Selenium nanoparticles (SeNPs) have been widely used as anti-inflammatory and anti-toxic agent. The present study used Bacillus tequilensis for biosynthesizing SeNPs from sodium selenite (Na 2 SeO 3 ) and investigated its ameliorative effects on acetaminophen (APAP) hepatotoxicity in male mice. The results indicated that Alanine transaminase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) significantly elevated in mice treated with APAP, while other liver markers (total proteins and albumin) did not change. SeNPs either alone or in combination with APAP showed ameliorative effects on liver enzymes to some extents where their activities decreased to be insignificant with those of normal group. A slight variation was shown in total antioxidant capacity (TAC). Histopathologically, the hepatocytes of the mice treated with APAP showed cloudy swelling and vacuolar degeneration, while those treated with SeNPs or both SeNPs and APAP appeared more or less histologically normal. In conclusion, SeNPs can be used to improve or replace today’s therapies of APAP hepatotoxicity.
Selenium nanoparticles (SeNPs) have been produced by many bacterial species like Pantoea agglomerans, Zooglea ramigera, Pseudomonas alcaliphila, Bacillus subtilis, Bacillus cereus and Duganella sp. [ 1 - 3 ]. Selenium respiring bacteria Sulfurospirillum barnesii, Bacillus selenitireducens and Selenihalanaerobacter shriftii also produced SeNPs in anaerobic conditions [ 4 ]. However, production in aerobic condition is much easier than in the anaerobic condition. Anaerobic biogenesis is a tiresome and difficult process [ 1 ].
Acetaminophen (APAP) is well-known drug inducing severe oxidative stress allowing mitochondrial permeability transition in liver cells. Liver cells eliminate free radicals and face apoptosis as a result of the drug hepatotoxicity [ 5 ]. Several studies tackled the hepatotoxic effect of APAP in male and female mice and rat [ 6 - 13 ].
In male albino rats, SeNPs have been used as an antioxidant against the hepatic damage caused by parasitism [ 14 ] and neural and renal APAP toxicity. SeNPs also showed anti-inflammatory and analgesic effects in irradiated rats [ 15 ]. It is therefore, necessary to investigate its ameliorative role against APAP hepatotoxicity in male mice.
Twenty male mice weighed 30 to 40 g were primarily divided into 4 groups (5 individuals per group). The animals were put in plastic cages, fed normally and left for acclimatization for 10 days in good ventilated room at Taif University of Western Saudi Arabia. The control mice administrated tap water. The second received APAP dissolved in water with the human therapeutic dose (75 mg/kg body weight). The third group was taken SeNPs (0.001 mol of 50 nm SeNPs dispersed in liter ddH2O) while the fourth group was subjected to a mixture of APAP and SeNPs. After 21 days administration, blood samples were obtained from the anesthetized animals while liver tissues were taken immediately after dissection. Liver enzymes; ALT, AST and ALP and the antioxidants super oxide dismutase (SOD), total antioxidant capacity (TAC) and malondialdehyde (MDA) were measured by kits prepared by authors cited in Dakrory et al. [ 16 ].
SeNPs were prepared by reducing selenium ions with Bacillus tequilensi. Bacterial strain was grown up in 100 ml nutrient broth incubated at 37˚C. After 18 hrs, the incubated culture was centrifuged at 7000 rpm for 10 min and washed three times. It was dispersed in 10 ml ddH2O at pH 7.0. Addition of sodium selenite (Na2SeO3) was undertaken in order to make Selenium concentration to be 1 mM. Further incubation was happened for 24 hrs. Observation of the biomass turning red was a marker of convenient formation of SeNPs [ 17 ]. Centrifugation at the same conditions followed by washing was conducted to collect the bacterial biomass. The precipitate was resuspended in 5 mL Phosphate Buffer at pH 7.4 followed by ultrasonication (130 W, 10 min, Vibracell VCX-130; Sonics and Materials Inc., CT, USA). The suspension was filtered with 0.45 mm and 0.2 mm pore size cellulose acetate filters (Advantec, Tokyo, Japan). Again, centrifugation of the filtrates was done at 10,000 rpm, 4˚C for 30 min and the pellet was suspended in 5 mL of ultrapure water for further study. Characterization of the formed particles was conducted [ 3 , 18 ]. The produced SeNPs were described by UV-Vis spectroscopy (Perkin Elmer, Lambda 25) with scanning range of 200 - 900 nm at 1nm resolution.
After centrifugation, a drop of the synthesized SeNPs was placed on the carbon coated copper grids and kept under vacuum desiccation overnight before loading onto a specimen holder. Size, morphology and composition were recorded by TEM operated at 120 k accelerating voltage (JTEM 1230, Japan, JEOL) with selected area electron diffraction. XRD analysis was conducted by an automated diffract meter (Philips type: Pw1840) at 0.02 step size, 20 in 2θ/min scanning rate and a 2θ range from 100 to 700. Powder patterns indexing and unit cell parameters least squares fitting using the software X’Pert High score Plus was undertaken.
For the histological preparations of liver tissues, fixation, preservation, embedding, dehydration, deparaffinization, rehydration was conducted as described by Amer et al. [ 19 ]. H&E staining was undertaken according to protocols of Bancroft et al. [ 20 ]. Photomicrographing [ 19 ] was also done.
The results were statistically manipulated by ANOVA packaged in SPSS version 11.0. LSD was conducted for the paired comparisons.
Observation of the culture incubated with (Na2SeO3) showed a change from yellow to red (
The biosynthesized SeNPs shown in TEM image were spherical and polydispersed with average diameter range of 10 - 55 nm (
Bacillus sp. MSh-1 produced spherical SeNPs with 80 nm diameter [ 3 , 18 ]. We consider this study the first that reported the formation of small SeNPs by Bacillus tequilensis. The XRD analysis for the biosynthesized selenium indicated three intense peaks in the whole spectrum of 2q values ranging from 5 to 80 (
As AL-Harabi et al. [ 22 ] concluded, liver was the main tissues clearly influenced significantly by nonoparticles treatment. ALP, AST and ALT were increased significantly (p < 0.01) in the group received APAP indicating the toxic effect of the drug. Several investigations accumulated similar findings [ 6 - 13 ].
The enzymes activity in the groups treated with SeNPs immediately after APAP supplementation or in combination with APAP showed normal enzyme activity with no significant difference to that of the control (
Liver histology of the control group appeared more or less normal (
Variable | C | APAP | SeNPs | APAP + SeNPs | f-value |
---|---|---|---|---|---|
ALP (U/L) | 23.03 ± 3.2 | 36.06 ± 3.3, ++, •• | 26.3 ± 1.2 | 17.8 ± 2.2 | 7.3** |
AST (U/L) | 86.1 ± 16.3 | 226.4 ± 8.9, ++, •• | 123.9 ± 27.2 | 84.9 ± 3.5 | 14.5*** |
ALT (U/L) | 75.6 ± 15.9 | 153.3 ± 6.9, ++, ¥ | 87.7 ± 11.2 | 117.1 ± 9.4$$ | 14.4*** |
Albumin (g/dl) | 4.1 ± 0.5 | 4.2 ± 0.1 | 3.5 ± 0.4 | 4 ± 0.1 | 0.74 |
Protein (g/dl) | 11.7 ± 1.4 | 10.2 ± 1.2 | 11.5 ± 0.7 | 10.4 ± 0.2 | 0.48 |
MDA (nmol/g) | 1289.5 ± 164.2 | 1965.8 ± 149.3 | 1658.8 ± 120.7 | 2229 ± 432♥ | 2.9 |
TAC (mM/g) | 1.5 ± 0.08 | 1.4 ± 0.05 | 1.6 ± 0.0‡ | 1.6 ± 0.01 | 3.6* |
SOD (U/g) | 1904.7 ± 47.6 | 7642.8 ± 829.7 | 6499.9 ± 4647.2 | 4071.5 ± 928.5 | 0.47 |
into hepatic lobules in which the central vein was centrally located and surrounded by hepatic columns. The hepatocytes appeared polyhydral with centrally located vesicular nuclei. Hepatic sinusoids were seen between the hepatic columns. Van kupffer cells were frequently seen. The hepatocytes of the mice treated with APAP had cloudy swelling and vacuolar degeneration (
kupffer cells. The liver of mice treated with SeNPs appeared more or less histologically normal (
In conclusion, SeNPs can be used to replace today’s therapies of APAP hepatotoxicity as the liver enzymes become normal after treatment with the synthesized nanoparticles. Meanwhile, liver histology showed an improvement by SeNPs administration.