Whey Protein and Nigella sativa Oil Mitigate Potassium Dichromate Induced Hepatic Injury, Oxidative Stress and Hematotoxicity in Rats

Department of Pharmacology, Medical Division, National Research Centre, 33 EL Bohouth st. (former EL Tahrir st.), Dokki, Giza, Egypt; bashandys@hotmail.com Biophysics Group, Department of Biochemistry, Genetic Engineering and Biotechnology Division, National Research Centre, 33 EL Bohouth st. (fFormer EL Tahrir st), Dokki, Giza, Egypt Department of Pathology, Medical Division, National Research Centre, 33 EL Bohouth st. (Former EL Tahrir st), Dokki, Giza, Egypt


Introduction
Human beings are subjected to a number of diverse chemicals that harm the liver. The liver has a noteable role in the metabolism of xenobiotics let this organ particularly liable to injury by chemicals to which we are exposed. The pathogenesis of most chemical-induced liver injuries is commenced by the metabolic transformation of chemicals into reactive intermediate species, such as free radicals, that can probably modify the structure and function of cellular macromolecules. Many reactive intermediate species can produce oxidative stress 1 .
One of these hazardous hepatotoxic complexes is potassium dichromate, it is a powerful reacting mediator exhibiting a noticeable attraction, when converted to trivalent chromium (Cr +3 ) through several cell reactions, to form a number of compounds with varied organic roots, together with nucleic acids 2 . Similarly, chromium triggered the production of Reactive Oxygen Species (ROS) which generate various poisonous properties, as well as DNA damage and phospholipid peroxidation that triggering hepatotoxicity 3 .
Whey protein is a mixture derived from milk, containing lactoferrin, beta-lactoglobulin, alpha-lactalbumin, and glycomacropeptide, reveals a wide range of immune-enhancing mechanisms besides the facility to play as a powerful antioxidant and scavenging agent for free radicals 4 . Also, whey protein is containing a great proportion of cysteine and methionine, which improve immune system via intracellular conversion to glutathione. In addition, Lactoferrin, plays as an iron-chelating glycoprotein, acting a major character as an antioxidant 4 . Nigella sativa kernels include 36-38% stable oils, alkaloids, proteins and (0.5-2.6%) volatile oil. Investigational literatures have verified that Nigella sativa concentrate has a variety of medicinal properties used in treatment of hypertension, type 2 diabetes, immuneregulative and liver defense. Nigella sativa can afford important improvement of the hepatotoxic consequences of rats linked to its reactive oxygen species hunting and antioxidant effects 5 .
Since whey protein and the Nigella sativa oil were appeared selected as essential supplements, the aim of our study is the evaluation of their proficiency to repairing liver injury and alleviating hematotoxicity accompanying potassium dichromate complications.

Drugs and Chemicals
Whey protein (WP) was brought out from Davisco Food International Company, USA, whereas Nigella sativa oil (NSO) was purchased from Mepaco, Cairo, Egypt, and potassium dichromate (K 2 Cr 2 O 7 )was bought from Sigma Aldrich, USA. Whey protein was administered orally, which suspended in distilled water.

Animals
Adult male Wistar albino rats weighing 130-140 g were obtained from the National Research Centre Laboratory (Dokki, Giza, Egypt) and were accommodated in standard polypropylene cages and kept under constant environmental conditions with equal light-dark cycles. Rats were adapted for 1 week and were served rat normal fat pellet diet and water ad libitum.

Ethics Statement
This experiment was carried out in according to recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH publication No. 85-23, revised 1996) and under regulations of Animal Care and Use of National Research Centre in Egypt. All surgery was performed under deep sodium pentobarbital anesthesia and all efforts were made to minimize suffering.

Induction of Hepatotoxicity using Potassium Dichromate and Experimental Design
70 rats were separated into 7 equal groups (Each group contained 10 rats) as follows: Potassium dichromate and whey protein were dissolved in water. The samples were collected 24 hours post potassium dichromate injection.

Multi-Component Spectrophotometric Method for the Simultaneous Determination of Four Hemoglobin Derivative Concentrations
Concentrations of Sulfhemoglobin (SHb), Methemoglobin (MetHb), Carboxyhemoglobin (HbCO) and Oxyhemoglobin (HbO 2 ) were measured by the multi-component method developed in our laboratory with some modifications. The hemolysate was prepared as described in this method. For absorbance measurements, about 30 μl of the purified hemolysate to 5 mL of temperature equilibrated (25°C) phosphate buffer (Na 2 HPO 4 27.50 mmol/L and KH 2 PO 4 13.16 mmol/L, pH 7.28) containing 0.4% Triton-X100. This technique has been applied in the simultaneous determination of SHb, MetHb, HbCO and the remaining functional Hb in the OxyHb form, with a conventional spectrophotometer. In this method, the measurements for the latter prepared extremely diluted Hb solutions were made, at four wavelengths (λ = 500, 569, 577 and 620 nm 9 ).

Plasma Collection for Analysis
Blood was collected in heparinized tubes from the retroorbital plexus of veins under brief sodium pentobarbital anesthesia and was centrifuged (700×g, 4°C, 15 min) to separate the plasma. Colorimetric kits were bought from Salucea Company, Netherlands to determine serum AST, ALT, GGT, ALP, and glucose. ELISA kits were purchased for the assessment of serum T3, T4, and TSH from R&D Systems, USA.

Liver Tissue Extract
After blood collection, rats were decapitated under a deep sodium pentobarbital anesthesia. Rats' liver was separated out, washed, weighed and homogenized in Phosphate Buffer Solution [PBS] [10%]. Tissue homogenate was centrifuged at 1500×g at 4C° for 20 minutes and the supernatant was collected and stored at -80C° for the direct assessment of parameters.

Histopathological Examination of the Liver
Liver samples from all groups were fixed in 10% formol saline, embedded in paraffin, and dehydrated in ascending concentrations of ethyl alcohol (70-100%). Subsequently, 5u m tissue sections were cut, mounted on slides, stained with hematoxylin and eosin (H&E) for liver histopathology.

Statistical Analysis
Values were stated as mean ± S.E. of 8-10 rats and the variances between groups were tested for significance using Analysis of Variance (ANOVA), followed by Tukey-Kramer posthoc test estimated by SPSS software, version 21.The level of statistical significance was at P<0.05.

Liver Function Tests
In our work, next the poisoning of animals with potassium dichromate, animals displayed a condition of hepatotoxicity which was established by a substantial up-shot of plasma AST, ALT, GGT, and ALP levels in relation with normal group. In the meantime, hepatotoxic groups treated with either both doses of whey proteinand/ orNigella sativa oil demonstrated an obvious enhancement in these factors in comparison with an control hepatotoxic group (Table 1).

Complete Blood Count and the Percentage of Hemoglobin Derivatives Parameters
Also, next to intoxication of rats with potassium dichromate, animals showed a significant alteration in complete blood count [except monocytes, MCV, MCH, and MCHC (%)] and an increase in the percentage of hemoglobin derivatives [except HbO 2 (%)] compared with normal control group. Unexpectedly, all treated groups with whey protein or Nigella sativa oil + K 2 Cr 2 O 7 revealed a notable improvement in most of these aforementioned parameters in comparison with K 2 Cr 2 O 7 group. (Table 2 and 3). Table 4 indicated important alterations in the antioxidant defense system of K 2 Cr 2 O 7 group as mirrored on the decrease of CAT, GSH and SOD with increase of MDA and NO levels compared with the normal control group. Values are means ± S.E of 6-10 animals. As compared with control (*), K 2 Cr 2 O 7 (#) groups, and the differences between groups were confirmed for significance using analysis of variance (ANOVA), followed by Tukey-Kramer posthoc test, at P<0.05.

Hormonal Parameters
Potassium dichromate group manifested a significant remarked decline in serum T3, and T4 levels with a subsequent elevation in plasma glucose, and TSH levels in comparison with the normal group as presented in Table 5. The rats treated with whey protein or Nigella sativa oil + K 2 Cr 2 O 7 revealed a substantial improvement in these aforementioned markers level compared with the K 2 Cr 2 O 7 group, nevertheless the result of high dose of whey protein plus Nigella sativa oil was extra pronounced and obvious than other treated intoxicated rats.

Histopathological Examination
Microscopic picture of the liver of control rat showed the normal architecture of liver, comprising several hepatic lobules, each hepatic lobule formed of radially organized cords of liver cells from central vein to the margin of lobule separated by blood sinusoids (Figure 1a). The section of liver treated with potassium dichromate at a dose of 30 mg/Kg revealed severe liver damage, including marked cytoplasmic vacuolar degeneration, Values are means ± S.E of 6-10 animals. As compared with control (*), K 2 Cr 2 O 7 (#) groups, and the differences between groups were confirmed for significance using analysis of variance (ANOVA), followed by Tukey-Kramer posthoc test, at P<0.05. Values are means ± S.E of 6-10 animals. As compared with control (*), K 2 Cr 2 O 7 (#) groups, and the differences between groups were confirmed for significance using analysis of variance (ANOVA), followed by Tukey-Kramer posthoc test, at P<0.05.
shrunken nuclei, sings of nuclear degeneration in the form of necrosis, pyknosis, and karyolysis as well as distortion of hepatic cells (Figure 1b). Furthermore, a minute of vacuolar degeneration in the bile duct around the dilated portal tract (Figure 1c). Animal treated with potassium dichromate plus a low dose of whey protein showed regular hepatic cords, the majority of hepatocytes around central vein appear healthy, however, slight dilation in central vein and blood sinusoids, sings of vacuolar degeneration as well as necrotic, pyknotic and karyolitic nuclei could be observed (Figure 1d).
On the other hand, the liver of the rats subjected to K 2 Cr 2 O 7 plus a high dose of whey protein showed few vacuolar and fatty degeneration around the portal area infiltrated by inflammatory cells, besides, sings of nuclear degeneration as necrosis, karyolysis, and karyorrhexis ( Figure 1e).
The histological picture of the liver of rats treated with potassium dichromate and Nigella sativa oil exhibited a normal architectural pattern, most of the hepatocytes appeared healthy expect some of the hepatocytes around peripheral zone appeared vacuolated (Figure 1f).

Discussion
Potassium dichromate is a formula of chromium and it has stayed used in the induction of hepaticinjury [10][11][12][13] . And it is described that acute contact encourageshepatic structural alterationin the hepatocytes and phospholipid oxidation in the liver 13 . Chromium produced metabolites were supposed to be induced by H 2 O 2 to form OHradicals 14 , with successive changes in amino acids, DNA, and lipids structure principal to changing cellular activities and itsstructure 14 .
For that reason, rats were challenged with Potassium dichromate showed a subsequent increase in the serum AST, ALT, GGT, and ALP levels with highly changes in liver histopathological investigation compared with normal group, via increase of Reactive Oxygen Species (ROS) that triggers liver tissue injury 15 . ROS produced through this way can cause damage to tissue amino acids, phospholipids, and DNA principal to oxidative stress 16 .
In the same way, the significant elevation in MDA and NO levels and the significant decline of GSH, SOD and CAT levels were are presentative markers for the elevated free radicals due to the stimulation of Inducible Nitric Oxide Synthase (iNOS), principal to excessive generation of NO and production of toxic peroxy-nitrite that indicated the overproduction of ROS 13,17 .
The elevation of plasma activity of ALT, AST, and ALP is indicative of hepatocellular damage since the disruption of the plasma membrane leak intracellular enzymes into the bloodstream 18 .
The increase in oxidative stress induced by potassium dichromate can explain hepatotoxicity and the alterations of CBC, the percentage of hemoglobin derivatives, glucose, and thyroid hormone levels. It was reported that chromium might elevate ROS production, trigger the Akt, NF-kB, and MAPK mechanisms alongside the increase Values are means ± S.E of 6-10 animals. As compared with control (*), K 2 Cr 2 O 7 (#) groups, and the differences between groups were confirmed for significance using Analysis of Variance (ANOVA), followed by Tukey-Kramer posthoc test, at P<0.05.  19,20 . Our results showed that potassium dichromate produced a toxic effect on hematological parameters such as total erythrocyte count, total leucocyte count, and hemoglobin value. Potassium dichromate altered the erythropoiet in factors signifying anemia as evidenced by the decrease in the count of erythrocytes and hemoglobin concentration. The decrease in hemoglobin concentrations can be attributed to structural alteration of heme or to the resistance of the enzyme mechanism convoluted in the synthesis of hemoglobin, as proposed previously with other heavy metals 21 .
It was reported that the ability of why protein to induce the expression of the enzymes associated with GSH synthesis might represent an important mechanism for the protective effect of whey protein 22 . Glutathione protects cells against exogenous and endogenous toxins, including reactive oxygen species and reactive nitrogen species 23,24 .
Also, whey protein complex showed a substantial antiinflammatory and antioxidant properties via elevating hepatic SOD, GSH, CAT and declining MDA, and NO through its ROS chelating effects which is evidenced from the improvement of liver biomarkers (AST, ALT, GGT, and ALP) 25 , CBC, the percentage of hemoglobin derivatives, glucose, and thyroid hormone levels.
Nigella sativa oil revealed an important enhancement in entirely affected markers since it has antioxidant scavenging activity 26 . The advantageous effects of this oil influence are to be linked to their cytoprotective and antioxidant activities, by their influence on inflammatory markers. Nigella sativa oil exhibited a hepatoprotective effect against carbontetrachloride 27 .
In our investigation, the rats given Nigella sativa oil and whey protein in combination indicated the best significant outcome compared to corresponding separared groups.

Conclusion
Our results reveal that protection with whey protein and/ or Nigella sativa Oil exhibited possible anti-oxidant and anti-inflammatory effects in albino rats, which they were capable to decrease chromium triggered hepatotoxicity and hematotoxicity. Nevertheless, the particular and full mechanistic effect of whey protein and Nigella sativa oil is not perfect in former literatures, so, we focused to spotlight on their action via diverse investigations to be added further explored in the forthcoming studies.