Comparative ameliorative actions of extracted bradykinin potentiating fraction from cobra snake venom and synthetic antioxidants on hepatic tissue of aflatoxicosed rats

ABSTRACT

Mycotoxins are secondary metabolites produced by Aspergillus, Penicillium and other genera which contaminate food and exhibit hazardous effects on humans and animals. Aflatoxins (AFs) caused liver necrosis, fibrosis. In the present study, we planned to evaluate the action of bradykinin potentiating factor (BPF) extracted from Egyptian cobra snake venom and synthetic antioxidants; butylated hydroxytoluene (BHT) and oltipraz (OPZ) on the hepatic tissue of aflatoxicosed rats. Aflatoxicosis caused a significant increase of nitric oxide (NO) and lipid peroxidation as malondialdehyde (MDA) associated with a significant decrease in the level of total thiols, glutathione (GSH), and the activities of glutathione peroxidase (GPx) and transferase (GST). In addition to a decrease in the counts of white blood cells (WBCs), lymphocytes (LYM), monocytes (MONO), eosinophils (EOS), red blood cells (RBCs) and an increase in neutrophil counts (NEUT) as well as a significant decrease in the gene expression of hepatic interleukin-1β (Il-1β). The previous changes were confirmed by histopathological alterations. Treatment of aflatoxicosed rats with any of BPF, BHT or OPZ resulted in amelioration of the oxidative stress parameters, blood picture and hepatic IL-1β with improvement in histological features. In conclusion, either BPF, BHT or OPZ can be used for the treatment of aflatoxicosis.

KEYWORDS:

• Aflatoxin B₁
• liver
• butylated hydroxy toluene
• bradykinin potentiating factor
• oltipraz

1. Introduction

Mycotoxins are secondary metabolites produced by Aspergillus, Penicillium and other genera which contaminate food and exhibit hazardous effects on humans and animals mainly malnutrition, immunosuppression, genotoxicity, carcinogenic and teratogenic effects due to inhibition of macromolecule synthesis and metabolism (Nassar et al. 1985; Megalla et al. 1990; Maresca and Fantini 2010; Adejumo and Adejoro 2014; Ashiq 2015; Tola and Kebede 2016). The AFs are referred to four different types and their metabolites contaminating the food of animals and humans (Nassar et al. 1982; Hafez et al. 1985; Rajendra et al. 2014). AFs caused liver necrosis, fibrosis and enhanced increasing the risk of hepatocellular carcinoma (Agag 2004; Rotimi et al. 2019).

Animal venoms including those from spiders, snakes, cone snails and scorpions contain components that cause rapid death of the animal prey (Yang et al. 2016). The Egyptian cobra (Naja haje haje) contains a number of non-toxic low molecular weight basic polypeptides such as bradykinin potentiating peptides (BPPs) that have physiological and immunochemical properties (Ferreira 1965; Caldwell et al. 2015; Xu et al. 2015). It was found that bradykinin potentiating factor (BPF) extracted from the Egyptian scorpion and cobra venom enhanced physiological functions as well as cellular growth of the uterus and the development of the ovarian follicle in mice (Nassar 1989, 1990; Abdel-Raheim et al. 1995). In addition, it generates thymus and spleen cellularity, recovered blood picture and accelerated the healing process in irradiated guinea pigs (Abu-Sinna et al. 2005; Salman 2009; Sarin 2015; Salman 2018).

Butylated hydroxy toluene (BHT) is an antioxidant phenolic acid that is used to prevent rancidity of food products containing fats and oils (Lundebye et al. 2010). Moreover, BHT has the potential to defend against oxidative stress and protect against cancer and cardiovascular diseases (Weisburger et al. 1977; Cohen et al. 1984; Jaouad and Torsten 2010). Oltipraz (OPZ) [4-methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione] has been widely studied as a cancer chemopreventive agent due to enhancement of necrosis factor-2 (Nrf2) and the consequent changes in target gene transactivation as well as enhancing the induction of the glutathione-S- transeferase (GST) A₂ gene (Ramos et al. 2001; Guise 2013). Moreover, OPZ has therapeutic effects on cirrhotic liver even in high doses (Dwyer et al. 1997; Cho et al. 2006).

Accordingly, this work has been designed in order to evaluate the therapeutic effect of BPF, BHT and OPZ against aflatoxicosis in female rats.

2. Materials and methods

2.1. Chemicals used

Butylated hydroxy toluene (W218405), oltipraz (O9389), thiobarbutric acid (T5500), naphthylethylene diamine dihydrochloride (N9125), 5,5- dithiobis-2-nitrobenzoic acid (D8130), sulphanilamide (S9251) and coomassie brilliant, blue R stain (B0149) were purchased from Sigma-Aldrich Company (St. Louis, MO), U.S.A. Interleukin-1β (IL-1β) taqman assay (Rn99999009_m1) was purchased from Thermo Fisher Scientific Company, U.S.A.

2.2. Extraction, purification and identification of AFB₁

AFB₁ was extracted from harvested media of growing Aspergillus flavus according to the method of Booth (1971), purified by TLC method of El-Kady and Moubasher (1982).

2.3. Extraction, purification and identification of BPF

Venom extraction and dialysis; the collected amount of the Egyptian cobra snake crude venom was dialyzed according to the method of Abdel-Raheim et al. (1995). Isolation and purification of BPF from the collected cobra venom dialysate by the chemical method of Ferreira (1965). Physiological examinations of the isolated BPF; isolated BPF was identified by its hypotensive effect on arterial blood pressure of rabbit by oscillograph in the central lab of Zoology Department, Faculty of Science, Assiut University. The amino acid content of the isolated BPF was analyzed by amino acid analyzer SW (Hydrolisate Separation Method) at Desert Research Center, Ministry of Agriculture and Land Reclamation, Cairo, Egypt.

2.4. Experimental animals

In this study, 120 female Wistar albino rats were used and obtained from the Animal House of the Faculty of Medicine, Assiut University, Assiut, Egypt with mean body weight of 130 ± 10 gm. Rats were housed in cages, kept at room temperature with normal 12 h light/12 h dark cycle and supplemented with standard commercial pellets for feeding, water, ad libtium. All of the animal procedures were performed in accordance with the guidelines for the care and use of experimental animals established by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and the protocol of the National Institutes of Health (NIH) (Ragab et al. 2015).

2.4.1. Design of the experiment

Rats were divided randomly into six groups of 20 rats each as follows:

• G I: Reference normal group.

• G II₀: Intoxicated group with AFB₁ for four weeks.

• G II₁: Self- recovery for the same period after intoxication.

• G II₂: Treated with BPF for the same period after intoxication.

• G II₃: Treated with BHT for the same period after intoxication.

• G II₄: Treated with OPZ for the same period after intoxication.

2.4.2. Route of administration of intoxicating solutions and the other treating agents

The animals of GII₀ were individually intoxicated by oral administration three times weekly (500 μg AFB₁ suspended in milk/kg b.wt) for successive four weeks according to Raisuddin et al. (1993). GII₁ animals after intoxication were left without treatment for self-recovery. GII₂ intoxicated animals were treated by intrapetionally three doses weekly for successive four weeks (1 μg BPF in PBS (PH 7.4)/kg b.wt) according to Omar and Meki (1997). GII₃ intoxicated animals were orally treated by 0.5 mg BHT in PBS (PH 7.4)/kg b.wt three times weekly for successive four weeks according to Hocman (1988). Similarly to GII₃, the animals of GII₄ were orally administrated by OPZ (2 mg in PBS (PH 7.4)/kg b.wt) according to Dimitrov et al. (1992).

2.4.3. Collection and preparation of the samples for biochemical determination and histopathological examinations

At the end time of the experiment, blood samples were collected from the heart under an aesthesia by ether and sacrificed by cervical dislocation for sampling of considered tissue organs. At the time of scarifying, the blood was collected in tubes and centrifuged after blood clotting at 4000 rpm for 10 min to separate serum. The liver was quickly removed, washed with saline solution and small slices were fixed in glutraldehyde for electron microscopic examinations and the remnant was imbedded in liquid nitrogen and kept frozen at −80 for complementary cDNA studies. 10% w/v homogenate of liver in 0.1M phosphate buffer (pH 7.4) was prepared, then the homogenate was preserved at −20°C for the subsequent biochemical indices.

2.5. Serological characters

Another portion of blood was immediately introduced into EDTA tubes for complete blood cell counting (CBC) by using automatic blood cell counter (Exigo PM800 blood analyzer) at the Pathology and Clinical Pathology Department, Faculty of Veterinary Medicine, Assiut University.

2.6. Biochemical determinations

Total protein content in the supernatant of hepatic tissue homogenates was performed according to the method of Lowry et al. (1951). Alanine aminotransferase (ALT) activity in serum was determined by ALT-Liquizyme (4 + 1) E.C.2.6.1.2 kit which purchased from Egyptian Company for Biotechnology. Nitric oxide (NO) was determined by Gries reagent according to Ding et al. (1988). Lipid peroxidation as MDA was evaluated according to the method of Wills (1969). Total thiols content was assayed according to the method of Ellman (1959). Glutathione (GSH) was estimated according to the method of Beutler et al. (1963). The activity of GSH-peroxidase (GPx) and transferase (GST) were assayed according to Habig et al. (1974). The interleukin-1β (IL-1β) gene expression in hepatic tissue was evaluated by Real time PCR at the Tissue Culture and Molecular Biology Center Lab in Assiut University according to Livak and Schmittgen (2001).

2.7. Histopathogical features

Semi-thin section and transmission electron microscope observations were done in Electron Microscope Unit (E.M.U) of Assiut University according to Bozzola and Russell (1991).

2.8. Statistical analysis

Statistical differences of parameters are presented as Mean ± SEM, statistical significance was determined using the one way ANOVA test in Graph Pad Prism 5.03 at P < 0.05 where ^a is a significant of aflatoxicosed GII₀ with respect to reference normal GI, ^b is a significant of treated groups GII_{1, 2, 3, 4} with respect to reference normal GI and ^c is a significant of treated groups GII_{1, 2, 3, 4} with respect to aflatoxicosed GII₀.

3. Results

3.1. Laboratory biosynthesized and collected pure sample of AFB₁

The biologically synthesized aflatoxins (B₁, B₂, G₁ and G₂) were characterized, identified and collected AFB₁ fraction in pure sample by TLC chromatographic separation by chloroform:methanol solvent (97:3) as presented in Figure 1.

Figure 1. TLC of aflatoxin fractions in the harvested mixture samples (b), matched with an authentic sample (a) and collected, purified AFB₁ fraction (c).

3.2. Identification of amino acid composition in the isolated BPF

The amino acid composition of the isolated BPF from the venom of Egyptian cobra venom (Naje haje haje) demonstrated high content of Asp, Lys, Cys, Arg, Thr, Glu, Ile and Gly and low content of Val, Phe, His, Ser, Pro, Ala, Met and then Leu, Tyr in a descending order with the absence of Trp, Hyp, Asn and Gln as it is shown in Table 1.

Table 1. Amino acid composition of the isolated BPF from the snake venom.

Amino acid	(ppm)	Percent %
ASP	62.36	13.7
LYS	52.68	11.6
CYS	50.45	11.1
ARG	48.54	10.7
THR	39.23	8.6
GLU	36.87	8.1
ILE	28.82	6.3
GLY	24.75	5.4
VAL	20.11	4.4
PHE	18.70	4.1
HIS	15.64	3.4
SER	12.97	2.8
PRO	11.31	2.5
ALA	10.87	2.4
MET	7.81	1.6
LEU	7.21	1.60
TYR	7.44	1.60
Total		100%

Aspartic (ASP), lysine (LYS), cysteine (CYS), arginine (ARG), threonine (THR), glutamic (GLU), isoleucine (ILE), glycine (GLY), valine (VAL), phenylalanine (PHE), histidine (HIS), serine (SER), proline (PRO), alanine (ALA), methionine (MET), leucine (LEU), tyrosine (TYR), tryptophane (Trp), hydroxyproline (Hyp), asparagine (Asn) and glutamine (Gln).

Identification of isolated BPF was confirmed by hypotensive effect on rabbit arterial blood pressure as shown in Figure 2.

Figure 2. An oscillograph changes initiated in a rabbit atrium contraction by the isolated BPF as indicator for hypotensive effect.

3.3. Serological characters

The blood cell counts in the tested groups showed that; WBCs, LYM, MONO, EOS and RBCs counts were significantly decreased in the intoxicated rats GII₀ and self-recovery GII₁ but the counts of NEUT was concomitantly increased with respect to the reference normal GI. All of these characters were returned back to be further approached to the reference normal GI in treated rats (BPF GII₂, BHT GII₃ and OPZ GII₄) as shown in Table 2.

Table 2. Blood cell counts in the tested groups.

Parameters	Groups
	GI n = 20	GII0 n = 13	GII1 n = 16	GII2 n = 17	GII3 n = 14	GII4 n = 15
WBC (1 × 10^3/µl)	14.60 ± 0.29	9.90 ± 0.07^a	10.20 ± 0.05^b,c	11.50 ± 0.13^b,c	11.10 ± 0.06^b,c	10.90 ± 0.05^b,c
LYM (10^3/µl)	8.10 ± 2.36	5.80 ± 0.11^a	6.07 ± 0.08^b,c	8.30 ± 0.11^b,c	7.78 ± 0.11^b,c	7.06 ± 0.06^b,c
MONO (10^3/µl)	0.99 ± 0.03	0.56 ± 0.01^a	0.68 ± 0.02^b,c	0.83 ± 0.02^b,c	0.71 ± 0.01^b,c	0.81 ± 0.01^b,c
NEUT (10^3/µl)	1.74 ± 0.03	2.85 ± 0.07^a	2.69 ± 0.03^b,c	1.74 ± 0.09^b,c	2.16 ± 0.02^b,c	2.53 ± 0.03^b,c
EOS (10^3/µl)	1.46 ± 0.02	0.58 ± 0.04^a	0.65 ± 0.01^b,c	0.97 ± 0.1^b,c	0.90 ± 0.06^b,c	0.79 ± 0.02^b,c
RBC (1 × 10^6/µl)	7.94 ± 0.03	6.25 ± 0.03^a	6.48 ± 0.10^b,c	7.20 ± 0.06^b,c	7.19 ± 0.06^b,c	7.54 ± 0.21^b,c

WBCs, White blood cells; LYM, Lymphocytes; MONO, Monocytes; EOS, Eosinophils; RBCs, Red blood cells; NEUT, Neutrophils.

^a Significant of aflatoxicosed GII₀ with respect to reference normal GI.

^b Significant of treated groups GII₁, ₂, ₃, ₄ with respect to reference normal GI.

^c Significant of treated groups GII₁, ₂, ₃, ₄ with respect to aflatoxicosed GII₀.

3.4. Biochemical indices

3.4.1. ALT activity (U/l) in serum of experimental groups

ALT in serum was significantly increased in the intoxicated rats (GII₀) in comparison to the reference normal (GI) and still elevated in the self-recovery (GII₁). However, in treated rats with BPF (GII₂), BHT (GII₃) and OPZ (GII₄), the levels were approximating to those of reference normal (GI) as shown in Table 3.

Table 3. ALT activity (U/l) in serum of experimental groups.

Groups	GI n = 20	GII0 n = 13	GII1 n = 16	GII2 n = 17	GII3 n = 14	GII4 n = 15
ALT (U/l)	15.7 ± 0.08	179.1 ± 4.17^a	142.8 ± 3.09^b,c	88.3 ± 2.18^b,c	77.9 ± 2.09^b,c	80.5 ± 2.15^b,c

Data presented as M ± SEM.

^a Significant of aflatoxicosed (GII₀, GII₀₀) with respect to reference normal GI.

^b Significant of treated groups GII_{1, 2, 3, 4} with respect to reference normal GI.

^c Significant of treated groups GII_{1, 2, 3, 4} with respect to aflatoxicosed GII₀.

3.4.2. Oxidative stress markers in hepatic tissue

NO and MDA in hepatic tissue were significantly increased in the intoxicated rats GII₀ in comparison to the reference normal GI and still elevated in the self-recovery GII₁. However in treated rats with BPF GII₂, BHT GII₃ and OPZ GII₄, the levels were approximating to those of reference normal GI. The chemical reducing agents; total thiols and GSH were significantly reduced by intoxication GII₀ and differentially increased during ageing GII₁ and treated with the three tested therapeutic agents; BPF GII₂, BHT GII₃ and OPZ GII₄. The hepatic tissue enzyme activities of GPx and GST were significantly reduced by intoxication GII₀ and also differentially elevated by self-recovery mechanisms GII₁ and treated by the considered three tested agents (BPF GII₂, BHT GII₃ and OPZ GII₄) as shown in Table 4.

Table 4. Oxidative stress markers in hepatic tissue of the tested groups (Mean ± SEM).

Parameters	Groups
	GI n = 20	GII0 n = 13	GII1 n = 16	GII2 n = 17	GII3 n = 14	GII4 n = 15
NO (nmol/ mg protein)	24.10 ± 0.80	39.40 ± 1.07^a	35.70 ± 0.65^b,c	30.30 ± 0.77^b,c	31.90 ± 0.97^b,c	31.80 ± 0.99^b,c
MDA (nmol/ mg protein)	1.72 ± 0.03	2.59 ± 0.04^a	2.23 ± 0.04^b,c	2.07 ± 0.03^b,c	2.14 ± 0.03^b,c	2.13 ± 0.02^b,c
T.thiols (nmol/mg protein)	63.20 ± 0.73	44.50 ± 0.72^a	49.60 ± 0.41^b,c	54.80 ± 0.68^b,c	54.70 ± 0.42^b,c	56.90 ± 1.77^b,c
GSH (nmol/ mg protein)	8.25 ± 0.25	6.21 ± 0.29^a	6.78 ± 0.20^b,c	7.12 ± 0.24^b,c	7.08 ± 0.20^b,c	7.02 ± 0.11^b,c
GPx (nmol/min/ mg protein)	16.80 ± 0.63	8.82 ± 0.62^a	9.60 ± 1.17^b,c	13.20 ± 0.89^b,c	12.40 ± 1.02^b,c	15.20 ± 0.64^b,c
GST (nmol/ mg protein)	2.19 ± 0.08	1.77 ± 0.13^a	1.87 ± 0.08^b,c	1.93 ± 0.04^b,c	1.92 ± 0.06^b,c	1.89 ± 0.08^b,c

MDA, Lipid peroxidation as malondialdehyde; NO, Nitric oxide; GSH, Glutathione; GPx, activities of glutathione peroxidase; GST, transferase.

^a Significant of aflatoxicosed GII₀ with respect to reference normal GI.

^b Significant of treated groups GII_{1, 2, 3, 4} with respect to reference normal GI.

^c Significant of treated groups GII_{1, 2, 3, 4} with respect to aflatoxicosed GII₀.

3.4.3. Gene expressions of hepatic IL-1β as % fold change:

It is illustrated in Table 5 that aflatoxicosis in GII₀ resulted in a significant reduction in the ability of gene expression for hepatic IL-1β as fold change compared with that of reference normal level GI. Elapsed period for 4-weeks without treatment (self-recovery GII₁) permitted the immunological status of the aflatoxicosed rats to enhance such factor for further increase of T-lymphocytic action. Furthermore, any of the treating agents (BPF GII₂, BHT GII₃ and OPZ GII₄) significantly increased IL-1β gene expression to be more promoted than that of the group of self-recovery GII₁.

Table 5. Gene expression of hepatic IL-1β as fold change parameter.

Parameters as percent %	Groups
	GI	GII0	GII1	GII2	GII3	GII4
Fold change to reference (GI)	99.0 ± 0.5	28.0 ± 1.1^a	50.0 ± 0.5^b,c	65.3 ± 2.6^b,c	61.0 ± 2.0^b,c	65.7 ± 2.3^b,c
Fold change to intoxicated (GII0)	346.0 ± 10.9	99.0 ± 0.5^a	116.0 ± 8.3^b,c	210.0 ± 5.7^b,c	165.7 ± 8.0^b,c	245.0 ± 10.4^b,c

^a Significant of aflatoxicosed GII₀ with respect to reference normal GI.

^b Significant of treated groups GII_{1, 2, 3, 4} with respect to reference normal GI.

^c Significant of treated groups GII_{1, 2, 3, 4} with respect to aflatoxicosed GII₀.

3.5. Histopathological features

xx.

4. Discussion

The obtained results for AFB₁ biosynthesis showed that the fungus of Aspergillus flavus efficiently produced the well-known four metabolites named B₁, B₂, G₁ and G₂ (Figure 1). Identification, isolation and purification of fraction AFB₁ was successfully performed by repeating collection of the chromatographed purified zones of AFB₁ according to El-Kady and Moubasher (1982).

The amino acid composition of the chemically isolated BPF presented in Table 1 was mostly similar to those detected by Fernandez et al. (2004) and Ianzer et al. (2004) who claimed that the structure of some bradykinin potentiating peptides (BPPs) must contain proline that facilitates inhibition of the ACE that prevents formation of angiotensin II (from the parent angiotensin I) initiating vasodilatation and hypotension of exposed animals. The tested hypotensive character of the isolated BPF in this work was practically achieved on rabbit arterial blood pressure as presented in Figure 2. Moreover, evaluation of BPF activity using isolated guinea pig ileum contraction was also effective in eliciting smooth muscle contraction mediated by endogenous BK which is responsible for the predicted hypotensive effect of the BPF. The results that resembling those detected by Ferreira (1965) in his work on Bothrops jararaca as well as that was isolated BPF from the Egyptian scorpion Buthus occitanus as it was published by Abdel-Raheim et al. (1995) and Sharma et al. (1996).

CBC of AFB₁ intoxicated rats (GII₀) and self-recovery (GII₁) groups indicated a significant decrease in white blood cells, lymphocytes, monocytes, eosinophils and erythrocytes, however, neutrophils were increased (Table 2). These results were similar to that published before by Corrier (1991) and Raisuddin et al. (1993) who found that aflatoxicosis is generally associated with immunosuppression in domestic animals, poultry and laboratory animals due to impairment of cellular mediated immunity functions. Moreover, the observed significant increase in neutrophils could be attributed to the elicited inflammatory response to aflatoxicosis (Donmez et al. 2011; Nicola and Jason 2018). Self-recovery group and treated groups by either BPF, BHT and OPZ showed recovery of CBC by increases of all CBC except neutrophils. This improvement in CBC could be returned to promotion of the activity of pro-inflammatory cascade reactions (Takahashi and Hiraga 1984; Cottrell et al. 1994; Petricevich and Pena 2002; Salman et al. 2017).

ALT activity was significantly increased in AFB₁ intoxicated rats (GII₀) in comparison to control one (GI) and still elevated in the self-recovery (GII₁), however, in treated rats with BPF (GII₂), BHT (GII₃) and OPZ (GII₄), the levels were elevated to those of reference normal (GI) and reduced against aflatoxicosis group as in Table 3. These results were similarly agreed before by many authors such as Groseva et al. (2014) and Rotimi et al. (2019) who reported that these changes represent the initial protective response of the mitochondria against the aflatoxin toxicity and the increased activity of these enzymes provided evidence for their release from damaged hepatocytes. In the present study, the reduction of serum ALT activity closed to normal levels after treated with BPF, BHT and OPZ suggested healing of hepatic parenchymal cells and regeneration of hepatocytes (Ashry et al. 2012; Dassarma et al. 2018; Giudice et al. 2019).

The hepatic tissue content of NO as in Table 4 was frankly increased by AFB₁ intoxication (GII₀) as a first responding agent against inflammation that recognizes oxidative damage of the tissue by providing the superoxide anion derived by AFs that accumulate ROS and RNS simultaneously (Massey et al. 1995; Anup and Hartmut 2018). All together of derived free radicals initiated what is determined as MDA as an expressing biomarker for accumulated LPO interpret the significant reduction in reducing chemicals; total thiols and GSH associated with reduced enzymatic actions of GPx and GST that respond against AFB₁ inflammation (Carlberg and Mannervik 1985; Sies 1997; Iciek et al. 2004). In the present study, the treatment from aflatoxicosis with BPF (GII₂) resulted in more improved characters against oxidative stress parameters (Table 4). These results were in a great agreement with those reported by Mikrut et al. (2001) after their trial to treat acute hypoglycemic animals with bradykinin agent. Moreover, Salman et al. (2016) reduced the intoxicating parameters initiated by HgCl₂ in living animals by therapeutic BPF isolated from scorpion venom Buthus occitanus. The antioxidative effect of BPF in both of acute or chronically inflammed animals be beneficially considered as well as promoting physiological activities and cellular tissue regeneration, proliferation and differentiation (Nassar et al. 1990; Nassar 1992; Jovcic et al. 1996; Őztürk 2001; Sancho et al. 2007; Ashry et al. 2012).

The synthetic antioxidants; BHT (GII₃) or OPZ (GII₄) showed powerful antioxidative stress action in treated aflatoxicosed rats (GII₀) (Table 4) due to their probable scavenging properties that stimulated the GSH-linked detoxification mechanisms as it was observed by others (Dimitrov et al. 1992; Klein et al. 2002; Nassar et al. 2014). Moreover, the detected hepato-protective effect of BHT is attributed to its predicted antioxidant activity that normalized the abnormal intracellular events involved in fat absorption and maintaining the integrity of the cell membrane of the liver and prevented accumulation of LPO as it was similarly observed by Barsha et al. (2018) and Dassarma et al. (2018) during their identification for hepato-protective effectors against hepatotoxicity in rats. In addition, the diminished GST activity by aflatoxication was mostly prevented by BHT treatment since reduction of GST activity is proportional to the GSH content in the biological system of the intoxicated animals as it was deduced by Philip and Anders (2007), Marcus et al. (2012) and Sohn et al. (2013). The observed therapeutic effect of OPZ in this study can be referred to its potential enhancing regulatory mechanisms for further genetic production of detoxificating enzymes through activation of the ARE- NrF2 pathway as it was detected by Dimitrov et al. (1992), Velayutham et al. (2005) and Giudice et al. (2019).

The gene expression of hepatic IL-1β (Table 5) was determined by real time PCR as fold change showed considerable reduction in AFB₁ intoxicated rats (GII₀). This reduction was slightly improved in self-recovery rats (GII₁) due to IL-1β could be served as a potent intermediate ligand between tissue injury and the resulting physiological indices of inflammation (Nicola and Jason 2018). In this study, it seems that induced aflatoxicosis suppressed the immune system of the exposed animals by negatively affected T-cell dependent immunity.

Dugyala and Sharma (1996), Batey and Wang (2002) and Miao et al. (2016) claimed that cellular components of the immune system which produce various cytokines play a key role in host resistance against intoxicating inflammatory actions. The tested isolated snake venom peptide BPF (GII₂) (ACE inhibitor) that potentiates the endogenous BK via activation of B₂ receptors; significantly increased the cytokine IL-1β against induced aflatoxicosis (Murakami et al. 1997; Fernandes et al. 2001; Abu-Amra et al. 2015). The authors recorded a profound cell signalling that initiated gene expression for TNFα, phospho-lipase A₂ and other pro-inflammatory immune response by such activated bradykinin. The therapeutic synthetic antioxidants BHT (GII₃) from phenol families and OPZ treated (GII₄) from other thiols significantly elevated the gene expressing affinity for production of IL-1β to activate the immunological respond against inflammatory action of AFs. The antioxidant BHT may enhance regulatory factors that up-regulated the appropriate gene expression of cytochrome-P₄₅₀ families to overcome the oxidative stress initiated by aflatoxicosis as it was announced by Nassar et al. (2014). Moreover, it was suggested by some workers (Ramos et al. 2001; Nisha and Peter 2017; Giudice et al. 2019) that chemoprotective action of OPZ against hepatic tissue injury or carcinogenesis could be predicted by enhancement of phase II genes that express the antioxidant responsive element necrosis factor-2 (ARE-NF2).

Normal liver rats (GI) showing the normal morphological structure of the hepatic lobule, hepatic cells (h), sinusoids (S) and central vein (C) as well as large vesicular nucleus (N), rough endoplasmic reticulum RER (er), mitochondria (m) as in Figure 3. The biochemical changes in intoxicated (GII₀) or self-recovery (GII₁) groups were confirmed by the histological observation in the hepatic tissue (Figure 4) which showed congestion of the portal vein, hyperplasia of the bile duct, increased collagen fibres in the portal area, presence of fat vacuoles and cell nucleus dysplasia. Similar findings were recorded by Preetha et al. (2006) and Devendran and Balasubramanian (2011) who returned these degenerative changes to the increased of oxidative stress and mitochondrial dysfunction by aflatoxin and its metabolites in hepatic tissue. Treatment of intoxicated rats by either BPF (GII_2), (Figure 5)_, BHT (GII₃) or OPZ (GII₄), (Figure 6) prominently reduced most of the previously detected histological observations due to reduction of oxidative stress and enhancement in mitochondrial function (Neal et al. 1976; Anup and Hartmut 2018). In addition, BPF (Figure 5) specifically enhanced a localized appearance of Kupffer cells in the hepatic sinusoids as well as other features of cellular improvements. It is known that Kupffer cells promote the resolution of inflammation and enhancement of hepatic wound healing which BPF acting as acceptable cell signalling peptide (Dixon et al. 2013). In previous work, similar BPFs enhanced cellular proliferation and differentiation of gonads in the intact animals (Nassar et al. 1990; Nassar 1992). In addition, an enriched α-neurotoxic peptide from scorpion with basic arginine in its sequence was assessed as a crucial amino acid residue responsible for its cellular receptor binding affinity (Kharrat et al. 1990). Moreover, the rich cysteine residues in the isolated BPF from venom of Bothrops jararaca initiate its physiological activity on cellular B₂ receptors of guinea pig ileum (Ferreira 1965). In this study, the most abundant content of amino acid residues of lysine and cysteine in the sequence of the tested BPF may considered as efficient condition for its probable cell signalling effect that was predicted by some workers (Omar and Meki 1997; El-Saadani 2004; Padrissa et al. 2009). Similarly, phenolic BHT (GII₃) or thiolic one OPZ (GII₄) (Figure 6) improved liver damage as direct antioxidant reagents. Sael and Pablo (2015) announced that BHT plays a role in injured hepatic tissue repair that could be revealed by antioxidants as well as workers like Liping et al. (2019) who reported that OPZ can potentiate activated collagenases that hydrolyze accumulated fibres in injured liver.

Figure 3. (a) Light micrograph of semi thin section of liver of normal rats (GI) showing the normal morphological structure of the hepatic lobule, hepatic cells (h), sinusoids (S) and central vein (C). T.B. stain. (b) T.E. micrograph of hepatic cell of normal rats (GI) showing large vesicular nucleus (N), rough endoplasmic reticulum RER (er), mitochondria (m).

Figure 4. (a) Light micrograph of semi thin section of liver of intoxicated rats with AFB₁ (GII₀) or that for self-recovery (GII₁) showing bile duct hyperplasia (b), congestion of the portal vein (v) and increases of collagen fibres in the portal area (x). T.B. Stain (The same histopathological features in GII₀ and GII₁). (b) T.E. micrograph of liver of intoxicated rats with AFB₁ (GII₀) or that for self-recovery (GII₁) showing increases of collagen fibre in the portal area (co), dysplasia of the hepatic cell nucleus (N) and presence of fat vacuoles in the hepatic cells (f) (The same histopathological features in GII₀ and GII₁).

Figure 5. (a) Light micrograph of semi thin section of liver of rats intoxicated with AFB₁ for one month then treated with BPF for one month (GII₂) showing the hepatic cells arranged in plats contain fat globuls with activation of Kupffer cells (arrow). T.B. Stain. (b) T.E. micrograph of hepatic cells of rats intoxicated with AFB₁ for one month then treated with BPF for one month (GII₂) showing presence of variable size fat globules (f) and large vesicular nucleus (N). (c) T.E. micrograph of hepatic cells of rats intoxicated with AFB₁ for one month then treated with BPF for one month (GII₂) showing presence of Kupffer cells (K) and collagen fibre (co) in Dise^,s space and RBC^,s in the sinusoid (S).

Figure 6. (a) Light micrograph of semi thin section of liver of rats intoxicated with AFB₁ then treated with BHT (GII₃) or OPZ (GII₄) for one month showing hepatic lobules are nearly of normal structures of central vein (CV) and hepatic cells (H) (The same histopathological features in GII₃ and GII₄). (b) T.E. micrograph of liver of rats intoxicated with AFB₁ then treated with BHT (GII₃) or OPZ (GII₄) for one month showing presence of collagen bundles (co) in the wall of the central vein (C) and the hepatic cells having vesicular nucleus (N) (The same histopathological features in GII₃ and GII₄).

5. Conclusion

Conclusively, any of the three tested components for injured hepatic treatment from induced aflatoxicosis; BPF from the cobra snake venom and the synthetic antioxidant animal food additives; BHT and OPZ each of which is a valuable therapeutic agent for hepatic tissue repair. Although, the metabolic pathway of BHT in rats and humans can depress growth and may cause lung damage or inflammation and bleeding but it is not classified as carcinogenic for humans. Concomitantly, hepatic metabolic derivatives of OPZ such as pyrrolo-pyrazine structures can induce microRNAs that affect gene transcription of hypoxia inducible factor (HIF-1α) in human colon cancer treatment as well as GST expression. OPZ can be stimulant for GST production as cancer chemopreventive agent. The chemically isolated BPF as a nontoxic and beneficial pharmacologically active peptide isolated from snake venom consisted less than twenty different amino acid residues that could be easily degraded by peptidases in exposed animal when it is used as a therapeutic agent against aflatoxicosis.

No conflict between the authors; the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Disclosure statement

No conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by Assiut University.