AZADIRACHTA INDICA OIL AS PROTECTOR OF CHRONIC OCHRATOXIN- INDUCED NEPHROTOXICITY

Ochratoxin A (OTA) mycotoxin affects protein synthesis, metabolic oxidative pathways and ROS mediation. In the present study, we investigate the antioxidative-therapeutic potential of Azadirachta Indica Oil (A. indica) against chronic (28 days) OTA-induced nephrotoxicity. Balb/c male mice were exposed to i.e., (1) controls; (2) A. indica treated (120 mg/kg b. wt. i.p., given every two days); (3) c) OTA treated (Isolate D2306, 1.25 mg/kg b.wt., i.p., given every two days); d) A. indica (120 mg/kg b.wt., i.p.) administered 2h prior to OTA-administration. Till the end of the 28 experimental days of chronic OTA-nephrotoxicity, the mortality rate (±0) was not observed in mice. Kidney tissue was subjected for the determination of biochemical indexes (the MDA ratio, and antioxidant capability of SOD, and GSH) and EPRROS productions. OTA-exposure resulted in twofold significant increases in SOD (p<0.004), GSH (p<0.05) and malondialdehyde (MDA) levels (p<0.00), compared to controls. Further, a nephro-cells experiment was performed to investigate the oxidative stress-protective action of A. indica. Our results showed that A. indica oil and A. indica +OTA combination inhibited OTAinduced nephrotoxicity via a significant reduction of lipid peroxidation (p<0.003), ROS production (p<0.005), and endogenous antioxidant activation. Thus, it can be concluded that A. indica treatment neutralized chronic OTA-induced nephrotoxicity, not only by reducing lipid peroxidation but also by improving antioxidant status. Through the present experiments, it was demonstrated that A. indica has protective potential in nephron-inflammations. UDC Classification: 604, DOI: https://doi.org/10.12955/pmp.v2.176


Introduction
Ochratoxins are cyclic pentaketides: dihydroisocoumarin derivatives linked to the L-phenylalanine moiety. Ochratoxin A (OTA) (R) -N -[(5-chloro-3,4-dihydro-8hydroxy-3-methyl-1-oxo-1H-2benzopyran-7-yl] carbonyl) -L-phenylalanine is a mycotoxin, contaminating cereals, legumes and dried fruits. OTA was found in an isolate of Aspergillus ochraceus, and since then several species of Aspergillus and Penicillium have been described as producers of this mycotoxin (Petrik et al., 2003). Ochratoxin A (OTA) is a ubiquitous nephrotoxic and carcinogenic mycotoxin that is thought to be involved in the etiology of Balkan endemic nephropathy (BEN). Several studies reported that BEN correlates with a very high incidence of otherwise rare urothelial tumors of the renal pelvis and ureters (Atroshi et al., 2000). OTA is found more often and/or in higher concentrations in the food and blood of residents of BEN areas than in other regions. The involvement of OTA in the development of BEN is not yet very well understood. Patients with chronic renal failure treated with dialysis have higher concentrations of OTA in their blood than healthy individuals. Moreover, no decrease in OTA concentration was observed as a result of the treatment (Fuchs & Peraica, 2005). BEN occurs without an acute phase and is accompanied by anemia of the aplastic or normochromic type and mild proteinuria without hypertension and edema. In blood samples there is a gradual increase in nitrogen and creatinine levels and in the advanced stages of the disease hypertension is reported (Varga et al., 2010). Histopathological changes in the early stages of the disease report degenerative and regenerative processes of the epithelial cells of the proximal tubules in the kidneys without a change in organ size. An increased amount of light eosinophilic, cell-free material is seen throughout the interstitium of the cortex. There are no signs of inflammation and glomerular lesions. In chronic cases, the size of the kidneys is significantly reduced and diffuse cortical fibrosis without inflammatory cells is the most important finding. BEN is more common in women than in men (Fuchs & Peraica, 2005). In recent years, the use of herbal preparations and active ingredients of medicinal plants in healthcare is becoming more common. Azadirachta indica (A. indica, Neem) is one of the most widely used plant extracts. A tonic is prepared from its bark, which is used as a medicine against various skin diseases (Silva et al., 2015). In addition, the bark of A. indica exhibits antitumor and interferon activity, and other parts of the plant have antibacterial, antifungal, antimalarial and anticancer effects. Neem oil has anti-fertility activity and stimulates a cell mediated immune response (Ezz-Din et al., 2011). In Asian countries, Azadirachta indica (A. Juss. Neem, Meliaceae) is used as a fertilizer and to control pests in cereals. In fact, in many regions, this fertilizer is a residue from the pressing of Neem seeds in oil extraction, which is used as a botanical insecticide and provides toxicity to maize caterpillars. In most pest control studies, the use of neem seed oil is for cereal protection purposes (Abireh et al., 2020). In the present study we investigated the antioxidant-therapeutic potential of Azadirachta Indica Oil (A. indica) in kidney tissue against chronic (28 days) OTA-induced nephrotoxicity. Kidney tissue was subjected to biochemistry analyses with ELISA methods to assay the Malondialdehyde (MDA) ratio, and antioxidant capability of Superoxide dismutase (SOD), and Glutathione (GSH). All biochemical results were compared to the levels of reactive oxygen species (ROS) productions measured with Electron Paramagnetic Resonance (EPR) spectroscopy.

Animals
Experiments were conducted in male Balb/c mice (30-45 g; n = 6 of each), maintained in the animal care facility at the Experimental Laboratory of the Medical Faculty, Trakia University, Stara Zagora, Bulgaria. The animals were fed a normal chow diet and water ad libitum and housed under standard conditions with a controlled temperature (~22 °C) and humidity (~60%) and were exposed to a 12/12h light/dark cycle. All biological assays were performed in accordance with the guidelines for the care and handling of laboratory animals as recommended by the Ethical Committee for Animals. All protocols were previously approved by the Directive 2010/63/EU/Ethical Committee for Animals of BABH and Trakia University, Stara Zagora, Bulgaria (131/6000-0333/09.12.2016). Mice were randomly divided into four groups as follows: (1) controls; (2) A. indica treated (120 mg/kg b.wt, i.p., given every two days); (3) OTA treated (Isolate D2306, 1.25 mg/kg b.wt, i.p., given every two days); (4) A. indica (120 mg/kg b.wt, i.p.) administered 2h prior to OTA-administration. Till the end of 28 experimental days of chronic OTA-nephrotoxicity, the mortality rate (±0) was not observed in mice.

Biochemistry analyses
At the end of experiment (the 28 th day), the mice were anesthetized with Nembutal (50 mg/kg i.p.) and sacrificed. The freshly isolated (un-extravasation with cold 0.9% saline) kidney tissue of all six animals from each group were collected in ice. After homogenization and the addition of solvents, the samples were centrifuged in 4000 rpm at 4°C for 10 min and 300µl of supernatant were stored at -40°C until further assay was done. Immuno-enzymatic methods Standard ELISA kits were used to determine the levels of the antioxidant enzymes: Superoxide Dismutase (SOD), and Reduced Glutathione Peroxidase (GSH-Px). The standard ELISA kit was used for Malondialdehyde (MDA) measurements. All enzyme-linked immunosorbent assays were performed according to the procedure described in the respective kit. Electron paramagnetic resonance (EPR) spectroscopy and ROS evaluation in kidney tissue EPR spectral processing was performed using Bruker Win-EPR and Sim-fonia Software. The ROS level measurement was according to Shi et al. (2005). The real-time formation of ROS in the serum was investigated by mixing the samples with PBN spin trapping. EPR measurements of all tested samples were conducted at room temperature (18-23⁰C) on an X-band EMX micro , spectrometer (Bruker, Germany), equipped with a standard Resonator. Quartz capillaries were used as sample tubes.

NO⋅ evaluation in kidney tissue
Based on the methods of Yoshioka et al, (1996) and Yokoyama et al. (2004) we developed and adapted the EPR method for the evaluation the NO radical (NO⋅) levels. The EPR spectrum of the spin adducts formed between the spin trap Carboxy. PTIO and generated NO⋅ was recorded. NO⋅ levels were calculated as double integrated plots of EPR spectra and the results were expressed in arbitrary units. The EPR settings were: 3505 G centerfield, 6.42 mW microwave power, 5 G modulation amplitude, 75 G sweep width, 2.5 x 10 2 gain, 40.96 ms time constant, 60.42 s sweep time, 1 scan per sample.

Asc ⋅ evaluation in kidney tissue
Ascorbate radical (Asc⋅) levels were studied according to Bailey (2004), with some modification. EPR settings were as follows: 3505.00 G center field, 20.00 mW microwave power, 1.00 G modulation amplitude, 15 G sweep width, 1x10 5 gain, 40.96 ms time constant, 60.42 s sweep time, 10 scans per sample.

Statistical Analysis
Statistical analysis was performed using Statistic 8.0 (Stasoft, Inc.), one-way ANOVA Student-Ttests to determine significant difference among data groups. The results were expressed as means ± standard error (SE). A value of p< 0.05 was considered statistically significant.

Results
In the present study we investigated the antioxidant-therapeutic potential of Azadirachta Indica Oil (A. indica) in kidney tissue against chronic (28 days) OTA-induced nephrotoxicity. The results were compared with mice treated with OTA alone and with combination A. indica + OTA. Based on literature data (Rodrigues et al., 2019), we selected the 120 mg/kg body weight dose as the "protector" dose for Azadirachta Indica Oil (A. indica). Figure 1 shows the level of ROS, Asc radicals and NO radicals in healthy untreated mice and mice treated with OTA, A. indica and combinations of A. indica and OTA. Figure 1: The levels of ROS, Asc⋅, and NO⋅ Source: Author The ROS level (Figure 1) in kidney tissue of mice treated with OTA were statistically significantly higher compare to the healthy controls (mean 3.38 ± 0.4 vs mean 1.36 ± 0.1, t-test, p=0.000) and the group treated with A. indica (mean 3.38 ± 0.4 vs mean 1.49 ± 0.08, t-test, p=0.000). The combination of A. indica + OTA showed a statistically significant decrease compared to the OTA group (mean 2.00±0.07 vs mean 3.38 ± 0.4, t-test, p=0.1). The results from the group treated with combination A. indica + OTA was higher, but not statistically compare to the controls (mean 2.00 ± 0.07 vs mean 1.36 ± 0.1, t-test, p=0.000), and A. indica group (mean 2.00 ± 0.07 vs mean 1.49 ± 0.08, t-test, p=0.000). Figure 1 presents a statistically significant increase in the NO levels in kidney homogenate from mice treated with OTA compared to control (< 0.001). The groups treated with combination OTA + A. indica show a statistically insignificant reduction compared to the OTA group, and a statistically significant increase in the NO levels compared to mice treated only with A. indica and control.

Asc⋅ levels in kidney tissue
The levels of Asc radicals in the group treated with OTA are statistically significant higher compared to the control group ( Source: Author Antioxidan enzyme -SOD levels SOD levels (table 1) were statistically significantly lower in the OTA treated group compared to the control (p<0.00), the A. indica group (p<0.00), and the combination A. indica + OTA group (p<0.00). A statistically significant increase was observed in the group treated with combination A. indica + OTA compared to control, as well as the group treated with A. indica alone. The levels of antioxidant enzyme SOD were statistically significantly higher in the group treated with combination A. indica + OTA compared to the group treated only with OTA (p<0.00).

Reduced Glutathione Peroxidase (GSH-Px)
The same statistically significant decrease is shown in the group treated with OTA compared to control, the A. indica group and the combination A. indica + OTA group. Also, statistically significantly lower was the result of the combination A. indica + OTA group compared to control (p<0.00) and the A. indica group (p<0.00), which were statistically significantly higher to the OTA group (p<0.00). Discussion OTA has been shown to be nephrotoxic to animals and to cause kidney nephrotoxicity in mice and rats Hoehler et al. (1997). OTA is accumulated in human food and animal feed, due to favorable weather conditions and climate, and/or to inappropriate food storage (Koszegi and Poor, 2016). According to Atroshi et al. (2000) OTA-induced ROS/RNS are elective but not mandatory apoptosis regulators. Moreover, ROS and NO have a major role in the cell damage mediation. Organism defense mechanisms attempt to minimize free radical production and harmful oxidation action; defense mechanisms include antioxidant enzymes -SOD, and GHS-Px, as well as natural antioxidants. Petrik et al. (2003) induced apoptosis with OTA in the kidneys of rats and reported that apoptosis was accompanied by oxidative stress, which manifested itself in elevated MDA levels, increased ROS, and statistically significantly reduced SOD activity. ROS and RNS levels may be further elevated in OTAtreated cells. Moreover, OTA promotes the expression of both the enzyme responsible for inducing nitric oxide synthase (iNOS) and the activity of dimethylarginine dimethylaminohydrolase (DDAH). According to Sorrenti et al. (2013) elevated levels of iNOS and DDAH further increase NO⋅ synthesis and nitrite / nitrate concentration. Therefore, high NO⋅ levels cause nitrosative stress due to its reaction with ⋅O2¯, which forms peroxynitrite (ONOO¯) and increases the level of nitrogen dioxide (NO2) and ⋅OH.
Elevated levels of NO⋅, NO2 and ⋅OH lead to a decrease in the levels of antioxidant enzymes SOD and GHS-Px. Mycotoxin-induced free radical damage is likely to be increased by lipid peroxidation chain reactions and expressed with elevated MDA levels. In addition, ROS is involved in the regulation of cell death by inducing apoptosis and increasing ⋅O2¯ formation. Our results (Table 1) show that combining OTA with an antioxidant (A. indica) prevents cell damage and statistically significantly increases the levels of the antioxidant enzymes -SOD and GHS-Px, while also statistically significantly decreases the levels of ROS, NO⋅, Asc⋅ and MDA. These results coincide with a study by Atroshi et al. (2000) which found that after 14 days, mice treated with OTA, showed a decrease in GSH activity. The authors treated the mice with combined antioxidants, which improved the liver's antioxidant/detoxification system, expressed in elevated glutathione levels. Abireh et al. (2020) investigated the therapeutic effect of aqueous leaf extract of Azadirachta indica on ibuprofen-induced nephrotoxicity in Wistar rats. The authors concluded that the administration of A. indica leaf extract led to the resolution of ibuprofen-induced kidney damage. Thus, it may serve as an option for the treatment of renal impairment resulting from the ingestion of ibuprofen, once the molecule responsible for this effect has been identified. Like most natural antioxidants, the extract of Azadirachta leaves and A. indica oil has a high content of polyphenols. A number of studies have reported the antioxidant effects of polyphenols associated with increased inhibition of lipid peroxidation and free radical scavenging.

Conclusion
In conclusion, the use of A. indica oil as a protector in OTA toxicity is a promising renal agent and this protective activity of A. indica oil may be due to its antioxidant activity, which normalizes impaired renal function.