0.5 mg of AA, carbonated soft drinks (CSD)+ 0.1 mg of AA, CSD + 1 mg of AA and CSD + 10 mg of AA, respectively for 21 non-consecutive days. At the end of the experiment, blood samples were collected in EDTA anticoagulant tubes, haematological parameters were evaluated, and data were analyzed. There was a dose-dependent increase (p < 0.05) in White Blood Cell counts of SB treated rats compared with the control group. The lymphocyte exhibited significant reduction (p < 0.05) in the groups treated with 1mg SB and 10mg SB/kg bodyweight of 67 ± 2.96 and 58 ± 4.18%, respectively. The mean corpuscular haemoglobin showed no significant difference at 95% confidence interval. However, mean corpuscular haemoglobin concentration, haematocrit and platelet were affected by an increase in the concentrations of SB. High SB concentrations increased the destruction of erythrocytes, which directly increased the catabolism of haemoglobin. However, AA administration mitigated the adverse effects of SB on the haematological parameters of the animal.


Introduction
Food safety is the control of recognized food hazards to achieve an acceptable level of food risk. The availability and consumption of safe food, which supplies the body's needs are essential for humans. Food and its active constituents have a significant effect on human health and nutrition because it is eaten to maintain life and stimulate growth. Activities of microorganisms or enzymatic reactions during packaging or storage result in food spoilage (Jay et al., 2005;Turkoglu, 2007). As a means of preserving food from deteriorating, several preservatives such as sodium benzoate (SB E211), ascorbic acid, aluminum silicate, butylated hydroxyl toluene (BHT), butylated hydroxyanisole (BHA), monosodium glutamate (MSG) and white sugar are added to keep its integrity (Kunkel and Barbara, 2004;Jay et al., 2005;Turkoglu, 2007;Abdulmumeen et al., 2012;Oloye, 2019;Femi-Oloye et al., 2019a, b).
Ascorbic acid is an antioxidant and water-soluble vitamin that is present in and outside of the cell as ascorbate (Chihuailaf et al., 2002). It is a natural antioxidant that stops the continuous creation of free radicals induced by oxidative damage to lipids and lipoproteins in various cellular sections and tissues (Costa et al., 2004;Adegoke et al., 2011. 2012;Musa et al., 2012). It is regarded generally as a critical first-line defensive agent that repairs or nullifies free radicals by donating a single electron, followed by a proton to yield a dehydroascorbic acid (Carr et al., 2000;Halliwell, 2001).
Sodium benzoate, on the other hand, is frequently used as a food additive to prevent the growth of microbes, including fungi and bacteria. Sodium benzoate, as a preservative, holds an important and significant status in food processing industries throughout the world. It is extensively used in a variety of foodstuffs including carbonated drinks, beers, jams, fruit juices, margarine, jellies, bakery products, cheese, pickles and sauces (Zengin et al., 2011) as well as the preservation of liquid medicines (Oyewole et al., 2012;Shahmohammadi et al., 2016). Although sodium benzoate is considered a harmless additive, yet its detrimental effects on human health have been stated (Yolmeh et al., 2014;Saatci et al., 2016), and it is associated with adverse health effects in consumers (Oyewole et al., 2012). Studies revealed that sodium benzoate could be metabolized in the body under irradiation to a derivative, benzene, which is capable of causing damage to mitochondrial DNA (Gardner and Lawrence, 1993;Oloye, 2019). It also has associated implications on health, which include liver and kidney dysfunction and gastrointestinal irritation (Gao et al., 2008;Saatci et al., 2016). A substantial connection with investigative value has been established between packed cell volume (PCV), haemoglobin concentration (Hb), red blood cells (RBC), and red blood cell indices (such as mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC)), in both human and rats (Yolmeh et al., 2014;Yadav et al., 2016). Alteration in haematological parameters could be an indication of anaemic conditions, or compromised immune status and poisoning (Ashaolu et al., 2011). The World Health Organization sets the acceptable daily intake of sodium benzoate as 5 mg/kg bw/day. Nevertheless, it is being used in higher concentrations in many food items (Yadav et al., 2016).
The massive increasing use of artificial preservatives in food, and consumers' desire to consume processed foods necessitate the need to continuously study the possible effects of preservatives on living cells. Furthermore, there is a possibility of two or more additives reacting together to form another product (Oloye, 2019). Assessments of blood parameters are essential to evaluate the health of many vertebrates (Tavares-Dias and Moraes, 2007). Through haematological constituents, the metabolic disturbances in animals could easily be assessed (Jamalzadeh et al., 2009). There is a shortage of evidence on the consequences of the interaction of food preservatives on haematological parameters. Thus, this study aimed to determine the effects of food additives (sodium benzoate and ascorbic acid) and their mixture on haematological parameters of Wistar rats.

Experimental animals
Forty-eight (48) male rats (albino) of 105 g mean weight were gotten from the Department of Animal and Environmental Biology, Adekunle Ajasin University, Akungba-Akoko, Nigeria.

Experimental procedure
Forty-eight rats were acclimatized for two weeks and were then allotted into 12 groups (n ¼ 4 per group) of Basal Control and other 11 groups orally administered 1.0 mg of SB, 10 mg of SB, 10 mg of AA, 0.2 mg of AA þ 0.5 mg of SB, 0.2 mg of AA þ 1 mg of SB, 0.2 mg of AA þ 10 mg of SB, 0.2 mg of SB þ 0.1 mg of AA, 0.2 mg of SB þ 0.5 mg of AA, carbonated soft drinks (CSD) þ 0.1 mg of AA, CSD þ 1.0 mg of AA and CSD þ 10 mg of AA for 21 non-consecutive days. The procedures were approved by the Department of Animal and Environmental Biology, Adekunle Ajasin University Akungba-Akoko, in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals.

Blood collection
After 21 days of exposure, rats were fasted overnight, and they were weighed before blood samples were collected. The rats were then sacrificed. All blood samples were taken between 7 and 9 am to avoid disparities due to circadian rhythm. Whole blood was obtained from a puncture of the retro-orbital sinus by the conventional method (Jay et al., 2005). This method was adopted to gradually introduce the tip of the micro-haematocrit (70 ml heparinized microcapillary tubes haematocrit) into the medial angle of the eye. Blood samples collected in EDTA (Ethylene Diamine Tetra-Acetic acid 8.5%) anticoagulant tubes were hurriedly mixed with the anticoagulant in the tube. All blood samples were labeled and immediately carried to the laboratory for examination.

Statistical analysis
Data were normally distributed and met the assumption for equality of variance. The differences among groups were analyzed by one-way Analysis of Variance (ANOVA) using SPSS 20.0, SPSS Inc., Chicago, Illinois, USA. The results were expressed as Mean AE Standard Deviation (SD). Duncan multiple range test was used for Post Hoc analyses, and p < 0.05 was accepted as significant.

Results
There was no significant difference (p > 0.05) in the WBC counts of Wistar rats administered commonly used food additives compared with the control group except for groups administered 10 mg AA/kg bw and CSD þ 0.1 mg AA/kg bw. These two groups showed a significant difference (p < 0.05) when compared to the control ( Figure 1). Figure 2 showed the effect of administered sodium benzoate, ascorbic acid, their mixture and AA with CSD on the LYM values of Wistar albino rats. The LYM showed no significant difference (p > 0.05) in the treatment groups when compared with the control group. However, LYM values in the groups administered 10 mg SB/kg bw and 0.2 mg AA þ 10 mg SB/kg bw showed significant difference (p < 0.05) when compared with the control group. The groups administered 10 mg SB/kg bw and 0.2 mg AA þ 10 mg SB/kg bw showed significant difference (p < 0.05) in the red blood cell (RBC) counts ( Figure 3) and haemoglobin concentrations ( Figure 4) when compared with the control group. The other test groups showed no significant difference (p > 0.05) in the RBC counts and haemoglobin concentrations when compared with the control group.
Mean corpuscular haemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) of Wistar rats administered commonly used food additives are shown in Figures 5 and 6, respectively. There was no significant difference observed at a 95% confidence interval in the MCH or MCHC levels in any of the test groups when compared with the control group. The haematocrit values in the control group and the group administered 10 mg AA/kg bw were both approximately 48 % of the blood (Figure 7). This value was relatively large compared to other groups. However, none of the groups was significantly different at a 95% confidence interval. Platelets showed a significant increase in values as the SB dosage increases (Figure 8). Nevertheless, other treatments showed no such differences.

Discussion
Sodium benzoate is a bacteriostatic and fungistatic agent in acidic environments which, after being absorbed into cells, interacts with the anaerobic energy production pathways and suppresses the proliferation and development of food-spoilage microorganisms (Pongsavee, 2015). An approximate SB concentration of 0.1% is usually enough for food preservations (Chipley, 2005). Additives are mostly used in combination with other types of preservation agents. The mixtures of two or more chemicals could lead to red or blue shifts in UV spectroscopy (Oloye, 2019), which indicates possible interactions of the substances. This interaction might lead to an adverse effect on the health of the organisms consuming the products. The results might differ among different species of organisms (Jacob Filho et al., 2018).
The values of the haematological parameters vary among different species of animals (Jacob Filho et al., 2018). Values of haematological parameters for the Control group in this study were within the normal ranges commonly stated in the literature for rats (Iranloye, 2002;Adebayo et al., 2005Adebayo et al., , 2010Gbore et al., 2010;Etim et al., 2014;Jacob Filho et al., 2018). Exposure to toxins can cause alterations and damage to the haematological profile and haematopoietic system in man and animals (WHO, 1995;Maaroufi et al., 1996;Costa et al., 2004;Adedapo et al., 2004Adedapo et al., , 2007Gbore et al., 2020). White blood cells are responsible for fighting infections or diseases in the body. Low counts of WBC may indicate that the body is immune-compromised. At the same time, too high WBC counts might be an indication of many underlined diseases or the introduction of a foreign body responsible for the upsurge of WBC counts.
Antioxidants are the type of molecules that nullify harmful free radicals, produced through a series of reactions (Joseph et al., 2009;Adegoke, 2011, 2012;Tanko et al., 2013), that are detrimental to living cells, spoil foods, damage materials such as rubber, gasoline, and lubricating oil. Antioxidants terminate these series of reactions through the elimination of free radical intermediates and inhibiting other oxidation reactions (Sies, 1997). The WBC count of rats in the control group was higher than 4.7 AE 0.40 and 4.51 AE 0.44 Â 10 9 /L reported by   Adedapo et al. (2004Adedapo et al. ( , 2007 and Barbeieri et al. (2001), respectively because WBC counts differ among species of an organism. In this study, the administration of 10 mg AA/kg bw reduced the WBC counts significantly in the rats compared with the control group. This finding suggests that AA (an antioxidant) might be capable of preventing excessive production of WBC (Liu, 1995;George and Adegoke, 2012). The significant increase in WBC count in 10 mg SB/kg bw group compared with the control group indicated that SB was recognized as a foreign entity. Hence SB at high concentrations has the potential of inducing WBC synthesis. The increased counts of WBC in rats treated with a high dose of SB may be due to the inflammatory response induced as a defence mechanism (Ekanem et al., 2015). A commercial fizzy drink (CSD) used for this experiment did not affect WBC count. However, the addition of a small quantity of AA with the CSD before consumption significantly reduced   WBC, which could be due to the anti-oxidative properties of AA (Akorede et al., 2020). Nonetheless, as the concentrations of AA increase in CSD, it loses its anti-oxidative property because of the possibility of AA forming aggregates instead of mixing with the food products to act as antioxidants.
Generally, it is expected to see a temporary rise in lymphocyte value after an infection because they help to fight diseases (Schalm et al., 1975) recognized by the body as foreign. Low lymphocyte values observed in the body could be as a result of cold or other infections, or it could be caused by intense physical exercise, severe stress, or malnutrition. The findings from the present study, which indicated that lymphocytes levels were low in 10 mg SB/kg and 0.2 AA þ10 SB mg/kg bw groups, could be attributed to the xenobiotic activity of SB, which is capable of eliciting immune response at a higher concentration. Thus, the lymphocyte levels of rats from other groups were not significantly different when compared with the control group.
Red blood cell (RBC) plays a vital role in the body. It aids in the transportation of oxygen throughout the body system and also removing carbon IV oxide from the body by transporting it to the lung for exhaling. Deficiency in RBC results in anaemic conditions (Cheesborough, 1991). In this study, the significant decrease in haemoglobin concentration along with reduced RBC count might be due to sodium benzoate intoxication, which suggests the initiation of an anaemic condition in the rats in groups administered SB. Furthermore, the reduction in RBC in the blood of rats in groups treated with SB may be attributed to multiple factors, one of which is the failure to supply the blood with cells from haematopoietic tissues, which might have suffered from a destructive effect of the foreign substance, according to the report of Tuormaa (2004). Also, the possible harmful effect of the xenobiotic compound (SB) on red blood cell membranes can result in hypoxemia. A high dose of SB increases the destruction of erythrocytes directly and increases the catabolism of haemoglobin (Linman, 1975;Tort and Torres, 1988). Generally, when the Hb concentration drops below the typical values (9-14 g/dL), the resultant medical condition is anaemia (Ashour et al., 2007). Specifically, toxin decreases haem-biosynthesis by inhibiting aminolaevulinic acid dehydratase and ferro-chelatase activities (Ashour et al., 2007). Thus, sodium benzoate might induce anaemia at a high concentration by meddling with haem-biosynthesis and weakening RBC survival. The increase in the level of SB decreased haemoglobin value, whereas, the addition of ascorbic acid was found to enhance the effects of the SB in this study. Similar results have been reported by Al-Hamdany (2010), who suggested that a significant increase in WBC counts and a decrease in Hb in rats exposed to xenobiotic due to inflammation could cause anaemia. A slight reduction in Hb was evident when AA was added to CSD, suggesting care must be taken to avoid adding AA to fast foods or drinks.
The average mass of Hb per RBC is called mean corpuscular haemoglobin (MCH). In the present study, the control MCH (23.20 AE 0.29pg) was within the ranges reported earlier (Maaroufi et al., 1996;Çay and Naziro glu, 1999). Mean corpuscular haemoglobin values of the test groups were not significantly different compared with the standard control, which suggested that the observed correlation in RBC and Hb, occurred through the rupture of red blood cells (Çay and Naziro glu, 1999). Mean corpuscular haemoglobin concentration increased with an increase in SB concentration. However, the haematocrit level decreased with an increase in SB dosage.
The increased PLT counts with increasing concentrations of SB might have been due to stimulation of erythropoietin by the high demands for oxygen and carbon IV oxide transport as a result of an increased metabolic activity or the destruction of the respiratory membranes causing damaged gaseous exchange (Zaki et al., 2008). Generally, the reduction in platelet count in rats could be associated with thrombocytopenia (Achukwu et al., 2009). Ognjanovic et al. (2003) have also shown that vitamin C played a significant part in preventing the deadly effects of cadmium on haematological values. Fox et al. (1971) had a similar result where they showed the protective effect of vitamin C on anaemia caused by heavy metals in rats. A significant reduction in the values of RBC and Hb with no variations in HCT in the treated groups indicated that SB could yield macrocytic hypochromic anaemia if administered orally for a, more extended period.

Conclusion
This study showed that sodium benzoate (SB) administered in low dosage caused little or no effects on haematological parameters (such as white blood cell count, lymphocyte value, red blood cell count, haemoglobin concentration, and mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haematocrit and platelet values). However, the administration of SB at a high dosage can induce an anaemic condition. The ameliorative potential of ascorbic acid (AA) helped to regulate the toxic effect caused by SB on hematological parameters.

Author contribution statement
Oluwabunmi P. Femi-Oloye: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.