Amany M. Salama¹, Seif E. Salem² and Ola F.A. Talkhan³

^1,2 Toxicology Unit, Biochemistry Dept., Animal Health Research Institute, Agriculture Research Center

³Chemistry Dept., Animal Health Research Institute-Shiben El- Kom lab., Agriculture Research Center

INTRODUCTION

The increased demand of the Egyptian population for low-cholesterol and high protein meat sources has led to high consumption of the poultry meat due to its affordability. Many heavy metals such as arsenic (As), lead (Pb), cadmium (Cd), mercury (Hg) and others are found in certain animal foods, or poultry feed additives (Jadhav et al. 2007). There is no standard definition for heavy metals. Generally, it is referred to a group of metals that are not required or required in trace amounts, they have high densities, atomic weights, or atomic numbers. Their bioavailability is controlled by chemical and physical factors like temperature, absorption, and physiological characteristics of the exposed species (Stern, 2010).Their contribution in industry, medicine and agriculture have resulted in their environmental bioaccumulation that raising concerns about their toxic effects on human health and environment. High exposure to heavy metals can occur through ingestion, inhalation or dermally causing several health problems such as neurological and neurobehavioral disorders, blood abnormalities, cancers, and cardiovascular diseases (Mehrandish et al. 2019).Also increasing the frequency ofchronic diseases like renal failure, liver cirrhosis, and anemias among Egyptian population (Salem et al.2000).There are several potential sources of toxic metals for poultry. Heavy metals may contaminate soil and water through sewage sludge application, industrial waste disposal, application of pesticides and fertilizers and atmospheric deposition (Adaikpohet al. 2005).

Arsenic compounds have been used for treatment of diseases like syphilis, amoebic diarrhea and trypanosome (Centeno et al. 2006). Arsenicis available in the form of oxides, sulfides or as a salt of iron, calcium, copper, etc. (Singh et al.2007). Phenyl arsenic acids are widely used as poultry feed additive to control coccidiosis, improves production, and imparts attractive color to the chicken flesh (Ghosh et al. 2012; Mondal, 2020). Although arsenic is essential for synthesis of methionine metabolites in poultry, but it is carcinogenic (Balos et al. 2019),Trivalent arsenic compounds are more toxic than the pentavalent forms. It produces toxicity when the concentration of arsenic compounds is 2-10 times higher than the recommended dose, which is usually 100 mg/kg complete feed. Most of the toxic effects arise from exposure to inorganic arsenic from commercial uses such as wood preservatives and herbicides. Its toxicity usually attributed to low methylating ability(Suganya et al. 2016). “Arsenic, however, is not a well-documented element when it comes to biota and particularly to birds” (Sánchez-Virosta et al.2015).

Lead is a highly toxic metal naturally present in the earth's crust, it has been disseminated throughout the environments by industries like battery manufacturing, motor vehicle repair, piping, paints, food wrapping, tobacco and as an additive in gasoline (Alloway and Ayres,1993).Lead is a “multimedia” contaminant, with sources include air, water, soil, dust, food, paint. Contamination of food can arise from food processing, food handling, or deposition of atmospheric lead on agricultural crops. Inorganic lead has been only detected in food (WHO, 2011).Lead poisoning can occur in domestic animals as horses, poultry, and dogs (Khan et al. 2008).(Suganya et al.2016)reported that diet contains as low as 1.0 ppm of lead can cause significant growth suppression in chickens and decline in blood Delta-aminolevulinic acid dehydratase.

Cadmium is a metal chemically similar to zinc and occurs naturally with zinc and lead in sulfide ores. It is a by-product of zinc production. Cadmium is mostly released to environment from industrial processes, waste disposal and cigarette smoke. Contamination with cadmium in the animal feed industry is mostly in conjunction with the use of zinc sulfate. Other sources include mining, discarded cadmium-chloride products, and use of phosphate and sewage sludge fertilizer. Cadmium is considered one of contaminants that are widely distributed in foods, can concentrate along the food chain reaching human body, it has been classified by the International Agency for Research on Cancer (IARC) as carcinogenic to humans (WHO, 2011).Cadmium exposure can induce oxidative stress, alter the activities of antioxidant enzymes of erythrocytes of the adult poultry birds (Kant et al. 2011).

Eliminating of these heavy metals from animal food supply is difficult because these metals are found in the air, water, and soil (AAFCO, 2019).Indestructibility with bioaccumulation of heavy metals contribute to the possibility of being as toxicants. The possible approach of their removal is their chelation as metals cannot be catabolized (Tokar et al. 2015).

Removal of toxic metals by microbial biomass is a promising inexpensive conventional method that has been introduced for decontamination of food (Zoghi et al.2014).Probiotics are live nonpathogenic, non-toxicogenic microbesused as feed supplements that beneficially affect the host animal by improving microbial balance (Fuller, 1989), according to WHO and FDA probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host (WHO/FAO, 2001). Probiotics can exert benefits to both human and animal health as proven in hundreds of scientific research.es, and they are marketed commercially indifferent brands all over the world (Soccol et al., 2010).Probiotic species like Lactobacillus, Streptococcus, Bifidobacterium, Enterococcus, Aspergillus and Saccharomyces have a beneficial effect on broiler performance (Ashayerizadeh et al.2009), and on modulation of intestinal microflora and pathogen inhibition(Higgins et al. 2007).

The populations of microorganism which are found within the gastrointestinal tract of poultry have of two types of microorganisms at their gastrointestinal tract. The first type called autochthonous bacteria which colonize the gut from the environment due to feeding behavior or other activities (Gusils et al.1999). The second type is allocthonous bacteria which are exogenous and introduced to gastrointestinal tract as a dietary supplement through the feed or drinking water as direct fed microbial (DFM) or probiotics (Chichlowski et al. 2007).Probiotics have great adhesive properties that significantly inactivate toxins by surface binding (Zoghi et al.2014). They have been investigated to have the capacity to bind targets like aluminium, arsenic, lead, and cadmium, and eliminate them out of body (Abdel-Megeed, 2020).In addition, Lactic acid bacteria are reported to remove heavy metals in vitro (Halttunen et al., 2008), as they can bind to cationic heavy metals, cadmium, and lead (Zoghi et al.,2014). Combinations of specific probiotic strains is thought to be more effective than one depending on the targets (Timmerman et al.2004).

Probiotics

were previously recorded to its powerful capacity to bind numerous targets and eliminate them with feces. These targets may be

aluminum, cadmium, lead, or arsenic

Probiotics

were previously recorded to its powerful capacity to bind numerous targets and eliminate them with feces. These targets may be

aluminum, cadmium, lead, or arsenic powerful capacity to bind targets like aluminum, arsenic, lead and cadmium , and eliminate them out of body(Abdel-Meg

This study is designed to investigate the biomodulationeffect of the probiotic; Bactozyme; to overcome the undesirable effects of feeding broilers with heavy metals contaminated feed.

MATERIALS& METHODS

1. Materials:

1.1  Broiler chicks: forty chicks at age of one day are raised on healthy feed till the beginning of the experiment at day 20 of age.

1.2  Probiotic: Bactozyme, obtained from MILES Chemical Solution, LLC, USA, consists of a synergistic blend from direct feed microbial (Lactobacillus acidophillus 40ⅹ10⁹ CFU, Aspergillus oryzae fermentation extracts 40GM, Bacillus subtilis fermentation extracts 50GM and Bifidobacterium bifidum 2ⅹ10⁹ CFU)in combination with enzymes and butyrate.

1. Broiler feed: Was purchased from local market, it contains protein (21%), fat (4.04%), 2.44% ash and energy 3000 kcal/kg ration.

1. Methods:

Chickens were randomly divided into 4equal groups; 1^st group received a balanced diet pre-examined to be free of heavy metals(Arsenic, lead& cadmium) using atomic absorption spectrometry; 2^nd group received balanced diet and Bactozyme (0.5 gm/ liter water); 3^rd group received contaminated diet that pre-examined and confirmed to contain high levels of heavy metals, as described in(Table1)and 4^th group received both the contaminated diet and Bactozyme (0.5 gm/ liter water).Birds were maintained at 24-h light schedule. Feed and water were supplied ad libitum throughout the entire experiment. All chickens were sacrificed at the end of 6^th week of age, fresh blood samples were immediately collected into sterile centrifuge tubes and centrifuged at 3000rpm for 15 minutes for sera collection. The clear supernatant serum was aspirated into dry sterile vials and stored at (-20ºC) until used for estimation of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase(ALP), total protein, albumin, globulin, and creatinine.

2.1              Both ALT and AST were determined colorimetrically according to Reitman and Frankel (1975) and Tietz et al.(1983), respectively, while ALP was estimated according to Henry et al.(1960).

2.2              Total protein was determined according to the method of Henry (1964) using Biuret Method, and albumin was determined colorimetrically according to Doumas et al. (1971).

2.3              Creatinine was determined according to Teitz (1986) using colorimetric method.

2.4              Samples from liver, kidney and muscles from each group, and their feed, were cut and preserved at -20°C for residual detection of heavy metals using atomic absorption spectrometry model No. A7942 according to AOAC(2002).

2.4.1    Feed pre-examination for residual detection of heavy metals:

Dried feed sample weighed 2.0g was placed in crucibles with Conc. nitric acid (1cm³) as ashing aid then pre-ashed by placing on a heater until the content charred, then transferred into a muffle furnace at a temperature of 480^oC for 2-3hrs. The cooled samples were dissolved by 5cm³ of 30% HCl and then filtered using Whatman filter papers. The filtrate was individually poured into 50cm³ volumetric flask and made up to the mark with deionized water. The sample solution was analyzed by atomic absorption spectrometry (Okoyeet al.2011).

2.4.2    Tissue preparation for residual detection of heavy metals:

one gram of each tissue sample was digested with10 ml of nitric / sulfuric / perchloric acids (8:  1:  1).  Initial digestion was made for 4 hours at room temperature followed by heating at 40-45°C for one hour in water bath then temperature was raised to 75°C until the end of digestion. After cooling at room temperature, the cold digest was diluted to 20 ml deionized water and filtered through 0.45 µl Whatman filter paper. The clear filtrate of each sample was kept in refrigerator till analysis using atomic absorption spectrometry (Okoyeet al.2011).

1. Statistical analysis

Data analysis was carried out using analyses of variance (F-test) followed by Duncan’s multiple range test. A probability at a level of 0.05 or less was considered significant.

RESULTS

This study examined the tissues (liver, kidney& muscles) of broiler chickens after feeding on contaminated diet by heavy metals with/without the probiotic; Bactozyme. The obtained results detected high levels of arsenic, lead and cadmium in broiler feed as shown in table (1). Regarding the effect on hepatic enzymes, AST, ALT and ALP are significantly increased (76.50±3.33, 12.83±0.49, 184.67±3.28)in the group received contaminated diet in comparison to control group (54.00±7.77, 9.00±0.53, 119.67±7.36)respectively, as recorded in table (2). These parameters are within control levels in groups received contaminated diet with Bactozyme or Bactozyme only. Total protein is high significantly decreased. Creatinine is significantly increased(0.65±0.1)in the group received contaminated dietin comparison to control group. Other groups showed normal levels of albumin, globulin and creatinine except total protein which is significantly increased(6.90±0.24)in the group fed on Bactozyme only and significantly decreased in group fed on contaminated diet with Bactozyme(4.98±0.22)in compare to control group(5.58±0.10)as shown in table (2).

Table (1)Preliminary pre-examination of broiler feed(contaminated feed) for heavy metal residues(ppm)& their maximum tolerance limit.

Table (2): Changes in parameters of kidney& liver function tests after feeding broilers with heavy metals contaminated diet with/ without Bactozyme in comparison to control:

Each value represents means ± S.E. Values with different letters in the same row represent significant difference at P ≤ 0.05 by Duncan multiple range test.

Table (3): The concentration of heavy metals; Cadmium; Lead and Arsenic in µg/gm tissue (ppm) in broilers fed on heavy metals contaminated diet with/ without Bactozyme in comparison to control& their maximum permissible limits according to E.O.S.Q.C and FAO/WHO:

Each value represents means ± S.E.

Values with different letters in the same row represent significant difference at P ≤ 0.05 by Duncan multiple range test.

In concern to residue concentrations of heavy metals in different tissues, cadmium, lead and arsenic are significantly detected in all examined tissues (liver, kidney& muscles) of the group fed on contaminated diet. While the groups received Bactozyme with/ without heavy metals contaminated feed, the residue concentrations are within control levels as shown in table (3).

DISCUSSION

Increases in economic activities and industrialization have led to excessive environmental pollution especially with heavy metals. Heavy metals toxicity is particularly dangerous to birds, due to their high metabolic rate and greater threat of accumulating toxic metals in different body organs (Felsmann, 1998).The results of the current study revealed significant increase of AST, ALT&ALPin the group fed on heavy metals contaminated diet that indicate alterations in hepatic tissue that supposed to be due to toxicity of heavy metals. These results agree with Irfan et al. (2013) who reported that cadmium adversely affect the enzymatic systems of cells. Djurasevic et al. (2017) observed high levels of AST& ALT in male rats received 1.25 mg of CdCl₂/100 g body mass/day over 5weeks. Also, high levels of AST, ALT& ALP were recorded in arsenic- intoxicated broilers (Khan et al. 2013); cattle (Rana et al. 2008) and birds (Halder et al. 2009). Castagnetto et al. (2002) described that Cadmium forms cystein-metallothionein complexin the liver that causes hepatotoxicity. Lead alter the haematological system by inhibiting the activities of enzymes essential for haem-biosynthesis, mainlyγ-aminolaevulinic acid dehydratase (ALAD)(WHO,2011).The ionic mechanism of lead toxicity occurs by replacement of lead metal ions to bivalent cations as Ca²⁺, Mg²⁺, Fe²⁺ and monovalent cations like Na+, so disturbs cell metabolism and causes changes in biological processes such as cellular signaling, protein folding, ionic transportation, enzyme regulation, and release of neurotransmitters (Flora et al. 2012).

The results showed that total protein and globulin are significantly decreased, while albumin is insignificantly decreased, creatinine is significantly increased in group received contaminated diet that may indicate toxic effect of heavy metals, while total protein is significantly increased, albumin, globulin and creatinine are within control limits in group fed on Bactozyme with/ without heavy metals contaminated diet which may attribute to the ameliorative effect of the probiotic. These results agree with the findings of (Siadati et al.2017&Yazhini et al.2018) who observed that plasma protein level was increased with probiotic supplementation in Japanese Quail and broilers, respectively. Mikulec et al. (1999) as well, described that lactic acid bacteria competitively exclude the pathogenic bacteria which reduce the breakdown of proteins to nitrogen and reduce the efficiency of dietary protein, so amino acids and proteins are efficiently utilized. In addition, Yazhini et al. (2018) reported that encapsulated probiotic increase villi height that could increase the protein absorption in broilers. Increasing protein level with probiotic supplementation may be due to increase of feed intake as observed by Samanta and Biswas (1995), and Jin et al. (1996). As well, Chiang and Hsiegh, (1995) reported probiotic improvement in weight gain: Feed ratio with reduction in ammonia production in broiler chickens fed diets mixed with six probiotics up to six weeks of age. In addition, Naik et al. (2000) and Safalaoh et al. (2001) reported the probiotic (Lactobacillus acidophilus, Saccharomycs cerevisiae and their combination) involvement in improvement of feed efficiency in broilers. However, the findings of Li et al. (2014) and Abdel-Hafeez et al. (2017) disagrees with our results as they observed significantly low serum total protein concentration in birds supplemented with probiotic.

Heavy metals can cause cell malfunction and toxicity by binding with protein sites and displacing original metals from their natural binding sites. Also, heavy metals bind to macromolecules as DNA and nuclear proteins resulting in oxidative distress (Flora et al.2008). Their ions bind to albumin, the most abundant protein in plasma. They bind to the free sulfhydryl group of terminal cysteine residues and to histidine residues (Ferguson et al. 2001), that causing malfunctioning of cell respiration, cell enzymes and mitosis especially by Arsenic (Gordon and Quastel, 1948). Moreover, cadmium stimulates the formation of beta 2-microglobulin in urine that results in renal tubular dysfunction (Suganya et al. 2016). As well, lead toxicity may be due to its affinity for thiol groups (–SH) and other organic ligands in proteins (WHO, 2011).

Examination of broiler tissues (livers, kidney, and muscles) after feeding on heavy metals contaminated diet with/without the probiotic; Bactozyme; showed significant levels of cadmium, lead and arsenic residues in all tissues after feeding on contaminated diets shown in table(3). Pb liver level (0.89±0.3) is by far the uppermost, followed by kidney(0.74±0.46)and the lower Pb concentration is detected in muscles (0.096±0.05). Cadmium concentration is the uppermost in kidney (0.19±0.05) followed by liver (0.178± 0.037) and muscles (0.126±0.028). Arsenic is highly increased in liver (6.82±3.16) followed by muscles (4.05±1.01), and kidney (3.69±1.08) respectively. According to European Union Regulation (EC) No (1881/2006), the maximum tolerance levels for lead and cadmium in liver, including poultry liver, are 0.5 ppm (mg/kg) for both (Ghimpeteanu et al. 2012). The maximum permissible limit for cadmium, lead and arsenic in different broiler tissues according to E.O.S.Q.C (2005), FAO (1992) and FDA (1992) are mentioned in table (3). Suganya et al. (2016) reported that chickens can tolerate up to 500 mg lead /kg, without affecting the rate of weight gain. Levels of accumulated lead in chickens are the uppermost in bone, followed by kidney and liver then skeletal muscle. Most of the studies showed that residues of arsenic, cadmium, mercury and lead are the most detected heavy metals in poultry liver. Lead is the most toxic elements, because it binds and inactivates essential enzymes (Baykov et al. 1996). Cadmium binds to cystein-rich protein as metallothionein. It can replace zinc present in metallothionein as they have the same oxidation states, so inhibit it from acting as a cell scavenger of free radical, causing hepatotoxicity in liver and circulates to the kidney, accumulates in the renal tissue causing nephrotoxicity (Castagnetto et al. 2002). Cadmium mainly accumulates in the proximal tubular cells and cause bone mineralization through bone damage or by renal dysfunction (Jaishankar et al. 2014). Arsenic is biotransformed in liver and kidneys (Ford, 2002), and the methylated metabolites are distributed throughout the body (Dopp et al. 2004). The amount of accumulated element in the organs, depends on the interval of exposure, the quantity of ingested element, as well as animal age and breed (Massányi et al. 2000). So, more deleterious changes to tissues are expected on long run exposure to heavy metals through contaminated feed as these effects are time-dose dependent. Maximum tolerable Cd level for poultry is 0.50ppm. The maximum permissible hygiene limits for Cd in meat (0.1 mg/kg) and liver (0.5 mg/kg) according to the Codex Alimentorum. High Cadmium (0.12-1 mg) in the poultry feeds may have bad environmental consequences if poultry manure is used to fertilize plants or to treat soil (Suganya et al. 2016).

In this study, all parameters that showed alterations in broilers fed on heavy metals contaminated diet were obviously improved in the group received either Bactozymeonly or with the contaminated diet. That may indicate the role of the probiotic in modulation of heavy metal toxicity in broilers. These results are concomitant with the findings of Djurasevic et al. (2017) who speculated obvious elimination of cadmium fromintoxicated rats by probiotic administration. Many studies support our findings as Dierck (1989) mentioned that probiotics act in poultry by improving feed intake. Probiotics can alter metabolism by increasing digestive enzyme activity; decreasing bacterial enzyme activity and ammonia production (Yoon et al. 2004); maintaining normal intestinal microflora by competitive exclusion and antagonism (Kizerwetter-Swida and Binek, 2009) and stimulating the immune system (Kabir et al. 2004).In case of heavy metals toxicity, probiotic can detoxify them by binding of metallic ions to cell wall of bacteria (Liu et al.2009). Zoghi et al. (2014) discussed that toxins removal by bacteria is not attributed to bacterial metabolism but to binding of protein and carbohydrate components in cell wall of either yeasts or bacteria to the toxic compounds. Beveridge and Murray (1980) illustrated that Bacillus subtilis can bind to lead that start with a stoichiometric reaction between metallic cations and surface binding sites, then inorganic deposition of more metal. Halttunen (2007) discussed that lactic acid bacteria have cationic binding sites for the removal of anionic As (V), and chemical modifications can increase As (V) removal.

Conclusion

There are increasing evidence of the beneficial effects of probiotics which induced by various mechanisms. This study examined the effect of a blend of probiotics; Bactozyme; on heavy metals intoxicated broilers. We conclude that probiotics potentially play an effective role in biomodulation of lead, cadmium, and arsenic toxicities in broilers, as one of the main sources of nutrition for humans. So, the study recommends incorporation of probiotics into broiler feeding to minimize any deleterious effects caused by incorporation of these heavy metals into broiler feed.