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Ameliorative Effects of Aspergillus awamori against the Initiation of Hepatocarcinogenesis Induced by Diethylnitrosamine in a Rat Model: Regulation of Cyp19 and p53 Gene Expression

Hepatocellular carcinoma (HCC) is the most common cancer in humans. Despite advances in its treatment, liver cancer remains one of the most difficult cancers to treat. This study aimed to investigate the ameliorative action and potential mechanism of Aspergillus awamori (ASP) administration against the initiation process of liver carcinogenesis induced by diethylnitrosamine (DEN) in male Wistar rats. Seventy-two male rats were divided equally into eight groups as follows, Group 1: untreated control; Group 2: DEN (200 mg/kg bw) intra-peritoneally for the initiation of HCC; Groups 3–5: DEN + ASP at a dose of 1, 0.5, and 0.25 mg/kg bw and groups 6–8: ASP at a dose of 1, 0.5, and 0.25 mg/kg bw. Supplementation of A. awamori significantly lightened the adverse impacts induced by DEN via restoring the leukogram to normal, lowering the elevated serum aspartate aminotransferase (AST), alanine transaminase (ALT), and γ-glutamyl transferase (GGT), and alkaline phosphatase (ALP). Furthermore, it enhanced the hepatic antioxidant capacity through increasing the reduced glutathione (GSH) level and catalase (CAT) activity with a marked reduction in malondialdehyde (MDA) level. In addition, it decreased the positive GST-P foci. Likewise, a significant alteration of DEN-associated hepatocarcinogenesis occurred through inhibiting cytochrome P450 (Cyp19) and activating p53 gene expression. In conclusion, supplementation of A. awamori counteracts the negative effects of DEN, inhibits the early development of GST-P-positive foci and could be used as a new alternative strategy for its chemo-preventive effect in liver cancer. To the best of our knowledge, the present study is the first to report the hepato-protective effect of A. awamori in induced hepatocarcinogenesis.

Ethics Committee, Kafrelsheik University, Egypt. All precautions were followed to diminish animal suffering during the experiment.

Aspergillus awamori Extract Preparation
A. awamori powder (25 × 10 4 cells per g.) was provided from Biogenkoji Research Institute, Kagoshima, Japan. A. awamori powder was suspended in saline with a concentration of 3 mg/6 mL and was used as a stock solution and prepared regularly by the same concentration.

Determination of the Total Content of Flavonoids and Polyphenols
For measuring the total flavonoids content, serial dilutions of A. awamori extract were analyzed colorimetrically according to the procedures of aluminum chloride method using rutin as a standard [25]. The total polyphenol content was determined following the Folin-Ciocalteu method using gallic acid as a standard [26]. The contents were expressed as mg/g equivalent of the corresponding standard for each method.

Experimental Animals
Seventy-two male Wistar rats of average weight 120 ± 15 g were used in the present study and obtained from the Animal House Colony of the Tanta Center. Rats were acclimatized for one week to laboratory conditions (temperature 22-25 • C, relative humidity 50-60%, and 12-h photoperiods with lights on 07:00-19:00 h) in stainless steel wire-mesh cages. The animals were provided with basal diet (Supplementary Table S1) and water ad libitum throughout the experiment.

Experimental Protocol
After the adaptation period, the rats were randomly divided equally into eight groups (9 animals each) and treated as follows: Group 1 Normal control untreated rats fed with standard diet and administered orally with 0.5 mL normal saline by gastric intubation. Group 2 Rats were injected with DEN to induce hepatocarcinogenesis by providing a single dose of intraperitoneal injection (i.p) of DEN (200 mg/kg BW) on the first day of the experiment [27].
Groups 3, 4 and 5 Rats treated with A. awamori after the administration of DEN at a dose of (1, 0.5 and 0.25 mg/kg BW respectively for seven days) orally by gastric intubation (DEN + ASP 1, DEN + ASP 0.5, DEN + ASP 0.25 respectively). Groups 6,7 and 8 Rats treated with A. awamori alone orally by gastric intubation daily for seven days at a dose of 1, 0.5 and 0.25 mg/kg BW respectively for seven days (ASP 1, ASP 0.5, ASP 0.25 respectively).
The experimental design is presented in Supplementary Figure S1. At the end of the experimental period, two blood samples were withdrawn from the retro-orbital venous plexus of each rat under anesthesia with intravenous injection of sodium pentobarbital (30 mg/kg BW). These samples were immediately divided as follows: one with anticoagulant for hematological examination and the other left to clot; serum was separated from clotted blood by centrifugation at 3000 rpm for 10 min for biochemical analysis. Then, the rats were sacrificed by decapitation. Livers were collected and rapidly excised from each animal, trimmed and divided into two parts; the first part was taken directly, snap-frozen in liquid nitrogen, and stored at −80 • C for evaluating lipid peroxidation, antioxidant status and real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. The second part was used for histopathological examination and immunohistochemistry.

Hepatic Oxidative Stress and Antioxidant Status Estimation
The liver from each experimental animal was dissected out, weighed, then washed with 50 mmol/L sodium phosphate-buffered saline (100 mmol/L Na 2 HPO 4 /NaH 2 PO 4 , pH 7.4) in an ice-containing medium, with 0.1 mmol/L EDTA to remove RBCs and clots. Samples were taken and homogenized in 5-10 mL cold buffer per gram of tissue and were centrifuged at 2000× g for 30 min. The resulting supernatants were subjected to biochemical determination of malondialdehyde (MDA), reduced glutathione (GSH), and catalase (CAT) according to the manufacturers' instructions (Bio-diagnostic Co., Giza, Egypt).

RNA Extraction and Real Time-PCR
Total ribonucleic acid (RNA) was extracted from the liver tissue of both control and treated rats according to a TRIzol reagent protocol using DNase treatment Kit (Invitrogen, Carlsbad, CA, USA) to determine the gene expression level of Cyp19 and P53. Firststrand cDNA was synthesized using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen Co., Carlsbad, CA, USA). Quantitative real-time reverse transcriptionpolymerase chain reaction (RT-PCR) analysis was performed using a SYBR Premix Ex Taq™ (Takara Bio Inc., Shiga, Japan). The primer sequences are listed in Table 1 [28][29][30]. The value of each sample was normalized with that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative gene expression concentrations were evaluated using the 2 −∆∆ct method as described by Pfaffl [31].

Histopathology
Tissue sections from the liver of each animal were taken and processed for histopathological examination. The sections were directly fixed in 10% formalin solution, dehydrated in alcohols, cleared in xylene and embedded in paraffin blocks. Sections of 5 µm thickness were obtained and stained with hematoxylin and eosin [32].

Immunohistochemical Analysis of Glutathione S-Transferase P
Immunohistochemical staining (IHS) of glutathione S-transferase placental form (GST-P) was performed on all hepatic tissue samples using 4-µm thick paraffin-embedded sections. IHS for GST-P (MBL Co. Ltd., Nagoya, Japan) and analysis of GST-P-positive foci were performed as described previously by [33]. The percentage of cells with positive labeling of GST-P was determined by counting 1000 cells observed in 10 high-power fields (×400) in the pre-neoplastic foci and the surrounding hepatic tissue.

Statistical Analysis
Data were analyzed with One-way ANOVA and Tukey's post hoc multiple range tests using GraphPad Prism 5 (GraphPad, San Diego, CA, USA). p value < 0.05 was considered statistically significant. All data are expressed as means± SD.

UPLC-PDA-MS/MS for Metabolite Analysis
The analysis of the data in the negative mode ion revealed the presence of 40 compounds. The major identified constituents are citric acid, citric acid isomers, and octadecenoic, octadecadienoic, and octadecanoic acid derivatives. The complete chemical profile is shown in Figure 1 and listed in Table 2. This assortment of such bioactive compounds can explain the exhibited biological potential of A. awamori in our study. UPLC-MS data were processed using Shimadzu's LabSolutions software. The compounds were identified based on the molecular weight determined from the most intense adduct ion found in the full MS spectrum of each compound, MS/MS fragmentation ions, or neutral losses found in full MS or MS/MS spectra and, whenever possible, maximum absorption wavelength from PDA spectra. MassBank and FooDB databases were used as references in identifying the eluted compounds in addition to the published data in the literature. Detailed description of all bioactive compounds is given in supplementary File 2.

Total Content of Flavonoids and Polyphenols
The total flavonoid content was measured as 6.78 mg/g equivalent to rutin and the total polyphenol content was measured as 7.36 mg/g equivalent to gallic acid.

Body and Liver Weights
In rats supplemented only by different concentrations of A. awamori, there was a non-significant effect on the body weight, liver weight, and liver weight/body ratio, except for a significant decrease in body weight in the group that received 1 mg/kg BW A. awamori. However, the DEN treated group exhibited a significant decrease (p < 0.001) in body weight when compared with the control untreated group (Table 3). While, a marked increase in liver weight and liver to body weight ratio was observed when compared with the control untreated rats. On the contrary, in the DEN injected groups supplemented with A. awamori (DEN + ASP1, DEN + ASP0.5, and DEN + ASP0.25), a significant improvement in the body weight, liver weight and liver to body weight ratio was noted. All effects on the body weight, liver weight, and liver weight/body ratio are shown in Table 3.

Hematological Examination
The erythrogram showed non-significant changes in RBC count, Hb, HCT, MCV, MCH and MCHC between the different experimental groups. However, the leucogram revealed that the DEN administration induced a significant increase of WBCs (p < 0.001), lymphocyte (p < 0.01), monocyte (p < 0.001) and neutrophil (p < 0.01) counts when compared with the control untreated animals. On the other hand, the groups supplemented with A. awamori in different doses along with DEN (DEN + ASP1, DEN + ASP0.5, DEN + ASP0.25) showed an improvement in the WBC, lymphocyte, monocyte and neutrophil counts compared with the group injected with DEN only as listed in Table 4. Mean values with different letters at the same column differ significantly at (p ≤ 0.05).

Biochemical Findings
Non-significant changes were observed in rats supplemented only with different concentrations of A. awamori, while rats administered with DEN showed a significant decrease (p < 0.001) in the levels of serum total proteins, albumin and globulins ( Figure 2I). Furthermore, a significant increase (p < 0.001) in the activities of ALT, AST, GGT and ALP ( Figure 2I) was seen when compared with the untreated control animals. Conversely, in groups supplemented with A. awamori (DEN + ASP1, DEN + ASP0.5, DEN + ASP0.25), there was a significant increase in serum total proteins, albumin, and globulin levels when compared with the DEN administered group. Moreover, A. awamori supplementation significantly reduced the elevated enzyme activities of serum ALT, AST, GGT, and ALP, which were induced by DEN administration as shown in Figure 2I.

Hepatic Oxidative Stress and Antioxidant Status
Non-significant changes were observed in the levels of MDA, GSH and CAT activities in rats that only received different doses of A. awamori. However, in the DEN treated group, the hepatic MDA levels were significantly increased (p < 0.001), while GSH levels and catalase activities were significantly decreased (p < 0.001) in comparison with control untreated rats. Inversely, in A. awamori supplemented groups (DEN + ASP1, DEN + ASP0.5, DEN + ASP0.25), the MDA levels were significantly reduced with a significant improvement in GSH levels and CAT activities when compared with the DEN treated group. Levels of MDA, GSH and CAT are presented in Figure 2II.

Genes Expression Level of Cyp19 and p53
Gene expression revealed significant up-regulation of Cyp19 mRNA (p < 0.001) in the DEN group as compared with the untreated control group. While, this was significantly down-regulated (p < 0.001) in rats given DEN and supplemented with A. awamori (DEN + ASP1, DEN + ASP0.5, DEN + ASP0.25) as compared with the DEN treated group. However, the expression level of p53 mRNA was significantly down-regulated (p < 0.001) in the DEN group as compared with the control untreated group. In groups supplemented with A. awamori (DEN + ASP1, DEN + ASP0.5, DEN + ASP0.25), there was a significant increase in p53 expression level in comparison with the DEN treated group ( Figure 2III).

Histopathology
Liver sections from the control untreated rats ( Figure 3I-A) and rats treated only with different concentrations of A. awamori ( Figure 3I-B) showed normal hepatocellular architecture comprising hepatocytes with typical cytoplasm and small regular nuclei radially arranged in a radial pattern around the central vein. In contrast, the animals treated with DEN showed loss of normal architecture with irregularly formed hepatocytes with increased nuclear to cytoplasmic ratio in addition to extensive steatosis cells with cytoplasmic vacuolation (Figure 3I-C). However, in rats supplemented with A. awamori and subjected to DEN (DEN + ASP1, DEN + ASP0.5, DEN + ASP0.25), there was a marked decrease in DEN's hepatic lesions and reversal of liver architecture ( Figure 3I-D-F).

Immunohistochemistry of GST-P Expression
Liver tissues from untreated control and A. awamori sole supplied groups had no positive cancer cells for GST-P. However, the immunohistochemical labeling of GST-P during the initiation of hepatocarcinogenesis induced by DEN revealed widely distributed GST-P-positive hepatocytes in a percentage of 43.33% ( Figure 3II-A). Interestingly, GST-Ppositive foci were significantly decreased (p ≤ 0.05) with A. awamori supplementation in a dose dependent manner, where it decreased to 16.33% in the DEN + ASP1 group, to 30.33% in the DEN + ASP0.5 group and to 36.6% in the DEN + ASP0.25 group ( Figure 3II-B-D). The immunoreactivity of GST-P is summarized in Table 5 and Figure 3II.

Immunohistochemistry of GST-P Expression
Liver tissues from untreated control and A. awamori sole supplied groups had no positive cancer cells for GST-P. However, the immunohistochemical labeling of GST-P during the initiation of hepatocarcinogenesis induced by DEN revealed widely distributed GST-P-positive hepatocytes in a percentage of 43.33% ( Figure 3II-A). Interestingly, GST-P-positive foci were significantly decreased (p ≤ 0.05) with A. awamori supplementation in a dose dependent manner, where it decreased to 16.33% in the DEN + ASP1 group, to 30.33% in the DEN + ASP0.5 group and to 36.6% in the DEN + ASP0.25 group ( Figure  3II-B-D). The immunoreactivity of GST-P is summarized in Table 5 and Figure 3II. Table 5. Scoring summary of GST-P immunolabelling in different experimental groups in the liver tissues in a rat model.

Discussion
The current work was planned to elucidate the modulatory effect of A. awomori against the initiation of DEN induced hepatocarcinogenesis. DEN is a ROS-generating carcinogen that results in formation of preneoplastic foci [8,34,35]. These generated ROS are usually deactivated by the endogenous antioxidants [36]. Consequently, antioxidants act as the cellular housekeepers via mopping up of free radicals before inducing DNA damage [8].
The current study revealed that oral supplementation of A. awamori alone in different concentrations did not have a significant effect on the body weight, liver weight, and liver weight/body ratio. Furthermore, non-significant changes in the erythrogram, leukogram, serum protein profile and liver injury markers indicated that dietary supplementation of A. awamori is safe and useful with no evidence of cellular oxidative damage. In the same context, numerous studies stated the beneficial effect of A. awamori regarding its antioxidant potential [23,37,38].
The observed reduction in the rat's body weight with significant increase of liver weight by DEN administration may be due to the enhancement in the metabolic activity of the body systems. The same findings were recorded in previous studies [39,40]. The increased hepatic weight may be due to cellular swelling (shift of extracellular water into the cells), which is the primary indicator for cell injury together with fatty changes of the liver [41].
Furthermore, our results demonstrated that DEN administration resulted in significant changes in the leucogram as compared with the control untreated group, which is in line with Holsapple et al. [42]. These changes in the leucogram were ameliorated with A. awamori supplementation. Furthermore, DEN/A. awamori supplementation restored the hepatic injury markers to normal with reduction of the MDA levels and a significant enhancement in antioxidant status. The increased level of MDA in HCC is an indicator for cell membrane injury and alterations in organ structure [43][44][45], which is clearly supported by our findings.
The antioxidant properties of A. awamori are related to its content of several bioactive compounds. Few data are available about the chemical principles of A. awamori [24]. For this reason, the current study aimed to investigate the metabolic fingerprint of this fungus. Citric acid was found to induce apoptosis in gastric carcinoma via the mitochondrial pathway [46]. Ren et al. [47] documented that citrate suppressed growth of different cell lines by inhibiting glycolysis. Moreover, citric acid, citric acid isomers and ascorbic acid are well recognized antioxidants, and reduced the hepatocellular injury induced by carbon tetrachloride in rats [48]. Additionally, Richard et al. [49] indicated that polyunsaturated fatty acids could act as antioxidants through their free radical scavenging ability.
In addition, the observed hepatoprotective effect of A.awamori in the DEN-induced HCC model may be attributed to its total flavonoid and total polyphenol contents. Polyphenols employ anti-proliferative and anti-angiogenic effects that are principally obvious in tumor cells [50,51].
Histopathological examination of the liver tissues from DEN treated rats showed marked alteration in the tissue architecture, indicating carcinogenicity, which is consistent with the results of Abdelhady et al. [8] who reported hypertrophy of centro-lobular hepatocytes, cytoplasmic vacuolation, and periductal oval cell proliferation with an increase in mitotic figures after one week from a single dose injection of DEN. In addition, Salah et al. [52] confirmed that the hepatic neoplastic changes induced by DEN are due to ROS generation. In rats post-treated with different concentrations of A. awamori after DEN administration, restoration of hepatic architecture with an obvious decrease in DEN's hepatic lesions was observed. Overexpression of GST-P in response to DEN indicates the initiation of hepatic carcinogenesis. These GST-P-positive foci were significantly decreased (p ≤ 0.05) with A. awamori supplementation in a dose dependent manner. Abdo et al. [35] detected an increase in the number and area of GST-P-positive preneoplastic hepatic foci induced by DEN. Abdelhady et al. [8] also recorded marked GST-P immune-labeling within the neoplastic hepatocytes after one week in a group treated with a single dose of DEN. Xin you et al. [53] observed that the GST-P positive foci increased in all rat groups treated with an intra-peritoneal dose of DEN (200 mg/kg BW) and decreased in a dose dependent manner with resveratrol treatment.
Cyp19 is a chief tumorigenesis enzyme that shares in chemotherapeutic activation in cancerous tissues [54]. Cyp19 in rat liver carcinomas was significantly up-regulated, which is linked to invasion and is a probable HCC predictor [55]. In the present study, DEN significantly up-regulated Cyp19, indicating tumor initiation. Attenuation of Cyp19 was observed in rats supplemented with A. awamori. These findings are firstly recording the effect of A. awamori on DEN cancer initiation through down-regulation of Cyp19.
The p53 gene is the genome guardian and mainly regulates the proliferation, development, and transformation of the cells [56]. P53 protein level is low in 50% of human tumors [57] and tends to be crucial for hepatocarcinogenesis [58]. Interestingly, genomic mutations in p53 have been seen in several phases of malignancy [56]. In the present study, significant down-regulation (P < 0.001) of p53 mRNA was observed in the DEN treated group, which is in line with [59,60]. Upon supplementation with A. awamori, a significant up-regulation of p53 expression level was noticed. This may be due to A. awamori promoting de novo synthesis of p53 protein or some other proteins to stabilize p53. To date, there are no previous reports about the effect of A. awamori on mRNA expression of Cyp19 and p53 in DEN-induced hepatocarcinogenesis. A. awamori achieved the equilibrium of cell proliferation and apoptosis through up-regulation of p53 together with down-regulation of Cyp19 to hinder tumor initiation and progression.

Conclusions
The current study proved for the first time that administration of A. awomori counteracts the adverse effects of DEN and initiated HCC in the Wistar rat model through restoring hepatic integrity by depletion of MDA level, thus lowering oxidative stress in addition to remarkable enhancement of hepatic antioxidant GSH level and CAT activities. Furthermore, reduction of GST-P positive foci, stabilization of some gene expression via down-regulating Cyp19 and up regulation of p53 gene occurred.
The presence of certain chemical constituents (citric acid, citric acid isomers, and octadecenoic, octadecadienoic and octadecanoic acid's derivatives), polyphenols and flavonoids can explain the biological activity of A. awamori, which may be considered as antioxidant and anticancer agents. Based on these findings, A. awamori could be used as a natural hepatoprotective agent against chemical induced carcinogenicity. However, detailed further studies are needed to assess the effect of A. awamori in a long duration study with a wide profiling of gene expression levels in HCC induced by DEN.