Potential hepatoprotective effect of empagliflozin on acetaminophen-induced liver cell damage in albino rat

INTRODUCTION

Drug-induced hepatotoxicity is one of the most frequent causes of restrictions on drug use, and withdrawal of approved drugs; this problem accounted for 0.00001% and 0.01% yearly.^[1,2] Acetaminophen (paracetamol) is a widely used safe painkiller at therapeutic doses, but patients may overdose due to individual differences. Furthermore, hepatotoxicity induced by the acetaminophen overdose occurs quickly within 1-2 days.^[3,4] Acetaminophen overdose toxicity accounts for about 50% of the cases of acute liver failure in a dose-dependent manner.^[5] Moreover, nearly 50% of liver transplantation cases caused by drug-induced hepatotoxicity in the United States are attributed to acetaminophen-induced hepatotoxicity (AIH).^[6]

Acetaminophen is essentially metabolized by the hepatic cells into several inactive ingredients that eventually become excreted by the kidneys.^[7] An overdose of acetaminophen decreases the hepatic levels of hepatic glutathione (GSH) that is required for the inactivation of N-acetyl-p-benzoquinone imine (NAPQI); the hepatotoxic metabolite of acetaminophen, which in turn induces liver cell damage associated with the generation of free radicals and oxidative stress.^[8] These biochemical changes induced by acetaminophen toxicity are reflected as functional and morphological secondary effects on the liver.^[9]

Since acetaminophen hepatotoxicity is by far one of the most common causes of acute hepatocellular failure, it seemed important to examine the cytopathic consequences of hepatocellular injury after acetaminophen toxicity.^[4] This was a particularly intriguing prospect; since molecular studies of the changes in the intracellular mitochondrial membrane potential had been used as an indicator of hepatocyte energy metabolism that provides further evidence for the thermodynamic control of metabolism inside hepatocytes, and that, the release of lactate dehydrogenase after experimental exposure to acetaminophen could be involved in ultimate stages of hepatic cell injury.^[10,11]

At present, N-acetylcysteine is considered the first-line antidote for AIH, but its effectiveness is restricted to the early stages.^[12,13] Hence, more effective and safe drugs to relieve AIH need to be urgently developed. Empagliflozin (EMPA) is used to improve blood sugar control in adults with type 2 diabetes mellitus.^[14] Previous studies have demonstrated that the antioxidant and anti-inflammatory actions of EMPA potentially mediated cardiovascular, renal, and cerebral protective benefits, in addition to amelioration of ethanol-induced hepatotoxicity.^[15,16]

The present study was therefore undertaken to trace the ultrastructural consequences associated with acetaminophen intracellular accumulation in rats. In addition, we aimed to investigate the potential hepatoprotective effect of EMPA on AIH.

MATERIALS AND METHODS

RESULTS

Morphologically, semithin sections of control liver specimens [ Figure 1a] revealed normal architecture of liver parenchyma; hepatocytes were arranged in cords and exhibited large, rounded vesicular nuclei. The homogeneous cytoplasm had moderate chromophilia with the presence of occasional cytoplasmic vacuoles. Hepatic sinusoids were hardly detected as they were very narrow. Under electron microscopy, the nuclei of control specimens exhibited a small amount of heterochromatin lining the inner aspect of the nuclear envelopes. Narrow membranous profiles of endoplasmic reticulum (ER) were interposed with mitochondria that packed the cytoplasm of hepatocytes [Figure 2a]. Under control conditions, glycogen formed dense aggregates of small-sized granules that were associated with some lipid droplets [Figure 2b].

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Figure 1:

Liver sections (Toluidine blue ×1000) showing effects of pretreatments with EMPA on the changes induced by oral administration of acetaminophen in albino rats. (a) A photograph of the control liver specimen showing vesicular nuclei (→) in most hepatocytes; some cells contained two nuclei (➻). The cytoplasm appears homogeneous with moderate chromophilic density and rare cytoplasmic vacuoles (⟼). Hepatic sinusoids (↣) are very narrow. (b) ACAM group II, as compared to control, sinusoidal spaces became dilated and congested (↣) with the appearance of few vacuoles (⟼) within the cytoplasm of some hepatocytes. Nuclei (→) are still vesicular. (c) ACAM group III, sinusoids are dilated (↣) and the cytoplasm of hepatocytes reveals enlargement of cytoplasmic vacuoles (⟼) and dilated dense irregular profiles of moderate size (▶) filled with dense chromophilic material. Some nuclei (→) appear dense. (d and e) ACAM groups IV and V respectively, marked increase of cytoplasmic vacuoles (⟼) inside the swollen hepatocytes, and the irregular cytoplasmic profiles (▶) increased in size. Sinusoids (↣) are markedly dilated with the appearance of prominent von Kupffer cells (). (f) EMPA group, showing normal liver architecture nearly like that of the control group despite the appearance of some dense irregular profiles (▶), sporadic cytoplasmic vacuoles (⟼), and some dilated sinusoids (↣). Vesicular hepatocyte nuclei (→).

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Figure 2:

EM of control liver (a and b; TEM ×5000), (a) The nucleus (N) contains little amount of peripheral heterochromatin and the cytoplasm is packed with mitochondria (M) that are separated by narrow membranous profile of endoplasmic reticulum (ER) (→). The perisinusoidal space of Disse (Sd) reveals multiple microvilli. Glycogen zones (✼). (b) A high-power electron micrograph showing the appearance of inclusions in the control liver; glycogen granules form condensed aggregates (✼) interposed among the few lipid droplets (L) of moderate size. Nucleus (N), bile canaliculus (Bc). ACAM group II (c and d; TEM ×6000); (c) non-membrane bound irregular vacuoles (✼) appear in the zones of glycogen aggregates in close vicinity to the space of Disse (Sd). Mitochondria (M). (d) reveals proliferation of smooth ER; it appears as swollen tubulo-vesicular interconnected profiles (S) that are relatively spaced. The magnified inset reveals spotty communications (⟼) of the perinuclear cistern of endoplasmic reticulum (Pc) with multiple oval membranous structures (Mm) of mitochondrial morphology. Nucleus (N), Mitochondria (M), and lipid droplets (L). (Magnified inset ×9000).

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Six days after acetaminophen administration, it appeared in semithin sections that the sinusoidal spaces became dilated and congested. Only some intracytoplasmic vacuoles appeared in some hepatocytes while their nuclei were still vesicular [Figure 1b]. Electron micrographs of the same group [Figure 2c] showed that the cytoplasmic vacuoles were irregular and non-membrane bound and they infiltrated the zones of glycogen aggregates. Mitochondria were still normal with regard to shape and matrix density. Plethoric microvilli projected into the dilated space of Disse. In addition [Figure 2d], smooth ER (sER) had proliferated, appearing as a widely-spaced network of interconnected tubules and swollen vesicles; the perinuclear cistern of ER evidenced clear spotty tubular communications with multiple mitochondria.

Ten days after ACAM administration [Figure 1c], sinusoids became highly dilated and congested, and the cytopathic effects became more prominent. The cytoplasm of hepatocytes revealed dilated irregular profiles of moderate size, containing a dense chromophilic material, and some nuclei became dense. Electron micrographs of the same group [Figure 3a and b] clarified that the dilated chromophilic profiles were nothing but dilated sER and rough ER (rER) structures that were differentiated based on the outer attachment of ribosomes. Vacuoles appeared in the glycogen areas.

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Figure 3:

ACAM group III (a and b; TEM ×5000), (a) Smooth endoplasmic reticulum (sER) appears as interconnected membranous profiles (S) that are more dilated than in group II, and vacuoles appear in the glycogen areas (✼) which are surrounded by many mitochondria (), Nucleus (N), Lipid droplets (L). (b) This electron micrograph reveals dilated rough endoplasmic reticulum membranous profiles (▶); they had Jigsaw outline, carrying many ribosomes. Cytoplasmic vacuoles (✼), Lipid droplet (L), and Nuclei (N) of hepatocytes. TEM ×3000. ACAM group IV (c and d; TEM ×8000); (c) HP showing dilated sER profiles (S) some of which are extensively dilated (✼) and are interconnected by membranous tubules (). (d) A high-power electron micrograph showing that some mitochondria (M) appear swollen with disrupted cristae. Glycogen zones reveal vacuolization (✼). Lamellar body (Lb), nucleus (N).

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Fourteen days after ACAM treatment [Figure 1d], the cytoplasm of hepatocytes revealed many cytoplasmic vacuoles, and the chromophilic dilated membranous profiles enlarged in size. Sinusoids became extensively dilated, and von Kupffer cells became more prominent. Electron micrographs of the same group [Figure 3c and d] revealed extensive widening of many of the dilated profiles of sER, which were interconnected by membranous tubules. Vacuolization appeared inside glycogen areas and in some dysmorphic mitochondria that appeared swollen with disrupted cristae architectures compared to normal mitochondria in the control group.

Eighteen days after ACAM intoxication, many dilated irregular membrane-bound vesicles [Figure 4a] were noticed very close to the nucleus; they had tubular communication with the perinuclear cistern of ER that led them directly to the nuclear pore. In addition, we noticed accumulations of unusual electron-dense granules making unique arrangements within dilated parts of the perinuclear cistern. These cytopathic changes were concomitant with the appearance of necrotic signs [Figure 4b], indicated by extremely heterochromatic nuclei and crumbled cytoplasm that was essentially divided into multiple autophagic compartments, containing degenerated cytoplasmic membranous profiles and glycogen granules.

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Figure 4:

ACAM group V (a and b; TEM ×10000); (a) Irregular dilated membranous bladder-like vesicles (V) appear close to the nucleus (N), one of them communicates through a long narrow irregular tubule (⟼) with the nuclear pore (→) and the dilated perinuclear cistern (Pc) that exhibits parallel arrays of electron-dense dots. (b) An apoptotic cell with crumbled cytoplasm revealing autophagic multiple vacuoles (V) of variable sizes, the nucleus (N1) is extremely heterochromatic. Normal nucleus (N2) of another hepatocyte. Liver of EMPA group (c and d); (c) Shows few swollen degenerated mitochondria (Dm) close to the space of Disse (Sd). Endothelium (E) of the sinusoid showed degenerative changes in the form of cytoplasmic vacuoles (V) and dense granules (), Nucleus of hepatocyte (N). (TEM ×4000); (d) The appearance of small vacuoles (✼) and a multivesicular body (MVB) among the glycogen granules, associated with large lipid droplets (L), Nucleus of hepatocyte (N). (TEM ×8000).

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Pre-treatment with EMPA (30 mg/kg/day) for 18 days reversed the previously described histopathological changes induced by oral administration of acetaminophen (500 mg/kg/day). However, occasional lipid droplets and a few dark spots were detected in the cytoplasm of some hepatocytes [Figure 1f]. Under EM [Figure 4c and d], minimal vacuolization of the glycogen zones and an increased number of associated lipid droplets were noticed, and mitochondria were minimally affected. On the other hand, the endothelium of the sinusoid exhibited degenerative changes as evidenced by multiple vacuoles and dense granules.

DISCUSSION

The liver is the principal organ where chemicals are metabolized. This fact accounts for the liver’s susceptibility to metabolism-dependent drug-induced injury. Drug-induced liver injuries are globally increasing and account for about half the cases of acute liver failure, and they mimic all forms of acute and chronic liver disease.^[22]

In the present study, histological examination of semithin sections revealed that prominent signs of tissue damage induced by acetaminophen in the liver have confirmed previous findings, as there was a time-dependent hepatocellular necrosis as manifested by swelling of the hepatocytes with the development of cytoplasmic vacuolations of dilated membranous profiles and hyperchromatic nuclei. Sinusoidal spaces became wider and congested with the prominence of von Kupffer cells. These results came in agreement with the results of previous researchers who announced that an overdose of acetaminophen produces centrilobular hepatic necrosis that would be fatal.^[23]

Using electron microscopy, the liver of acetaminophen-intoxicated animals showed some hepatocytes with hyperchromatic nuclei and dysmorphic mitochondria; some of them were distorted and revealed electron-dense matrix, and others appeared with rupture of their membranes. The exact molecular mechanism of AIH is still obscure, but it is believed that mitochondria play an important role in acetaminophen-induced hepatocellular cytotoxicity.^[24]

These results suggest that acetaminophen induces a direct effect on mitochondrial function before cell injury develops and add further evidence to the role of mitochondria in drug toxicity.

Previous histochemical and ultrastructural studies have confirmed that structural damage to subcellular components, including the mitochondria, is associated with the late stages of liver necrosis.^[25] They explained that acetaminophen elicits an early direct change in the membrane potential of the mitochondria, which is followed by ATP depletion and cell death. Acetaminophen administration selectively alters membrane permeability and decreases the efficiency of oxidative phosphorylation, which depletes mitochondrial GSH and produces local toxicity. This makes mitochondria more vulnerable to oxidative damage, especially during increased free radical production.^[26]

In this study, six days after acetaminophen intoxication, we noticed spotty tubular profiles communicated the dysmorphic mitochondria with the perinuclear cistern of ER. Later, unusual electron-dense granules were seen within dilated parts of the perinuclear cistern, with the concomitant appearance of necrotic cytopathic signs. Opening of the mitochondrial membrane permeability transition pore leads to hepatic necrosis, and this triggers the collapse of the membrane potential with cessation of ATP formation. Mitochondrial swelling results in the rupture of the outer mitochondrial membrane with the release of proteins from the intermembranous space and subsequent karyorrhexis.^[27]

Proliferated ER, dilatation of sER, and destructed rER were observed in this study. This came in agreement with the results of previous research workers who stated that the acetaminophen toxicity induced disruptive distributions of cytoplasmic organelles, mild dilatation of rER and sER, and the development of cytoplasmic myeloid bodies.^[28]

Acetaminophen hepatotoxicity is induced through a toxic metabolite, NAPQI, which is normally detoxified by hepatic GSH to form the acetaminophen-GSH conjugate.^[29] During exposure to an overdose of acetaminophen, the total hepatic GSH is depleted and NAPQI is significantly increased in the liver, interacting with a wide range of cellular proteins, disrupting their function, and causing damage to hepatocytes that finally results in liver failure.^[30]

The results obtained in the present study demonstrated that pre-treatment with EMPA offered significant protection against the aforementioned liver histopathological changes induced by acetaminophen. This was visibly evident in the histological examination of the liver and was demonstrated by the decrease in the previously mentioned degenerative and necrotic hepatocellular changes induced by acetaminophen intoxication. The hepatoprotective effect of oral doses (30 mg/kg) of EMPA was confirmed from the histological appearance of the liver of rats in group IV, as the liver restored its normal architecture, hepatocytes appeared with normal arrangement, and mitochondria appeared normal with closely packed cristae. However, minor degenerative changes were noticed, including minimal cytoplasmic vacuolation in hepatocytes and sinusoidal endothelium.

The preventive effects of EMPA against acetaminophen toxicity are mostly based on its antioxidant activity. This idea coincides with the results of recent researchers who reported that EMPA acts as an antioxidant, and that it increases the levels of non-enzymatic antioxidant GSH.^[31-33] Other investigators have considered that the beneficial effects of EMPA are mediated by its antioxidant defense ability and the scavenging of free radicals; furthermore, they stated that EMPA is multiple times more potent as an antioxidant compared with Vitamin E due to its anti-inflammatory property. In addition, EMPA can inhibit nuclear factor κB-mediated transcription of inflammatory cytokines.^[34,35] Accordingly, the amelioration of acetaminophen-induced liver damage by EMPA, as demonstrated in the present results, is suggested to be attributed to its antioxidant and anti-inflammatory properties, as it neutralizes free radicals, which are highly unstable molecules that can damage sub-cellular structures through abnormal oxidative reactions.

CONCLUSION

In summary, our results demonstrated that EMPA protected the mitochondria of hepatocytes. This drug ameliorated oxidative stress and decreased apoptosis in hepatocytes through the stabilization of mitochondrial structure and functions, which preserved hepatic architecture and inhibited the cytopathic effects. Low doses of EMPA may be effective in the earlier stages of AIH before the onset of liver cirrhosis and failure, because mitochondrial damage occurs quickly after hepatocyte injury. These results may provide new insights into a probable anti-fibrotic mechanism of EMPA. This study has many limitations that would be considered in extended research that may provide a more comprehensive understanding of the mechanisms involved in liver protection; increasing the number of animals per group to improve the statistical robustness of the findings; measurements of liver enzymes, and oxidative stress markers.