Baluchistan Gerbil hepatotoxicity as a new biomonitor of heavy metal pollution

Experimental Animal’s Collection
 
Baluchistan gerbils were captured in spring season during three months (March-May). Animals were collected from an area 35 kilometers east of Al-Ahsa (N 25°46.748 × E 49°15.850) near a cement factory and Al-Asfar lake. The area is surrounded by highways (Al-Oqair road) and the circular eastern road (Fig 1). Gerbils captured using small mammal live traps (31×12.5×12.5 cm). Twenty traps were randomly placed each in between 10m. They were placed and opened before sunset between 16h and 17h, then gathered after dawn. Control animals were collected from area 90 Km to the west of the shooting area as reference area. The captured gerbils (weight 30-35g) were identified and transported to the laboratory for further processing (Fig 2). 
 

 

 
Blood Samples Assay
 
Whole blood (5ml) from each animal (n=10) was collected after sacrifice on the day of capture in two specimens. The first (1ml) was collected in heparinized tubes for measurement of the hematological indices as Complete Blood Count using SysmexKX 21 automated hematology analyzer, Germany. It is a double capillary instrument which built on reliable Sysmex technology. The second portion (4ml) was collected into dry glass centrifuge tubes, left to clot and then centrifuged at 5000 rpm for 10 min; the separated sera was used for the determination of aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT) and alkaline phosphatase activity (ALP) were estimated by commercial kits (ASAT; REF: GOT111120; ALAT; REF: GPT1131001 and ALP; REF: ALP101050, BIOMED Diagnostics, Germany).
 
Liver Tissue Assays
 
Every liver tissue sample was divided into four specimens. The first was fixed in 10% neutral buffered formalin for 24 hours, dehydrated in ascending series of ethanol, cleared in xylene then embedded in paraffin wax. Sections were stained with conventional hematoxylin and eosin (H&E) dye and examined using a light microscopy according to Banchroft and Gamble (2008). The second specimen was fixed in 3% phosphate buffered glutaraldehyde (pH 7.3) and processed for preparation of semi thin sections (1µm). Ultrathin sections were prepared and stained with uranyl acetate and lead citrate, then examined by transmission electron microscope (1011 TEM-JEOL). The third was dried for 2 days at -80°C and 0.5 g of each dried tissue sample was fine powdered and digested with nitric acid (65%) and hydrogen peroxide (30%) mixture (5:1) (v:v). The solution was filtered and diluted till 10 ml with ultra-pure water (Zarrintab et al., 2016). Selected heavy metals Pb, Cu and Zn were measured using the flame atomic absorption spectrometry (iCE 3000 Series AAS, Thermo Scientific, England). The fourth specimen was used for bioassays kits of Superoxide Dismutase (SOD), Catalase (CAT) and Malondialdehyde (MDA) according to the manufacturer’s protocol (SOD; CAT. No: EIASODC, CAT; CAT. No: MBS006963 and MDA according to Ohkawa et al., (1979). 
        
All the experimental protocol conforms to the ethical standards of the National Institutes of Health (NIH Publication No. 85-23, revised 1996).
 
Statistical analysis
 
Data were expressed as means ± SE. Statistical analysis was done by independent samples T- test at significance level p<0.05 using IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp.

The present study showed significant increase in liver concentrations of Cu and Pb and significant decrease of Zn in gerbils’ inhabited polluted site as compared to the control values (Table 1). Heavy metals can enter the food chain and threaten human health (Stankovic et al., 2014). In addition, (Zarrintab and Mirzaei, 2017) reported the ordered elevated tissue concentrations of Pb, Zn, Cd; hair > liver > kidney > muscle, in gerbils from different polluted areas. The serum enzymes activities of ALAT, ASAT, and ALP were significantly higher in G. nanus of polluted areas comparing to the control values (Table 1). Liver CAT and SOD enzymes activities were significantly decreased in tested animals as compared to control values. As regard to the lipid peroxidation end product (MDA) data recorded significant elevated level in tested animals than controls (Table 1). A prior study in one of Riyadh polluted areas with Cd, Cu, Hg, Ni and Pb showed that Libyan jird acquired liver damage in view of elevations in ALT and AST activities compared with reference jirds (Adham et al., 2011). The liver OS status recorded in our study in line with Al-Otaibi et al., 2018 who reported that Pb accumulation in different organs of wild Meriones. Libycus accompanied by elevated MDA and declined reduced glutathione in liver and kidney tissues. Also, Liua et al., (2018) reported the role of Pb in hepatotoxicity due to OS and impaired antioxidant defense. Overload of Cu can induce a set of toxicological effects in liver through elevated liver enzymes, lipid metabolism alteration and changes in gene expression involved in the hepatocytes necrosis (Yang et al., 2010; Gaetke et al., 2014; Tang et al., 2018).
 

 
        
The hematological parameters (RBC, Hb, HCT, MCV, MCH and MCHC) recorded significant decrease in animals inhabited the polluted area as compared with the corresponding control values (Table 1). These data are in agreement with the study of Al-Otaibi et al., (2018) outcomes. Moreover, the hematological impairments due to the impact of pollution in various mammalian species; such as wood mice, Algerian mice, house mice, Mus spretus, and Mus musculus were previously discussed (Sánchez-Chardiet_al2008). Lead previously reported to inhibit hemoglobin synthesis and induce changes in the RBCs’ membrane proteins and lipids (Valko et al., 2005). Also, Pb liver accumulation induced erythropoiesis defects and anemia (Yuan et al., 2014). That is in line with the recorded significant decrease in Hb in our tested gerbils. This result may be explained through the effect of decreased liver Zn contents. Zinc functions as the catalyst in iron metabolism and has a role in heme synthesis and erythropoiesis homeostasis by serial development of hematopoietic stem cells and mega-karyocytes (Osawa et al., 2002; Hacibekiroglu et al., 2015).
        
Data expressed as means ± SE. All data are expressed as mean ± standard error (SE) and independent samples T- test performed at significance level of p<0.05 represented by superscript (a) between control and sample means.
        
The control liver sections H&E stained revealed normal histological features as represented in (Fig 3 a). Tested sections revealed necrotic changes in liver cells, cytoplasmic vacuolation, congestion and central venous bleeding with severe damaged blood sinusoid. Increased inflammatory Kupffer cells and bleeding around hepatocytes also observed (Figs 3b and 3c). This feature of damage may be a response against heavy metals toxicity (Jadhav et al., 2007). The abnormality of liver structure in our study may be due to copper toxicity as shown in many previous studies. Sub lethal dose of Cu to Asian sea bass showed liver alterations; including vacuolization, hypertrophy and necrosis (Maharajan et al., 2016). High dose of copper induced lysosomal inclusions, irregularly shaped cell nuclei and abundance of mitochondria (Cholewiñskaet_al2018) and long-term exposure of copper to rats resulted in necrosis and apoptosis (Aburto et al., 2001).
 

        
Ultrastructural results revealed normal hepatocytes with rounded euchromatic nuclei with nuclear membranes and nucleoli. The control liver section showed normal hepatocytes with cytoplasm-riched organelles such as mitochondria, rough endoplasmic reticulum and lysosomes (Fig 4). Polluted gerbil’s liver section showed cytoplasm with large vacuoles (Fig 5); heterchromatin nuclei; swollen endoplasmic reticulum and mitochondria and heterogeneous lysosomes (Fig 6). Another pronounced irregular nucleus and degenerating cytoplasm was seen in tested liver section (Fig 7); also shrinking nuclei and hepatic sinusoids were appeared (Fig 8). The present study clearly demonstrated that the liver is an important target organ for copper toxicity in the tested gerbil. This result agreed with the study of Maharajan et al., (2016) that recorded hepatic histopathological alteration in Asian sea bass under sub lethal dose of Cu. The loss of regular cytoplasmic compartments and the degree of hepatocyte cell damage may explain through copper accumulation in hepatocytes that activate the vacuolation process. Hence, excessive liver vacuolation volume affects negatively the cell and even leads to its death (Gupta et al., 2016; Aghamirkarimi et al., 2017).