Mitigation Effects of Vitamin C and E Against Copper Toxicity in Broiler Chickens, With Reference to Biochemical, Genotoxicity and Pathological Studies

Copper (Cu) is necessary for biological utility, nevertheless when existing in abundance; it can produce plentiful injurious impacts. This enquiry was carried out to explore the eciency of individual or combined doses of vitamin C (Vit C) and vitamin E (Vit E) in ameliorating some biochemical, genotoxicity and pathological changes in the liver of copper sulphate (CuSO 4 )-intoxicated chickens. One hundred-one day old broiler chicks were haphazardly divided into 5groups of 20 chicks each. The broilers were fed on basal diet only (control, gp.1 ) or supplemented with 300 mg CuSO 4 /kg diet (Cu, gp.2 ), CuSO 4 + 250 mg Vit C /kg diet (Cu+ Vit C, gp.3 ), CuSO 4 +250 mg Vit E /kg diet (Cu+ Vit E, gp.4 ) and both vitamins C + E (Cu+ Vit C+ Vit E, gp.5 ) for six weeks. The results displayed that CuSO 4 - intoxicated birds (gp.2) had signicantly ( p<0.05 ) dwindled body weight, gain and feed consumption with increased feed conversion rate from week 2 till the 6 th week compared with control group. Serum aminotransferases (ALT, AST), and alkaline phosphatase (ALP) were signicantly ( p<0.05 ) augmented in CuSO4- exposed group (gp.2) with signicantly ( p<0.05 ) drop in serum total protein (TP), albumin, globulins, triglycerides (TG), total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C), very low density lipoprotein-cholesterol (VLDL-C), and high density lipoprotein-cholesterol (HDL-C) levels compared to control. Concomitantly, histopathological and DNA changes were perceived in liver of CuSO 4 -intoxicated birds. Co-supplementation of Vit C, and Vit E single-handedly or incorporation to CuSO 4 -intoxicated chickens displayed an enhancement in performance traits and abovementioned changes, especially with those given combination of vitamins. From the extant enquiry, it could be established that supplementation of vitamin C and E were benecial for alleviation the harmful effects of CuSO 4 on performance and other studied parameters in broiler chickens.


Introduction
The proper activities of iron metabolism-related metalloenzymes are maintained by copper (Cu), an important microelement involved in poultry diets (Samanta et al. 2011). Despite the need for unlike metabolic processes and enzyme activities, chronic over-exposure to copper caused some adverse effects (Spatari et al. 2002). In poultry, the extreme type of Cu poisoning is long-term ingestion of Cu compounds from different sources. Metabolism of Cu is regulated primarily by the liver, where it can be released into the circulatory system or excreted via the bile (Linder and Hazegh-Azam 1996). It accumulates steadily in the liver during chronic Cu toxicity without causing any noticeable signs or symptoms. It can lead to hepatocellular lesions when the hepatic Cu storage capacity is surpassed, and thus, the release of Cu from the liver into the blood stream causes hemolysis, jaundice, and renal disease (Liu et al. 2020). Aforementioned studies have designated that excessive exposure to Cu can induce oxidative stress in the brain tissue of chickens (Li et al. 2010), reduce the activities of copper-zinc superoxide dismutase and glutathione peroxidase, and increase the contents of malondialdehyde and hydroxyl radical in the liver of ducklings (Zhao et al. 2008). An discrepancy between reactive oxygen species (ROS) production and the body's ability to detoxify these intermediate species is considered to indicate oxidative stress. Supplementation with Cu up to 200 ppm is used for growth promotion (Thompson 2007), but excess amounts of Cu in the diet depress indices of performance as it result in low digestibility of Cu salt which does not exceed 20% and absorption in poul try which led to ampli ed elimination of fecal matter and environment pollution (Świątkiewicz et al. 2014).
Few reports have displayed that supplementation of antioxidants such as vitamin E, vitamin C, zinc, betacarotene, alpha-lipoic acid and polyphenols has a protective impact against the toxicity of Cu (Gaetke and Chow 2003; Ajuwon and Idowu 2010). However, only restricted studies have been achieved on vitamin C effects on Cu-induced oxidative stress in broilers; no studies have analyzed the effects of vitamin E single-handedly or incorporation with vitamin C on performance, biochemical markers, DNA damage or pathological ndings in broilers exposed to Cu. Consequently, the ambition of the existent inquest was to assess the effects of dietary supplementation with Cu on these parameters and then to appraise the protecting effects of vitamins C and/ or E against excess dietary supplementation with Cu, individually or in combination.

Materials And Methods
Experimental animals, diet and protocol One hundred, 1-day old commercial Cobb broiler chickens were attained from Al-Kahira Poultry Company, 10 th of Ramadan City, Sharkia Governorate, Egypt. The chicks were given starter diet from day 1 until 14 days of age and grower diet (15-28 day) followed by a nishing diet up to 42-days age. Ingredients and chemical composition of diets were demonstrated in Table 1. As designated by the Ross management manual (Aviagen 1999), experimental diets were formulated. Chicks were reared under identical husbandry circumstances (14 h light: 10 h dark, relative humidity 60% and 25-32 o C) in standard battery cages with automatic nipple drinkers and standard feeding trough. The diets and water were offered ad libitum. All birds were vaccinated at 7 and 18 days old against Newcastle disease and at 15 days old for Gumboro disease (Giambrone and Clay 1986).
The chicks were haphazardly divided into 5groups of 20 chicks each. The birds were fed on basal diet only (control or negative group), or supplemented with 300 mg CuSO 4 /kg diet only (Cu or positive group), The body weight (BW) of each chick and feed consumption (FC) of each group were weekly veri ed, starting from 1day-old, and weight was certi ed to the bordering 1 g. Mortality was recorded and growth performance was appraised in relations of live body weight gain (BWG), feed intake and feed conversion ratio (FCR). The FCR was calculated according to Wagner et al. (1983). The enquiry was ended at the 6 th week of the experiment.

Sampling:
Samples of blood were collected from the wing vein of 10 aimlessly selected birds in each group at termination of 3 and 6 weeks post-supplementations and centrifuged (1200 g for 15 min) immediately for separation of serum, which is stored at −20°C in deep freeze until biochemical analysis (Hashem et al. 2019).
The chicks were sacri ced by manual cervical dislocation and two portions of liver tissues were separated, and blotted dry. The rst part was put in ice-cold PBS (phosphate buffer saline) for comet assay determination and the 2 nd part was xed in ten percent formalin for histopathological inspection.

Detection of DNA damage
The liver DNA damage was measured using a single-cell gel electrophoresis technique (also known as comet assay) as previously de ned by Singh et al. (1988). Comet assay is a quick, accurate and simple method for detecting DNA damage. In this method, 0.5 g of crushed samples was transferred to 1ml icecold PBS. This suspension was stirred for 5 min and ltered. The cell suspension (100 μl) was mixed with 600 μl of low melting agarose (0.8% in PBS). A 100µl of this mixture was spread on precoated slides. The coated slides were immersed in lysis buffer [0.045mol/l Tris/Borate/EDTA (TBE), pH 8.4, containing 2.5% Sodium dodecyl sulphate (SDS)] for15 min. The electrophoresis conditions were 2 V/cm for 2 min and 100mA. The slides were then washed 3times, for 5minutes each, with neutralization buffer [0.4 Mol/L Tris (pH 7.5)]. Finally, the slides were stained with 50 AL of ethidium bromide (2 mg/ml) and covered with a cover slip. The DNA fragment migration patterns of 100 cells at 400 magni cations with the Optika Axioscope uorescence microscope were calculated for each dose level. The length of DNA migration (tail length) on PX was calculated for each cell from the center of the nucleus to termination of tail. By calculating the total intensity ( uorescence) in the cells, which was taken as100%, the DNA% in the tail was determined and deciding what percentage of this total intensity corresponded to the intensity only measured in the tail. The tail moment was expressed in arbitrary units. Although any image analysis device may be su cient for SCGE data quanti cation, Comet 5 image analysis software developed by Kinetic Imaging Ltd (Liverpool, UK) linked to a CCD camera has been used to determine the degree of quantitative and qualitative DNA damage in the cells by measuring the length of DNA migration and the % of migrated DNA. Finally, the program calculated the tail moment. Generally, 50-100 randomly selected cells are analyzed per sample.

Histopathological investigations
Tissue samples were taken from the liver of euthanized chicks by manual cervical dislocation and xed in formalin of 10 %. The samples preserved with formalin are dehydrated and embedded in para n. Fivemicron thick para n slices were set and tainted with hematoxylin and eosin (H& E) and inspected microscopically (Suvarna et al. 2018).

Statistical analysis:
The ANOVA analysis for all data was accomplished using SPSS (IBM, 2013) for windows version 22. The data was expressed as mean and the data variability was expressed as standard error of mean (SE). The Duncan's multiple range tests were used to assess the differences in signi cance between groups, p < 0.05 suggesting a difference in signi cance.

Results
Clinical signs and body performance: No clinical signs or mortality were found in all of the supplemented birds during the experimental times except CuSO4-intoxicated group (2) which showed mild diarrhea (few cases), decrease appetite, and pale comb.
As illustrated in Fig. 1 (a, b, c), the BW, BWG and FC of broilers were signi cantly (p<0.05) declined in all groups from the 2 nd week till the termination of experiment (6 th week) compared to control (Figs. 1-3).
However, these changes were minimized in birds supplemented with vitamins (gps. 3-5) matching with gp. (2), but not return to control values. FCR was signi cantly (p<0.05) high in CuSO 4 -intoxicated broilers (gp.2) at 2 nd to 6 th week of age related to control (Fig.1 d). Comparatively with CuSO 4 -intoxicated broilers (gp.2) FCR was insigni cantly decreased in those fed with vitamins (gps. 3-5) from the 4 th week of age and go back to near normal control values at the 6th week.
Effect of CuSO 4 and vitamins on serum levels of liver biomarkers As shown in Tables 2-6, CuSO 4 induced hepatotoxicity as re ected by statistically (p < 0.05) augmented serum activities of ALT, AST and ALP, whereas serum TP, albumin, globulins, TG, TC, LDL-C, VLDL-C and HDL-C levels are reduced (p ≤ 0.05) after 3 and 6 weeks compared to normal control values. The reduction in serum albumin and HDL-C was only at 6 th week period.
On the other hand, co-supplementation of copper with either vit. C or vit. E had signi cantly (p ≤ 0.05) declined serum enzymes and enlarged serum TP, albumin, globulins, TG, TC, LDL-C, VLDL-C and HDL-C levels in birds of gps. 3&4 matching with CuSO 4 -induced hepatotoxicity group (gp.2). However, coadministration of CuSO 4 + Vit C+ Vit E (gp.5) restored the changes in liver biomarkers levels back to near the normal control values.

Effect of CuSO 4 and vitamins on DNA damage
The data in Tables 6&7 and Fig. 2 (a-f) revealed that CuSO4 intoxication produced a signi cant (p ≤ 0.05) elevation in comet %, %DNA in tail, tail length, tail moment and olive tail moment at the end of 3 rd and 6 th week parallel to control group (1). On contrary, co-administration of CuSO4+Vit C, CuSO4+Vit E or their combination (CuSO4+ Vit C+ Vit E) to birds ensued in an enhancement in the results of comet assay performance, showing a substantial decrease in previous parameters relative to the CuSO 4 -intoxicated group but not return to values of normal control. It is obvious that the combination regimen produced the most marked reduction as related to CuSO 4 -intoxicated group.

Histopathological ndings
The livers of control rats displayed the typical histological arrangement of hepatic lobules. In contrast, the livers of chicken from CuSO 4 -intoxicated group showed hyperplastic, and necrotic biliary epithelium with various degenerative and necrotic changes at third week (Fig.3 a). In addition cholestasis, necrotic bile duct epithelia, beside portal lymphocytic aggregation and broblast proliferation were encountered on termination of 6th week (Fig.3 b). Liver from chicken of Cu+ Vit C supplemented group for 3weeks viewed moderate enlargement of hepatic cells and hyperplastic kupffer cells (Fig.3 c), as well as partially contracted hepatic cells proliferative kupffer cells and dilated sinusoids after six weeks (Fig.3 d). Liver from chicken of Cu+ Vit E supplemented group revealed portal lymphocytic aggregations within apparently normal hepatic parenchyma on termination of third week ( Fig.3 e), while intense hyperplasia of kupffer cells and cloudy swelling of hepatic cells with a few lymphocytic aggregations were seen on 6 th week (Fig.3 f). Liver of Cu+ Vit C+ Vit E supplemented chicken (gp.5) displaying little portal and interstitial lymphocytic aggregations within apparently normal hepatic parenchyma on termination of third week (Fig.3 g), mild portal lymphocytic aggregation, normal hepatic parenchyma and dilated blood vessels were observed after 6weeks (Fig.3 h). The growth parameters were upgraded meaningfully in Cu+ Vit C and Cu+ Vit E-supplemented groups compared to Cu-intoxicated group, however, better results have been found in birds-taken both Vit C and Vit E (gp.5). Many reports showed better performance with feeding of Vit C or Vit E to broiler chicks Birds supplemented with excess CuSO 4 (gp.2) revealed an elevation in serum transaminases (ALT&AST) and ALP activities correlated with diminished serum TP, albumin and globulins levels equated to the control group, suggesting marked liver damage (hepatotoxicity), which was further con rmed by hyperplastic, and necrotic biliary epithelium, hepatic degeneration and necrosis in the histopathological ndings. The height transaminases activities in birds of gp. (2)  Beside, Vit C and Vit E may preserve the hepatic cellular membrane and protect hepatocyte from copper's toxic effects, which may minimize the enzymes leakage into the blood stream (Prabu et al. 2011). In dissimilarity, supplementation broilers-exposed to toxic levels of cadmium with vitamins C and E did not improve transaminases activity (Cinar et al. 2011).

Discussion
Co-supplementation of copper with Vit C and Vit E alone or in combination had signi cantly (p < 0.05) elevated serum TP, albumin, globulins. The enhancement in the protein pro le in Cu-exposed birds fed with Vit C and Vit E alone or in combination may  (Pearce et al. 1983). The reduction in LDL level was related to copper toxicity because Cu is an effective catalyst of LDL-C oxidation to an atherogenic form (Steinberg 1997) or to alkoxyl radicals (Burkitt 2001). The reduction in HDL-C is due to hypocholesterolemia and hypoproteinemia in this enquiry as more than 40% of HDL-C value represents cholesterol value and the remaining proteins (Ganong 2005). The same ndings were obtained by El-Ghalid et al. (2019) in broiler chickens with dietary supplementation of CuSO4 at levels 50 and100 ppm for 5 weeks. However, earlier studies exposed dissimilar outcomes, for example rises of total lipid, cholesterol and LDL-C with no variation HDL-C level in Cu-exposed quail (Almansour 2006)  Dietary supplementations of CuSO4-intoxicated birds with Vit C and Vit E alone or in combination reduce the opposing effects of copper on lipid pro le. The treatment of Cu toxicity with Vit C and Vit E combination is more effective than the use of each alone. This can be contributed to capacity of Vit C to regenerate Vit E by removing free radicals bound to it (Niki, 1987). A combination of the two vitamins protects lipid structures against peroxidation (Prabu et al., 2011). Vit C guards against Cu toxicity by preventing excess Cu absorption by minimizing copper absorption from the intestine by reducing soluble Cu-levels in the small intestine (Kies and Harms 1989;Van den Berg et al. 1994). In addition, Vit E can prevent cholesterol-related endothelial dysfunction, preventing functional impairment induced by ROS (Gaetke and Chow 2003).
Although the micronucleus assay was informative and its usefulness should be considered (Corona-Rivera et al. 2007), comet assay is a more sensitive method to assess genotoxicity (Zhong et al. 2001;Andrade et al. 2004). The realistic comet assay has been used to test genotoxic agents (Andrighetti- ). Free Cu causes ROS and many forms of DNA damage to be generated, such as base alteration and DNA strand breaks, which can cause massive cell death (Hayashi et al., 2000). A copper-induced high ROS production consequence in oxidative destruction to a single DNA base and sugar phosphate and breaks DNA strands (Bjelland and Seeberg 2003). In addition, copper reduces DNA-binding cell viability, resulting in cell death (Tchounwou et al. 2008).
The existing inquiry revealed that supplementation of CuSO4-intoxicated chickens with Vit C and Vit E alone or in combination exert a partial genoprotective effect against DNA damage induced by excessive concentrations of copper which was proved by the decrease of comet%, tail length and moment. A combination of vitamins is more effective in reducing genotoxic effects of copper than use of each vitamin alone. The genoprotective effect of Vit C and Vit E could act as free radical scavengers and antioxidants ( These unsuited fallouts may be credited to variation in the types of test materials, experiment animals, and test duration. Long-term exposure of birds to high levels of Cu, hazardous impacts would occur (Zhou et al. 2020).
In contrast, the CuSO 4 -intoxicated rats supplemented with Vit C and Vit E alone or in combinations reduced the histopathological ndings with apparently normal liver architecture in some cases. Our results are supported with other nding presented that addition of vitamins (C&E) in feed has been successful in offsetting arsenite's toxic effects in broiler chicken (Khatun et al. 2020).

Conclusions
In conclusion, long-term exposure to CuSO 4 caused signi cant alterations the liver evaluation biomarkers, genotoxicity and histopathology. Dietary supplementation of Vit C and E showed a fall in the harmful effects induced by CuSO 4 , especially with their combination which cause an improvement in the growth performance; return the levels of some biochemical parameters in close to normal values, with subsidence the histopathological changes and DNA degeneration. Overall, the protective roles of vitamins C & E and their synergistic action against the toxic effects of Cu are clearly seen in our research, but further studies are still needed to understand the complete potential of vitamins.

Funding
This study does not have a funding source.

Data Availability
All author stated that all information produced or analyzed during this work is included.     Lipid pro le in all groups of chickens at the end of 3 rd week (Mean ±SE, n= 10).