Nanocurcumin and curcumin prevent N, N'-methylenebisacrylamide-induced liver damage and promotion of hepatic cancer cell growth

Acrylamide (AC) is an environmental contaminant with cancer-promoting and cytotoxic properties, while curcumin (Cur.) is a phytochemical with documented anticancer and cytoprotective efficacy. Nanoparticle formulations can increase the efficacy of phytochemicals, so we examined the anticancer and hepatoprotective efficacies of nanocurcumin (N.Cur). Curcumin and nanocurcumin reduced HepG2 and Huh-7 cancer cell viability and increased apoptosis in the presence and absence of AC, while AC alone promoted proliferation. Furthermore, the anticancer efficacy of nanocurcumin was greater than that of curcumin. In mice, AC greatly increased hepatic expression of CYP2E1, P53, cleaved caspase-3, and COL1A1 as well as serum alanine aminotransferase and aspartate aminotransferase activities. These effects were reversed by nanocurcumin and curcumin. Nanocurcumin also reduced the histopathology and fibrosis caused by AC, and reversed AC-induced glycogen depletion. Nanoparticle formulation can increase the anticancer and hepatoprotective efficiencies of curcumin.

MTT assay. After drug treatment, growth medium was exchanged for 200 µL of drug-free medium and 50 µL of MTT (2 mg/ml). The formazan produced from MTT by viable cells during 3 h of incubation was dissolved in 200 µL DMSO and absorbance measured at 570 nm and reference wavelength of 630 nm as an estimate of viable cell number. The results are expressed as a percentage of vehicles (DMSO)-treated control cell number 25  Animal experiments and treatments. Fifty healthy male Swiss albino mice (Mus musculus) weighing 25-30 g was purchased from the animal house Theodor bilharziainstitute (Giza, Egypt). Mice were divided into five groups, control, vehicle control, AC, AC + Cur, and AC + N.cur, and the indicated drugs were administered daily for four weeks. The control group received distilled water, the vehicle control group Tween-80, the AC group 3 mg/kg oral AC 21 , and the AC + Cur and AC + NCur groups 7 mg/kg oral Cur. or N.Cur. 26 30 min prior to 3 mg/kg oral AC.
Western blot analysis. Changes in hepatic cytochrome P450 (CYP2E1) and P53 were measured by immunoblotting as described 27 . Liver samples were lysed by homogenizer at 4 °C in 500 µl of RIPA lysis buffer (1% Nonidet-P40, 1% Triton X-100, 0.5% Na deoxycholate, 150 mM NaCl, 1 mM PMSF, 5 mM EDTA, 10 mM EGTA, 50 mM Tris-HCl, and 1% leupeptin/pepstatin protease inhibitor cocktail). Centrifugation at 10,000×g for 10 min at 4 °C was used to remove insolubilized tissue debris. The protein concentration in the supernatant was measured. SDS-PAGE (10%) was utilized to resolve protein aliquots, which were transferred onto a nitrocellulose membrane. The membranes were blocked with 5% skim milk in TBS containing 0.05% Tween 20 and then incubated with primary antibodies anti P53 and P450 (1:1000) overnight at 4 °C. Subsequently, the membranes were incubated with the suitable HRP-conjugated secondary antibodies (1:10,000) in the blocking solution for 1 h at 24 °C. Chemiluminescent substrate kit was used to see the immunoreactive bands. For equal loading confirmation, an anti-actin goat polyclonal antibody was utilized. The data are expressed as mean ± SE from at least three separate experiments; the statistical software version of Fiji/Image J was used to estimate each band's optical density in relation to the corresponding actin band.
Tissue immunohistochemistry. Liver tissues were embedded in paraffin, sectioned at 3 µm, deparaffinized, rehydrated in gradient ethanol (100-70%), heated in 10 mM sodium citrate buffer (pH 6.0) for antigen retrieval, developing sections in 3% H 2 O 2 for 10 min, washing with wash buffer (1X PBS) for 5 min, and then blocking each section at room temperature with 100-400 µL blocking solution for 1 h. the cleaved caspase 3 primary antibody was added (1:10) after the blocking solution was removed. The sections were then treated with secondary antibody (1: 5000) for 2 h, washed, stained with 3, 3′-diaminobenzidine (DAB) for 2-3 min, and counterstained with hematoxylin The reaction was immediately quenched in distilled water. A light microscope was used to visualize the stained sections 28 .
Quantitative real time RT-PCR. Liver tissue was homogenized and total RNA isolated using Triazole reagent (Invitrogen). RNAs were reverse transcribed using the High-Capacity cDNA Reverse Transcriptase kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). Quantitative RT-PCR was then performed in 25 µL reaction mixtures containing 1 µL template cDNA, SYBR green PCR Master mix (Applied Biosystems), 10 pmol of Primers for collagen type I alpha 1 (COL1a1) (forward, TCT GCG ACA ACG GCA AGG TG and reverse, GAC GCC GGT GGT TTC TTG GT) and for GAPDH as the internal control (forward, AAC TTT GGC ATT GTG GAA GG and reverse, GTC TTC TGG GTG GCA GTG AT) 29 .
Liver function parameters. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in blood plasma were determined using kits (Boehringer Mannheim, Mannheim, Germany) according to manufacturer procedures.
Histopathological examination. For  Statistical analysis. Data are presented as the mean ± standard error of the mean (SEM) from at least three independent experiments. Treatment group means were compared by one-way analysis of variance followed by post hoc Student Newman-Keuls T tests using Graph Pad Prism 3 (Graph Pad Software Inc., USA).

Results
Morphological and physicochemical properties of N.Cur.. The size distribution of curcumin nanoparticles is shown in (Fig. 1a). Particles were of various globular forms and sizes but generally smaller than 100 nm in diameter. The absorption of N.Cur. peaked at 432 nm (Fig. 1b). The size distribution and zeta potential as determined by DLS for hydrated N.Cur. are shown in (Fig. 1c).
Cur. and N.Cur. reduced hepatic cancer cell viability. Both Cur. and N.Cur. demonstrated time-and dose-dependent cytotoxicity on HepG2 and Huh-7 cells as measured by MTT assay. Furthermore, in the presence of AC, there was non cytotoxicity in cell viability of HepG2 and Huh-7 cell lines after 24-48 h incubation. N.Cur. significantly reduced the cell viability and increased the cytotoxicity of both cell lines than Cur.. It was found Huh-7 cells were more resistance to N.Cur (Fig. 2).
Cur. and N.Cur. induced hepatic cancer cell death. In principle, these effects on viable cancer cell number could reflect inhibition of proliferation or induction of cell death. Therefore, we also conducted dual EB/ AO staining for apoptosis and necrosis. Most control HepG2 and Huh-7 cells appeared to have green nuclei with intact structure. Similarly, after 24 h of incubation in AC, most HepG2 ( Fig. 3a,b) and Huh-7 cells (Fig. 4a,b) were viable and no apoptotic staining was detected. However, incubation with Cur. or N.Cur. caused morphological and staining changes indicative of early apoptosis, late apoptosis, or necrosis (e.g., chromatin condensation, orange nuclei, and nuclear fragmentation). Further, N.Cur. prevented the effects of AC on proliferation and increased the proportions of cells in late apoptosis and necrosis.
Cur. and N.Cur. reversed AC-induced upregulation of the CYP450 and tumor suppressor P53. Immunoblotting revealed that AC increased the protein expression levels of CYP450 and P53 in mouse liver compared to controls (by 216.06% and 148.37%, respectively), while co-treatment with Cur. or N.Cur. completely reversed these changes (Cur. by 44.95% and 62.36%, respectively, and. N.Cur. by 66.21% and 47.85%, respectively, versus the AC group) (Fig. 5). Thus, N.Cur. was more effective than Cur. at reducing CYP450 induction while Cur. was more effect at reducing P53 expression.
Cur. and N.Cur. regulate apoptosis via controlling C.caspase-3. Immunohistochemistry for the apoptosis effector cleaved revealed that AC substantially enhanced apoptotic activity in liver (by 195.2%) compared to control mice (Fig. 6a,b and e). Conversely, cleaved caspase-3 immunoexpression was downregulated by both Cur. (37.8% reduction compared to the AC group) and N.Cur. (57.5% reduction compared to the AC group) (Fig. 6c-e). Thus, nanocurcumin was more effective than curcumin at inhibiting AC-induced apoptosis in mouse liver.
Cur. and N.Cur. protected mouse liver from AC-induced degeneration and inflammation. Liver sections from control mice exhibited typical hepatic tissue architecture as revealed by HE staining (Fig. 8a), while AC treatment induced focal necrosis, pyknotic nuclei, inflammatory leucocytic infiltration, and the formation of lipid droplets (Fig. 8b). In some specimens, hepatocytes exhibited giant nuclei or vacuolization indicative of hydropic degeneration, while blood sinusoids were dilated and the central vein showed signs of congestion (Fig. 8c). Pretreatment with Cur. reduced the number of lipid droplets as well as the number and size of focal necrotic areas (Fig. 8d), while N.Cur. treatment restored normal tissue histology, although proliferation of bile duct cells and dilatation of the portal vein were still observed (Fig. 8e). Quantitative assessment using Heijnenʼs score confirmed that the histopathology induced by AC (score of 126.4% vs. control) was markedly reduced by Cur. (by 34.01% compared to the AC group) and by N.Cur. (by 50.3% compared to the AC group) (Fig. 8f).
Cur. and N.Cur. protected mouse liver from AC-induced liver fibrosis and glycogen depletion. Picrosirius . 9a), while AC treatment induced marked collagen fiber accumulation among hepatocytes and surrounding dilated and congested blood sinusoids (Fig. 9b). Cur. pretreatment reduced the number of collagen fibers surrounding hepatocytes, the central vein, and blood sinusoids (Fig. 9c) while N.Cur. appear to reduce collagen fiber density even further (Fig. 9d). Morphometric analysis of collagen deposition confirmed these qualitative observations, with AC increasing deposition by 126.9% compared to control mice (Fig. 9e) and both Cur. and N.Cur. reducing deposition by 42.9% and 55.0%, respectively, compared to the AC group. The rich distribution of red-stained glycogen particles in control liver (Fig. 10a) was markedly reduced in AC-treated liver (Fig. 10b), while both Cur. and N.Cur. treatment restored these glycogen stores ( Fig. 10c and d).

Discussion
In the current work, nanotechnology was used to break curcumin into nano-sized particles for increased bioavailability and enhanced membrane permeability. Acrylamide significantly increased the proliferation and viability of HepG2 and Huh-7 hepatic cancer cells while reducing apoptosis rate, while co-treatment with Cur. or N.Cur. increased apoptosis and mitigated the proliferative effects of AC. Further findings provided additional support for the antioxidant, immunomodulatory, and anti-inflammatory properties of nano-curcumin 33 .
There are many pathways involved in apoptosis induction, including the death receptor-dependent (extrinsic) and mitochondria-dependent (intrinsic) pathways 34 . While curcumin prevented AC-induced apoptosis, in accord with a previous study 35 , it had a little inhibitory effect on the proliferation of HepG2 cells, likely due to its poor water solubility 36 . In contrast, nano-curcumin exhibited a potent cytotoxic effect in both hepatic cancer cell lines tested and a greater antiproliferative effect than curcumin, likely due to enhanced cellular uptake as demonstrated by 37 . Curcumin has been shown to reduce ROS generation in various cell lines, which may account for reduced cell proliferation 38 .
Overexpression of CYP450, P53, and cleaved caspase-3 in response to AC may enhance tissue sensitivity to AC toxicity. Cur. significantly inhibited AC-induced CYP2E1 overexpression in the liver possibly via free-radical scavenging and antioxidant activities 4 . The p53 protein plays a central role in eliciting cellular responses to DNA www.nature.com/scientificreports/ damage, hypoxia, and aberrant proliferative signals, such as oncogene activation 39,40 , and nano-curcumin also significantly reduced the AC-induced increase in hepatic p53 expression, in agreement with previous reports 41 . Acrylamide-induced toxicity is linked to oxidative stress, and long-term exposure to AC can induce mitochondrial dysfunction and apoptosis 42 . Activation of the mitochondria-mediated intrinsic apoptosis pathway was enhanced by nano-curcumin in HepG2 cancer cells in our finding may be the result of up regulation of pro-apoptotic Bax, down regulation of anti-apoptotic Bcl-2, and promotion of cytochrome c release from mitochondria 43 . Conversely, nano-curcumin significantly decreased cleaved caspase-3 in AC-treated liver, consistent with the observed antiapoptotic activity. Basniwal et al. investigated the effect of curcumin nanoparticles and their anticancer activities in cancer cell lines from the lungs (A549), liver (HepG2), and skin (A431). In aqueous circumstances, curcumin nanoparticles were found to have a far better effect on cancer cells than native curcumin 44 .
Sun et al. found that curcumin solid lipid nanoparticles (CUR-SLNs) exhibited increased cell uptake and growth inhibition in cancer cells, as well as better drug dispersibility and chemical stability 45 . In breast adenocarcinoma cells, CUR-SLNs were tested for antitumor efficacy (MDA-MB-231). In comparison to native curcumin, CUR-SLNs had higher solubility, biocompatibility, and toxicity. Furthermore, CUR-SLNs triggered cancer treatment by inducing considerably greater apoptosis in MDA-MB-231 cells 46 . Thus, nano-curcumin has dramatic reciprocal effects on cancerous and healthy hepatic cells, suggesting clinical promise as an anticancer agent without associated side effects due to non-target toxicity 43 .
Curcumin and nano-curcumin also reversed the AC-induced increase in COL1A1 mRNA expression. According to 47 , AC may induce hepatic gene expression abnormalities that result in the accumulation of collagen, a major pathological feature in a variety of liver disorders. A previous study has also found that curcumin and nan-ocurcumin can reduce collagen deposition 48 , suggesting therapeutic utility in fibrotic liver diseases.
Administration of AC also induced significant increases in plasma AST and ALT, liver-specific enzymes that are released from damaged hepatocytes and thus serve as biomarkers of hepatocellular injury. This finding may be explained by previous studies showing that AC increases hepatocyte membrane permeability and induces cellular transformation 41 , responses that may be attributed to the bipolar nature of AC (hydrophobic interactions and hydrogen bonds) 49 . The ability of curcumin and nanocurcumin to enhance endogenous antioxidant activity, resulting in reduced lipoperoxide formation 50 , may also contribute to the reductions in AST and ALT release.
Acrylamide administration caused degeneration, necrosis, hyperemia in interstitial vessels, cell vacuolation, nuclear pyknosis, and inflammatory cell infiltration in mouse liver. These hepatocyte vacuoles may protect hepatocytes 49 by sequestering injurious substances and preventing them from interfering with biological activities. However, Mahmood et al. (2015) reported that AC can interfere with liver detoxification and excretion of toxic materials 51 .
The protective effects of curcumin against AC-induced damage have been attributed mainly to its antioxidant and radical scavenging properties 4 . However, curcumin may also have immunomodulatory activity, and acrylamide increased hepatic inflammation, in accord with 52 , who reported inflammation in multiple organs, as well as elevated neutrophil counts following AC treatment 25 also reported that the bioavailability and controlled release of nano-curcumin could be responsible for increased cellular immune responses.
Many cells in the AC-treated liver demonstrated a weak PAS reaction indicative of reduced glycogen content, which suggests that AC induces glycogen breakdown into glucose 53 . Co-treatment with nano-curcumin completely restored glycogen content, suggesting that nano-curcumin could be a potential therapeutic agent for sustaining metabolic integrity under stress.

Conclusion
Collectively, these findings support the application of nano-curcumin for combating oxidative damage to hepatic cells induced by AC and down-regulating genes involved in fibrosis pathways, thereby preserving liver function and preventing fibrotic disorders. In addition, nano-curcumin may also be an effective therapeutic agent against hepatic cancer without the non-target toxicity of many other anticancer agents (Fig. 11).

Data availability
All data generated or analyzed during this study are included in this published article [and its supplementary information files].
Received: 9 February 2022; Accepted: 6 May 2022 Figure 11. Graphical abstract shows the potential protection Cur. and N.Cur against acrylamide-induced toxicity, and oxidative stress via histopathological, biomarker, and molecular mechanism of some proteins in liver. Additionally demonstrates its cytotoxicity efficiency against HepG2 and Huh7 cell lines.