INTRODUCTION:

Acetaminophen (APAP) overdose is one of the most frequent reasons for acute liver injury^1,2. APAP is metabolized in liver by conjugation with glucoronidate and sulfate in reactions mediated by uridinediphosphate-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs), which are members of phase II drug metabolizing enzymes. The conjugation products are nontoxic and excreted without causing harms.

A dose dependent fraction of APAP is metabolized by cytochrome P450 enzyme family, specifically CYP2E1, producing the highly toxic intermediate N-acetyl-p-benzoquinone imine (NAPQI). The highly reactive NAPQI covalently binds to vital cellular proteins forming cytotoxic protein adducts leading to hepatocellular necrosis. Although NAPQI can be reduced by GSH then excreted in bile, APAP high dose leads to saturation of glucoronidation and sulfonation pathways and depletion of GSH exposing the liver to injury and may be failure^3,4.

Hepatocyte nuclear factors (HNFs) are a group of phylogenetically unrelated transcription factors that are highly expressed in liver. Hepatocyte nuclear factors have been shown to play important roles in liver development and hepatocyte differentiation^5,6. HNF4α and HNF1α are two important HNFs involved in regulation of many UGTs, SULTs and CYP450 family enzymes in liver and kidney^7,8,9. A regulatory role for HNF4α and HNF1α in modifying acute phase response gene expression was reported by Bauzá et al.¹⁰. HNF4α plays an important role in inflammation through regulation of acute-phase protein expression and cytokine induced inflammatory responses. Moreover there is a negative regulatory role for cytokines on expression levels and transcriptional activity of both HNF4α and HNF1α^11,12,13.

Galectin-3 (Gal-3) is a β-galactoside-binding lectin that regulates macrophage activation and is a mediator of inflammatory cytokines production¹⁴. Gal-3 is an important modulator of both acute and chronic inflammation^15,16. Modified citrus pectin (MCP) is obtained from citrus pectin via pH and temperature modifications, which break its bulky structure into shorter, unbranched, galactose‑rich carbohydrate chains. MCP is able to tightly bind with Gal‑3 carbohydrate recognition domain, which inhibits galectin‑3 bioactivity ^17,18. There may be a relation between Gal-3 and both of HNF4α and HNF1α through inflammation mediators, so this study aimed mainly to evaluate the impact of Gal-3 inhibition on HNF4α and HNF1α gene expression and to investigate if there is any effect against hepatotoxicity due to APAP high dose administration as a secondary goal.

MATERIALS AND METHODS:

Chemicals:

Acetaminophen (APAP) was purchased from Sigma Aldrich Co., St. Louis, Missouri, United States, and suspended in 20% Tween-80. Modified citrus pectin (MCP) was purchased from EcoNugenics Inc., Santa Rosa, CA, USA. Any other chemicals mentioned were purchased from Sigma Aldrich Co., St. Louis, Missouri, United States unless reported otherwise.

Animals and ethical consideration:

Sixty four male Wistar rats, weighing 90 –110 g each, were purchased from the National Research Centre (NRC), Cairo, Egypt. The animals were housed in the NRC animal house and maintained under standardized environmental conditions at 12 h light/dark cycle under a constant temperature of 25±1 ^◦C. Rats were fed on basal diet¹⁹ and water was supplied ad libitum. Animals were acclimated to laboratory conditions one week prior to experiment beginning. All experimental procedures were performed according to guidelines of the institutional committee of animal’s care and use of the NRC. The study protocol was approved by the Medical Ethical Committee of the NRC, with approval no.17/140.

Experimental design:

The rats were divided into four groups; two of them were divided into 3 subgroups (8 rats per each undivided group and subgroup) as follows:

Group A: normal control.

Group B: Rats received 1% MCP dissolved in drinking water daily for 3 days¹⁷.

Group C: rats were fasted for 12 h before receiving orally single dose of APAP (3.5g/Kg b.wt)²⁰ then divided into 3 subgroups C1, C2 and C3, which were sacrificed at 24 hours (h), 48h and 72h after APAP administration respectively.

Group D: Rats were fasted for 12 h before receiving a single dose of APAP (3.5g/kg b wt) orally then received 1% MCP dissolved in drinking water. After APAP administration, the rats were divided into 3 subgroups D1, D2 and D3, which were sacrificed after 24h, 48h and 72h respectively.

Collection of blood and liver samples:

At the end of the experiment, Rats were anesthetized and blood samples were collected using the orbital sinus technique of Stone²¹. Blood samples were left to clot in clean dry test tubes, and then centrifuged at 4000rpm for five minutes. The serum was then separated and frozen at -20ºC for the biochemical analysis. After blood collection, rats were sacrificed by decapitation and the whole liver of each animal was rapidly dissected, thoroughly washed with isotonic saline and plotted. Small parts of each liver were cut and weighed for extraction of RNA and preparing liver tissue homogenate for biochemical analyses, the rest of each liver was fixed in 10% formaldehyde buffer for histological examination.

Preparation of tissue homogenate:

Liver tissue samples were homogenized in ice cold PBS buffer (pH 7.4, 4⁰C) contains 100mM Tris, 1mM EDTA, 1% Triton X-100, and Protease inhibitor cocktail by using Fisher brand 850 homogenizer, Pennsylvania, USA. Homogenates were centrifuged at 12,000 x g for 10 minutes using Sigma 2K15 centrifuge, Osterode, Germany. The supernatants were separated for parameters determination by ELISA. Meanwhile other liver tissue parts were homogenized in ice cold PBS buffer (pH 7.4, 4⁰C), centrifuged; then supernatants were separated for determination of reduced glutathione (GSH), glutathione reductase (GR) and glutathione peroxidase (GPx)²².

Biochemical analyses:

Liver Galectin-3 (Gal-3) and tumor necrosis factor alpha (TNF-α) levels were determined by ELISA technique using kit purchased from Elabscience Co., Texas, USA; Liver CYP2E1 level was determined by ELISA technique using kit purchased from Cusabio Co., Houston, USA according to the methods described by the manufacturers and using Stat Fax 2100 Microplate Reader, Awareness Technology Inc., Florida, USA. Liver reduced glutathione (GSH) level, glutathione reductase (GR) and glutathione Peroxidase (GPx) activities were estimated using commercial kits purchased from Biovision Inc., California, USA based on the methods described by the manufacturer.

Serum alanine and aspartate transaminases (ALT and AST), alkaline phosphatase (ALP) activities and total bilirubin levels were estimated using kits from Salucea BV Co., Haansberg, Netherlands according to the methods described by the manufacturer, on UVD-3500 spectrophotometer, Labomed Inc., California, USA.

RNA Isolation and reverse transcription:

Liver tissue parts were homogenized using Tissuelyser and Qiagen Stainless beads (5mm) (Qiagen, Hilden, Germany), then manual extraction of total RNA from homogenized liver tissue was done using RNeasy kit™ (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The purity of extracted RNA was checked using NanoDrop2000™ spectrophotometer (Thermo Scientific, Massachusetts, USA). Complementary DNA (cDNA) was obtained from the extracted RNA using the High Capacity cDNA Reverse Transcription Kit™ (Applied Biosystems, Massachusetts, USA) according to the manufacturer’s instructions on Veriti thermal cycler (Applied Biosystems) with conditions of 25°C for 10 minutes, 37 °C for 120 minutes and 85°C for 5 minutes to terminate the reaction.

Gene expression analysis of HNF4α and HNF1α using qPCR:

Levels of HNF4α and HNF1α gene expression were evaluated by using Taqman assays (ID no.s are Rn04339144_m1 and Rn00562020_m1 respectively) and Taqman gene expression master mix (Applied Biosystems). Expression levels of target genes were normalized to the endogenous control beta actin (ID no. Rn00667869_m1). QuantStudio 12K Flex Real time PCR equipment was used with cycle conditions: 95°C for 15 min, followed by 40 cycles of 95°C for 15 s, 60 °C for 30 s, and 72°C for 30 s. Relative expression levels were calculated with the 2^−ΔΔCt method and represented as fold change.

Histopathological examinations and quantitative measurement of liver damaged areas:

The liver of rats in all groups were removed and fixed in 10% formal saline, 5µm thick paraffin sections were stained with haematoxylin and eosin stains²³ and examined by digital light microscope, Leica Qwin 500, Leica Microsystems, Wetzlar, Germany. Quantitative measurement of liver damaged areas using the device accompanied Image Analyzer software. Five non overlapping fields were chosen from each rat from groups treated with APAP alone or accompanied with MCP, and the mean values were obtained.

Statistical analysis:

Statistical analysis was performed using SPSS version 21.0. Data obtained in the present work are represented as average mean±standard deviation. Statistical analysis was evaluated using the ANOVA test and Tuckey post-hoc test for multiple comparisons between groups. P values less than 0.05 were considered statistically significant²⁴.

RESULTS:

Liver function parameters:

Acetaminophen (APAP) high dose administration resulted in hepatic damage represented by significant increase (P<0.05) in serum activities of alanine and aspartate aminotransferase (ALT and AST), alkaline phosphatase (ALP) enzymes and level of total bilirubin in comparison with the control group. This toxic effect was strongly obvious after 24 h of APAP administration (group C1), then it diminished gradually after 48h (group C2) and almost disappeared after 72h (group C3) as shown in Fig. 1.

Inhibiting Galectin-3 (Gal-3) improved levels of the parameters mentioned above towards normal control values. That occurred especially after 24h of APAP administration (group D1) which showed significant change (P<0.05) in all the parameters when compared with its time correspondent group (C1) which received APAP only (Fig. 1).

Quantitative gene expression levels of HNF4α and HNF1α:

APAP administration led to significant depression (P<0.05) in expression level of liver hepatocyte nuclear factor HNF4α after 24 and 48 h of administration in comparison with control group. Meanwhile there was significant depression (P<0.05) in expression level of liver HNF1α after 24 h and notable decrease after 48 h compared to control group. Inhibiting Gal-3 induced significant increase in both liver HNF4α and HNF1α expression levels after 24 h of APAP administration compared to its correspondent group without MCP treatment (group C1) (Fig. 2).

Gal-3, TNF-α, CYP2E1, GSH levels, GR and GPx activities:

In comparison with normal control group, APAP overdose led to significant elevation (P<0.05) in liver levels of galectin-3 (Gal-3) and tumor necrosis factor-alpha (TNF-α) (Fig. 2), along with significant depression (P<0.05) in CYP2E1 level, and significant decrease (P<0.05) in reduced glutathione (GSH) level and glutathione reductase (GR) and glutathione peroxidase (GPx) activities (Fig. 3). All these changes occurred after 24 h of APAP administration and withdrew gradually after 48 h and almost disappeared after 72 h which showed insignificant difference versus control group except for higher TNF-α level.

Inhibiting Gal-3 improved the previous parameters levels towards normal control values. Again this was most obvious after 24 h of APAP administration which showed significant change (P<0.05) in all the parameters and 48h with significant change (P<0.05) in some of the parameters compared with their time correspondent groups (C1 and C2) that received APAP only.

Figure 4: Liver damaged area per five non overlapping fields per rat in groups treated by APAP only or accompanied with MCP. The data are represented as mean + Standard deviation (error bars). Values with different letters (a, b, c, d) are significantly different at P<0.05 using ANOVA test. APAP: acetaminophen. MCP: modified citrus pectin.

Histopathological results:

Histopathological examination of liver sections from control and MCP only receiving groups shows normal hepatic lobule structure, in which hepatocytes are radiating from the central vein and separated by sinusoids (Fig. 5.A and B respectively).

Administration of acute high dose of APAP resulted in disturbance of the structure of hepatic lobule, appearance of focal necrosis of hepatocytes and vacoular degeneration, which were obvious clearly after 24 h (Fig. 5.C1), still present after 48 h (Fig. 5.C2) and decreased relatively after 72h (Fig. 5.C3).

On the other hand, liver sections of Gal-3 inhibited rats that received APAP high dose showed reduction in dispersion and area of necrosis. This was noted after 24, 48 and 72 h (Fig. 5.D1, D2 and D3). Furthermore there was restoration of normal hepatic lobule structure, which indicates regeneration of hepatocytes, after 72 h (Fig. 5.D3).

Liver damaged area appeared after administration of APAP high dose. It was highest after 24 h then decreased after 48 and 72 h. However Gal-3 inhibition resulted in significant reduction (p<0.05) of hepatic damaged areas in the treated groups when compared with their time correspondent groups without MCP treatment (Fig. 4).

DISCUSSION:

This work studied the effect of acetaminophen (APAP) acute toxic dose on rat liver hepatocyte nuclear factors HNF4α and HNF1α expression levels. And also the effect of inhibiting Gal-3 on limiting the toxic effect of APAP high dose was evaluated. The study was applied through time course of 72 h after APAP administration at time intervals of 24, 48 and 72 h. APAP high dose induces hepatorenal toxicity in Wistar rats including hepatocellular necrosis and inflammation ^4,^20,^{25, 26, 27, 28, 29}. Additionally, this study hypothesized proinflammatory cytokines as link between Gal-3 and the mentioned HNFs. APAP high dose induces inflammation in rats’ liver as reported by Eakins et al.²⁶ and Mahmoud et al.²⁷.

Serum alanine and aspartate transaminases (ALT and AST), alkaline phosphatase (ALP) activities and total bilirubin level increased significantly after APAP administration by 24 and 48 h when compared with normal control group, which agrees with Amin et al.³⁰, Ozcelik et al.²⁵and Ilavenil et al.⁴, meanwhile their levels decreased after 72 h of APAP administration and became closer to control levels which can be attributed to liver tissue regeneration after regression of APAP toxicity. ALT, AST and ALP are normally found inside cells, especially ALT which is found mostly in hepatocytes, the increase in their serum levels indicates hepatocyte necrosis. Also increased serum bilirubin level indicates biliary tubules damage. After reduced glutathione (GSH) depletion, N-acetyl-p-benzoquinone imine (NAPQI) covalently binds with vital cellular proteins led to disturbance in hepatocellular functions followed by necrosis, which agrees with our histopathological results. Meanwhile inhibiting Gal-3 led to a significant improvement in serum ALT, AST, ALP and total bilirubin levels especially 24 and 48 h after APAP administration in comparison with their correspondent groups without MCP treatment. These results are supported by significant decrease in liver damaged area. This could be the positive effect of Gal-3 inhibition on GSH production through protecting HNF4α expression level and transcriptional activity, explained later in the discussion, which together enhanced hepatic defence against NAPQI.

Administration of APAP led to significant increase in liver Gal-3 and TNF-α levels after 24 h and 48 h compared to control group, then Gal-3 level decreased to slightly above control group’s level while TNF-α still significantly higher after 72 h. APAP toxic dose induces inflammation in rats as reported by Mahmoud et al.²⁷ and Eakins et al.²⁶. Also these results agree with Dragomir et al.¹⁴ who mentioned that NAPQI covalently binds to critical hepatocellular proteins leading to necrosis and tissue injury which stimulates macrophages to secrete TNF-α ³¹. There is a link between tissue injury, Gal-3 and inflammation. Tissue injury stimulates recruitment of macrophages to injury site which in turn start to secrete Gal-3 and pro-inflammatory cytokines in extracellular matrix. Gal-3 mediates recruitment of more macrophages to injury site which release more Gal-3 and cytokines in a feedback loop effect ^{15, 32, 33}. Meanwhile MCP administration lessened both liver Gal-3 and TNF-α levels in the treated groups in comparison with their correspondent groups without MCP treatment accompanied by significant decrease after 24 h of APAP administration. This is parallel with Kolatsi-Joannou et al.¹⁷ who reported that MCP reduces Gal-3 expression in experimental acute kidney injury. This can be explained as a direct effect on Gal-3 function in stimulating inflammatory cytokines secretion, so by inhibiting Gal-3 functionality, MCP could also have role in suppression the mutual secretion loop of Gal-3 and inflammatory cytokines in injured tissue.

Liver HNF4α and HNF1α relative expression levels decreased significantly due to APAP administration after 24 h which extended to 48 h for HNF4α when compared with normal control group. Wang and Burke³⁴ and Babeu and Boudreau¹²reported that proinflammatory cytokines have inhibitory effect on HNF4α expression level and transcriptional activity. Meanwhile Qian et al.¹³ reported that proinflammatory cytokines have inhibitory effect on HNF1α expression level in hepatocytes. Also it is reported that HNF4α positively regulates HNF1α expression ³⁵, so down regulation of HNF4α can negatively affect HNF1α expression. Inhibiting Gal-3 resulted in significant increase in liver HNF4α and HNF1α relative expression levels after 24 h and obvious increase after 48 and 72 h of APAP administration in comparison with their correspondent groups (without MCP treatment). This may be related to the anti-inflammatory role of inhibiting Gal-3 functionality, which reduced inflammation, so consequently improved HNF4α and HNF1α expression levels.

APAP administration significantly reduced hepatic CYP2E1 level after 24 and 48h compared to the control group, then it partially recovered after 72 h. APAP toxic dose administration led to reduction in CYP2E1 expression as reported by Papackova et al.³⁶. This can be related firstly to the induced inflammation, because inflammatory cytokines were reported to inhibit CYP2E1 expression level ³⁷. Secondly to the reduction occurred in HNF1α expression level, which regulates CYP2E1 expression ³⁸. Inhibiting Gal-3 caused significant increase in CYP2E1 level after 24 and 48 h compared to their correspondent untreated groups. This can be related to the higher expression level of liver HNF1α and reduced inflammation in the MCP treated groups versus the untreated ones. Both HNF4α and HNF1α induce expression of cytochrome p450 enzymes including CYP2E1.

Also, liver GSH level decreased significantly due to APAP administration and returned to its normal level after 72 h. Ilavenil et al.⁴ reported significant decline in GSH level due to APAP induced liver toxicity. Irreversible covalent binding occurs between NAPQI and GSH molecules leading to GSH depletion. However Gal-3 inhibition resulted in significant increase in liver GSH level 24 h after APAP intoxication in comparison with the correspondent group. Zhang et al.³⁹ reported that HNF4α affect GSH production and HNF4α deficient mice suffered reduction in GSH production. Although HNF1α activate expression of CYP2E1 enzymes which mainly mediates the oxidation of APAP to produce the toxic metabolite NAPQI, both HNF4α and HNF1α also promote expression of phase II detoxifying enzymes including uridinediphosphate-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) that consist the principle pathways of APAP non-toxic metabolism and excretion, especially UGT1A9 ^{40, 41, 42}. This supports the non-toxic pathways of APAP excretion and lessens NAPQI formation and consequently GSH consumption rate. Since HNF4α and HNF1α expression levels were higher in MCP treated groups than the levels in their correspondent groups which received only APAP; so this can be assumed to contribute in the higher GSH level in MCP treated groups.

Liver glutathione reductase (GR) and glutathione peroxidase (GPx) activities decreased significantly because of APAP administration after 24 h, improved after 48 h and returned to normal activities after 72 h. GPx activity was reported to decrease significantly after high doses of APAP ⁴³. The conjugate of GSH and NAPQI has inhibitory effect on GR activity ⁴⁴. HNF4α deficiency leads to decrease in GPx activity ⁴⁵. Inhibiting Gal-3 led to an improvement in hepatic GR and Gpx activities, especially in GR which restored its normal activity after 24 h of APAP administration in comparison with the untreated correspondent group. These results can be related to the effect of Gal-3 inhibition on HNF4α expression level and activity preservation as mentioned before. This may enhanced GPx expression and decreased the probability of GSH-NAPQI conjugate formation due to promoting UGTs and SULTs expression.

Acetaminophen administration resulted in great damage to the rats’ liver represented by focal necrosis accompanied with vacuolar degeneration of hepatocytes and disturbance of liver lobule structure. These results are parallel with Eakins et al.²⁶and Mahmoud et al.²⁷. This also comes in line with our biochemical analyses results. Inhibiting Gal-3 resulted in an improvement in the liver architecture after APAP induced injury, which is represented by significant reduction in areas of necrotic damage compared with corresponding APAP only receiving groups. This result supports our biochemical analyses results.

CONCLUSION:

In conclusion, APAP induced acute toxicity after high dose administration caused reduction in HNF4α and HNF1α gene expression levels, which may be through the elevation in levels of inflammatory cytokines that inhibited their expression. By inhibiting functionality of Gal-3, which is an important mediator of inflammation, an improvement observed in HNF4α and HNF1α gene expression levels. Additionally, improvements were observed in other liver function and APAP detoxification system parameters toward normal control levels. So Inhibition of Gal-3 maintains liver HNF4α and HNF1α gene expression levels, which can be helpful against APAP induced acute liver toxicity.

ACKNOWLEDGEMENT:

The authors gratefully acknowledge the financial support of the Science and Technology Development Fund (STDF), Egypt, through Capacity Building program, under grant No. 4880 (Digital Molecular Biology Research Laboratory, Medical Research Centre of Excellence, NRC).

FUNDING INFORMATION:

This work is partially funded by the National Research Centre, Egypt (grant no. 7/8/10).

CONFLICTS OF INTEREST:

The authors declare no conflicts of interest.

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Received on 08.09.2021 Modified on 27.10.2021

Research J. Pharm. and Tech. 2022; 15(6):2747-2755.

DOI: 10.52711/0974-360X.2022.00460