Liver toxicity and repair evaluated by histopathology and electric modulus

Abstract

Detoxification is one of the most important liver functions. Therefore, liver is the front line of defense when the biosystem faces drug overdose, toxins, and anything that may cause harm. Some famous antibiotics are known for their side effects on liver; one of them is amoxicillin, AM. This work has investigated the toxic effect of amoxicillin on rat’s liver with overdose (90 mg/kg) and has studied the ameliorative role of protective and therapeutic Ashwagandha seeds extract (ASE) at doses (100, 200, and 300 mg/kg) against this toxicity. To achieve this work, the authors used two modalities; the first is liver histopathology to figure out the amoxicillin and ASE effects and to detect the sensitivity of another modality; the electric modulus, and its related thermodynamic parameters of liver tissue. Histopathological examination showed that the role of therapeutic ASE in reducing amoxicillin (AM) toxicity was more effective than the protective one. Also, most dielectric and thermodynamic results achieved the same result. Histopathology confirmed the liver injury by amoxicillin and the partial repair by the biosystem using ASE. Moreover, electric modulus, related dielectric parameters, and their thermodynamic state functions showed different changes in their values under the effect of amoxicillin. Using ASE helped the biosystem to restore these changes near their control values.

Introduction

The liver is considered one of the largest organs in the human body (about 1,500 g). The liver makes further than 500 metabolic functions; it is summarized in the synthesis of products such as clotting factors, glucose resultant from glycogenesis, plasma proteins, and urea which are directed into the bloodstream. Liver parenchyma can store many products such as fat, fat-soluble vitamins, and glycogen. Furthermore, it is responsible for the production of bile which helps remove toxic substances.¹

Amoxicillin, AM, is an antibiotic drug that belongs to a class of Penicillins. It can fight a wide range of gram-positive and gram-negative bacteria in both humans and animals. Amoxicillin is used to treat a lot of diseases such as infections of the middle ear, throat, larynx, pharynx, bronchi, lungs, urinary tract, and skin.² Amoxicillin is combined with clavulanate to avoid degradation by enzymes like beta-lactamase, but, unfortunately, this drug is one of the drugs that can cause hepatotoxicity. Some hepatotoxic symptoms associated with amoxicillin toxicity are jaundice, mild-macular, non-petechial rash, increasing levels of lipid peroxidation (MAD) and liver enzymes; aspartate aminotransferase (AST), and alanine aminotransferase (ALT), in addition to cellular necrosis and cirrhosis.^3–6

Amoxicillin/Clavulanate is the third most optional antibiotic in the United States.^5,7

Therefore, in this work, the authors used the herbal supplement, Ashwagandha or W. somnifera, as a protective and therapeutic agent to ameliorate the hepatotoxicity of amoxicillin. The main bioactive components of Ashwagandha are called “withanolides”; these are mainly steroidal lactones with ergostane skeleton. Further than 40 withanolides, 12 alkaloids, as well as infrequent sitoindosides were discovered in this plant. Therefore, Ashwagandha is considered a neuro-protective herbal medicine, antistress, improves thinking ability and memory process, decreases pain and inflammation, and prevents aging effects. Moreover, it contains numerous natural antioxidants such as polyphenols that can preserve the liver enzymes, treat inflammation and fevers, and protect the immune system. Therefore, Ashwagandha helps in the therapy of several diseases such as asthma, bronchitis, leukoderma, Parkinson’s disease, and chronic liver diseases.^8–16

The impact of Ashwagandha root powder on liver marker enzymes in ammonium chloride-induced hyperammonemia was detected by Harikrishnan et al.¹⁷ Ammonium chloride caused a significant rise in the levels of liver enzymes, while these parameters were significantly reduced in rat groups treated with Ashwagandha root powder and ammonium chloride. The authors returned that to the existence of free radical scavengers; alkaloids, withanolides, flavonoids, and polyphenols in Ashwagandha root powder. Sultana et al.¹⁸ observed the effect of Ashwagandha root extract on gentamicin-intoxicated rats. They noticed that Ashwagandha extract reinstated the enzymes of the liver; AST and ALT to normal levels in the gentamicin-intoxicated group. They reverted this effect to the ability of Ashwagandha to scavenge the free radicals. Additionally, Manvitha et al.¹⁹ proved the protective role of Ashwagandha and selenium against chlorpyrifos (CPF) induced changes in hemato- biochemical factors and liver histopathology in rats.

From the previous works, we noticed that most studies focused on the effect of Ashwagandha root extract on several diseases, so in this work, we’d like to concentrate on the influence of Ashwagandha seed extract on amoxicillin-induced liver injury as an example of one of the problems that threaten human life. This was investigated by using simple measurements; histopathological examination and dielectric measurements of the liver which later proved their ability to investigate and diagnose different diseases in previous studies.^20–30

Materials and method

Drug preparation

An Amoxicillin drug was obtained from a local pharmacy. Amoxicillin (AM) capsule was dissolved in distilled water to form a dose of 90 mg/kg. b. wt.³¹ and gavage orally to rats once daily for twelve days.

Plant material

Ashwagandha seeds were obtained from the Department of Horticultural Crops Technology in our institute.

Preparation of Ashwagandha seeds extract

Aqueous extract of Ashwagandha seeds was prepared according to the method of Maheswari and Manisha,³² and Elnasharty et al.³³ After that, the dry extract was stored at −20C until used.

Experimental animals

Male Sprague–Dawley rats, about 120 g, were obtained from the lab of Animal House. The rats were kept in cages at a temperature of 25 ± 3C and given access to food and water. The handling of animals and all experimental procedures followed the ethics of animal care and approval from Medical Ethical Committee, No. (05440723).

Experimental design

The animal’s number used was 56 rats. They were divided randomly into eight groups (7 animals each) as follows:

Table 1.

Group	Treatment
Control (G1)	Only on basal diet was fed to animals.
Amoxicillin (G2)	Rats received a dose of 90 mg/kg amoxicillin for 12 days orally.
Protected groups with ASE (G3-5)	Rats were treated as G2. In addition, from the first day of amoxicillin administration, the groups (G3-5) received, orally, ASE (100, 200, and 300 mg/kg) respectively, two hrs. After the amoxicillin treatment, daily for 12 days.
Therapeutic groups with ASE (G6-8)	Animals were orally given amoxicillin dose of 90 mg/kg daily for 12 days. After 24 h. From the last amoxicillin dose, the groups (G6-8) were administrated, orally, with ASE (100, 200, and 300 mg/kg) respectively, for 7 days.

Histopathological examination

Tissue samples of the liver were separately collected after abdominal incision and fixed in 10% formaldehyde solution for 24 h then dehydrated through alcohols, 50%, 70%, 90%, 95%, and 100%, cleared in xylene, and then embedded in paraffin wax. Sections (5 μm thick) were stained with haematoxylin and eosin for histopathological examination, then processed using a standard protocol of Bancroft and Gamble,³⁴ then, the samples were examined under a light microscope.

Dielectric measurements

Sample preparation

Liver samples were collected after incision and fixed in formaldehyde, 10% solution. Samples were immersed in distilled water for 24 h. to remove formaldehyde, then they were dehydrated by a series of concentrations of ethyl alcohol; 50%, 70%, 90%, 95%, and 100%. Samples were positioned in alcohol for 30 min in each concentration respectively. Then dielectric measurements were performed.

Dielectric measurements

Dielectric measurements were performed on liver tissue using a Broadband dielectric spectrometer, Concept 40, from Novo Control, Germany. Using two brass electrodes the diameter of the small one was 10 mm and placed within the cell of the alpha impedance analyzer. The frequency range investigated from 0.1 Hz to 20 MHz, and 1 V_rms were applied. The temperature range of (10–40 C), was controlled, by the Quatro of Concept 40 system, within the measuring cabinet using N2 gas under vacuum with a temperature accuracy of 0.1 C.

High-loss materials have too much conductivity that conceals a relaxation process(es) between the material and the electric field. This renders information about the permittivity relaxation of material under study impossible by this way; from permittivity data. In such case comes the electric field modulus which is the inverse of the permittivity. Electric modulus unveils permittivity relaxation enabling extracting information from measured data.

The dielectric calculations of modulus were carried out by the formula:

(1)

where, M* is the complex electric modulus, is static permittivity, are shape parameters, is the dc conductivity relaxation time, is the angular frequency, is .

Thermodynamic state functions:

A] Helmholtz free energy change,

(2)

where,, and , are the Helmholtz free energies as functions of temperature, , when the electric field, , is switched on or off respectively and is the permittivity of free space.

B] Entropy change,

(3)

where,, , are the entropies as functions of temperature, , in the presence and absence of an electric field.

C] Internal energy change,

(4)

where,, and , are the internal energies as functions of temperature, , in the presence and absence of the applied .^{19,23,26,27,35,36}

Results and discussion

Liver histopathology

The control group (G1) shows the normal structure of liver tissue with normal hepatocytes arrangement, and portal area (thick arrow).

Amoxicillin group, AM, (G2) displays mild granular or vacuolar degeneration of hepatocytes (arrow), hepatic cord disarrangement, and hemorrhage in hepatic parenchyma (star).

Amoxicillin +ASE L.L (G3), or protected group with 100 mg/kg of ASE, displays portal vein with mild fibrous connective tissue proliferation in the portal area (triangle), atrophied hepatocytes (yellow arrow), and Kupffer cells (curved arrow).

Amoxicillin +ASE M.L (G4), or protected group with 200 mg/kg of ASE, demonstrates congested central blood vessels (star) with mild mononuclear cell infiltration (curved arrow), vacuolar degeneration of hepatocytes (arrow), mild fibrous connective tissues proliferation (yellow triangle) and Kupffer cells (thin arrow).

Amoxicillin +ASE H.L (G5), or protected group with 300 mg/kg of ASE, shows disarrangement of hepatic cords, vacuolar degeneration, and necrosis of hepatocytes (arrow), with hemorrhage in hepatic parenchyma (star).

ASE L.L (G6) or treated group with 100 mg/kg of ASE, displays hemorrhage in hepatic parenchyma (star), disarrangement of hepatic cords, vacuolar degeneration, and necrosis of hepatocytes (arrow).

ASE M.L (G7) or treated group with 200 mg/kg of ASE, shows vacuolar degeneration of hepatocytes (arrow), atrophied hepatocytes, the proliferation of Von Kupffer cells (curved arrow), and severe fibrous connective tissues (triangle).

ASE H.L (G8) or treated group with 300 mg/kg of ASE, shows atrophied hepatocytes, dilated congested portal vein (star), and proliferation of Von Kupffer cells (curved arrow).

The liver histopathology (Fig. 1) showed that a toxic dose of Amoxicillin 90 mg/kg caused various disorders in liver tissue. These disorders were observed in the degeneration of hepatocytes, disarrangement of hepatic cords, and hemorrhage in hepatic parenchyma.

Fig. 1.

Photomicrograph of liver of control, amoxicillin, protective and therapeutic groups.

Omodamiro, Ferreira et al., Khanna et al., Elnasharty et al., and Elizalde-Velazquez et al.^3,5,6,33,37 concluded that Amoxicillin can cause hepatotoxicity. One of the studies explained how amoxicillin can induce liver injury³⁸; they discovered that cytokines are up-regulated with Drug-Induced Liver Injury (DILI), especially, Tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ), by using a model of human hepatocytes with recognized three hepatotoxic drugs; amoxicillin, flucloxacillin, and isoniazid. According to that, they assumed that IFN-γ may induce the TNF-α formation, instigating cellular injuries and then cell death. Moreover, in other studies,^33,39 the effect of amoxicillin toxicity on different organs (liver, brain, and testes) was indicated by the elevation of malondialdehyde (MDA) and lowering of total antioxidant capacity (TAC).

On the other hand, protective ASE showed no remarkable protective effects on liver tissue at all doses, this was noticed in vacuolar degeneration and necrosis of hepatocytes, mild fibrous connective tissue proliferation, congested portal veins, and blood vessels, hemorrhagic patches in hepatic parenchyma and disarrangement of hepatic cords. In the case of therapeutic ASE, liver tissue showed various damages at 100 mg/kg of ASE such as hemorrhage in hepatic parenchyma, vacuolar degeneration and necrosis of hepatocytes, and disarrangement of hepatic cords, while at doses 200 and 300 mg/kg of ASE, they exposed less necrosis of hepatocytes and more intact hepatic parenchyma. However, the improvement of tissue was not enough to be healthy. So, the liver tissue still needs more time to recover and/or a higher dose of ASE. So, from these results, it can be said that the effect of ASE as a therapeutic agent at doses 200 and 300 mg/kg was better than its effect as a protective agent. However, the previous studies^33,39 indicated that ASE use, as a protective and therapeutic agent, improved the MDA and TAC values of liver pointing out two facts; the first is the occurrence of tissue toxicity upon the use of amoxicillin treatment, and the second was that the use of ASE managed to reduce the amoxicillin toxicity for both protection and therapeutic modalities.

The weak influence of ASE may be a result of three probabilities (Fig. 1): First is the interaction between Ashwagandha and Amoxicillin. In another study, the authors discovered drug interactions between Ashwagandha, alprazolam, and propranolol.⁴⁰ This may explain why the ASE effect, in our study, was more observable in the therapeutic case, meaning after preventing the consumption of amoxicillin drug and consequently stopping the interaction between ASE and amoxicillin drug. The second probability is that ASE caused a liver injury as mentioned in previous studies that worked on patients who developed cholestatic jaundice after consuming a herbal or chemical supplement containing Ashwagandha for a long time.^41–45 But the second probability is unlikely because it needs a long time of ASE consumption and this wasn’t the case in our study. The third is that utilizing the seed extract of Ashwagandha may be less effective on hepatotoxicity than the roots and leaves extract adding to the severe liver damage caused by overdose of amoxicillin.

Dielectric measurements

The electrical modulus and thermodynamic state functions were used before in other studies to investigate various biological systems; molecules, cells, and tissues.^{21–30,33,45} They proved to be good biosensors to biosystem cases in health, injury, and repair. However, this is the first case of the authors’ knowledge that studies the liver tissues by these parameters.

Electric modulus of liver

The use of electric modulus analysis overcomes the high conductivity of biological tissues which masks permittivity relaxation.^21–24,28 As shown, the upper part of (Fig. 2) depicts the complex electric modulus relaxation of the liver tissue, L, of control group, G1L. The modulus relaxation extends from 5 kHz to 20 MHz. The relaxation fitting at 35C as an example, using the Havriliak-Negami, HN, model for one peak, 1P, is represented in the lower part of (Fig. 2). Havriliak – Negami model was used to account for all accountable variations to the relaxation.

Fig. 2.

Upper part: Electric modulus, M,” of liver control at temperature range (10–40C) versus frequency. Lower part: Fitting of modulus real (M’) and imaginary (M”) parts of control at 35C.

The dielectric parameters extracted from fitting are relaxation time, and static permittivity. The thermodynamic state functions presumed from static permittivity are Helmholtz free energy, entropy, and internal energy. This fitting was calculated for all groups (G1L-G8L) using the equations (2–4).^{19,23,26,27,35,36}

Relaxation time of liver

The relaxation time (τ) represents the time requested for reinstating the original status of investigated material, approximately. Figure 3A and B shows relaxation time, Y axis, of amoxicillin group (G2L), protected groups of ASE (G3L-G5L), therapeutic groups (G6L-G8L), compared to control (G1L) with temperature range (283–315 K), X axis.

Fig. 3.

Relaxation time of liver with temperature (283–315 K) in response to amoxicillin, G2, and ASE: A) protected groups (G3-G5), and B) therapeutic groups (G6-G8) compared to control, G1.

As shown in (Fig. 3), Amoxicillin (AM) interaction with cellular components (G2) caused hindrance of relaxation leading to an increase in relaxation time, (Fig. 3A). This can be associated with the alteration of cellular formation shown in the histopathological examination of liver.

Concerning the ASE groups, the intermediate protection dose, G4, had better relaxation time than the lower dose; G4 is closer to control, G1. Data of the highest protection dose was not fitted, (see 2.6). Figure 3B, indicates that the highest therapeutic dose of ASE (300 mg/kg) was the best one in retaining the relaxation time to the control level followed by the intermediate dose (200 mg/kg). So, we can see that the intermediate protective and higher therapeutic doses of ASE have the best results approaching control values, then the intermediate therapeutic dose. Physically they were able to eliminate most of the amoxicillin hindrance for the relaxation process. These findings agree with that of liver histopathology.

Permittivity and free energy change of liver

Figure 4A and B signifies the static permittivity, ε_s, on the left Y axis and change in free energy, on the right side, of amoxicillin group (G2L), protected groups of ASE (G3L-G5L), therapeutic groups (G6L-G8L), compared to control (G1L) with temperature range (283–315 K).

Fig. 4.

Permittivity (ε_s) and change in free energy (ΔF/E²) of liver in response to amoxicillin, G2, & ASE: A) protected groups (G3-G5), B): Therapeutic groups (G6-G8) compared to control, G1.

Figure 4A displays that the amoxicillin dose, G2, decreased both parameters, permittivity, and change in free energy at lower temperatures (283–298 K), and had a non-significant effect in the normal body temperature range. ASE protection doses had a stronger effect on both parameters within the normal body temperature range except the lowest protective dose, G3, which was nearer to control. Figure 4B shows that in the case of therapeutic use of ASE following amoxicillin treatment; lower and intermediate doses, G6 and G7 respectively, had closer results to control. A high ASE therapeutic dose, G8, led to the elevation of both permittivity and free energy change to high levels that were far from control values.

Consequently, the lower protective dose of ASE gave better results for permittivity and free energy change; was able to reverse the amoxicillin effect and lower both parameters slightly below the control level at physiological temperatures. The protective intermediate dose added more stress on the liver tissues raising both parameters above the amoxicillin level. This again assures that ASE has another effect on the cells and tissues other than helping tissues reverse the amoxicillin effect, which should be studied separately. This outcome was mentioned in other studies,^40–44 where Ashwagandha supplement caused some liver injuries including jaundice. This result can also support the possibility of drug-Ashwagandha interaction that was pointed to previously.

Entropy change of liver

Entropy represents the disorder degree of the studied system. Figure 5A and B shows the entropy change in response to amoxicillin treatment and ASE as a protective (G3-G5) and therapeutic (G6-G8) agents.

Fig. 5.

Entropy change of liver in response to amoxicillin, G2, & ASE: A) protected groups (G3-G5), B): Therapeutic groups (G6-G8) compared to control, G1.

In (Fig. 5A), data showed that amoxicillin treatment (G2) raised the entropy change compared to control, while the use of protective doses of ASE, especially the lower dose (G3), helped to reduce this change. Therapeutic use of ASE subsequent amoxicillin treatment revealed that the intermediate dose (G7) had the closest results to the control, (Fig. 5B). Where the highest ASE dose (G8) had the least entropy change, even than control.

Entropy changes indicate high disturbance caused by amoxicillin treatment. Using ASE along with amoxicillin as a protective natural supplement decreased the disturbance produced by amoxicillin. The therapeutic use of ASE raises a question about its effect on the liver tissue from entropy’s point of view, which requires another investigation, of whether this decrement of entropy change is due to more stability brought to the biosystem by the supplement or more stress applied to the liver cells leading to an entropy decrement.

Internal energy change of liver

Change in the internal energy is the sum of both heat transfer and performed work. Figure 6A and B revealed the internal energy change in response to amoxicillin treatment and ASE (protective and therapeutic doses).

Fig. 6.

Internal energy change of liver in response to amoxicillin, G2, and ASE: A) protected groups (G3-G5), B): Therapeutic groups (G6-G8) compared to control, G1.

In (Fig. 6A), amoxicillin treatment caused an increase in internal energy change. On the other hand, the protective ASE doses decreased this change; the lower dose (G3) had the most effect. Therefore, ASE protective doses helped the liver tissues to decrease amoxicillin effects.

Therapeutic role of ASE in the internal energy change was different, (Fig. 6B); the lowest ASE dose was not as effective as it was when used for protection. The therapeutic effect of ASE required the highest dose (G8) to approach the control level of internal energy change.

Data of internal energy change show that amoxicillin treatment, 90 mg/kg for 12 days, applied a lot of stress imposing more work and heat on liver cells to detoxify amoxicillin interactions within cells. The lower protective ASE dose was better. As more of ASE, the intermediate dose, caused internal energy change to move further away from control. On the other hand, the lower therapeutic dose was not enough to get the internal energy close enough to the control. The intermediate therapeutic dose achieved better results. The high therapeutic dose returned the internal energy to a lower level than that of the control. These data tell us that the ASE has its own stress on the liver tissue, which needs further investigation.

Modulus, M,” spectra of liver tissues

All previous fitting data have been extracted from the relaxation peak of the liver Modulus. In this part we have shown this peak of all groups in (Fig. 7), where G1 showed the control group, G2 displayed the effect of amoxicillin on the modulus relaxation where the low-frequency part of the peak was condensed and peak height was slightly decreased. As for protection groups, G3, low and intermediate ASE doses, expressed little condensation of the left half of the modulus peak that increases with G4. Both G3 and G4 decreased the height of the modulus peak. High ASE dose, G5, split the modulus peak into two adjacent peaks and widened the frequency range of the modulus relaxations to extend from about 6 Hz to 20 MHz. G5 showed also a greater decrease in peak’s height. At low temperatures up to 30C, peak height declines much more than the physiological temperatures, 35–40C, and the two peaks are more separated. At physiological temperatures, the two peaks come closer together, with temperature, and peak height increased but still less than that of the control. This is mostly due to the kinetic energy gained by charge carriers from the higher temperature which energizes them all so their speeds become closer. This splitting of the peak is not noticed either for amoxicillin alone or for the same therapeutic ASE dose, 300 mg/kg in the absence of amoxicillin. This means the splitting effect is either due to high stress on the liver tissue due to both amoxicillin and high ASE dose and/ or an interaction between amoxicillin and ASE components that affected the liver tissues and led to the splitting of the modulus peak. This suggestion agrees with the study of Inagaki et al.³⁹ which confirmed the interaction of Ashwagandha with different types of drugs. On the other hand, therapeutic ASE showed slight condensation of the lower frequency part of modulus relaxation peak, and also, the peak’s height decreased with the ASE doses.

Fig. 7.

Imaginary part of the modulus, M,” for control (G1), amoxicillin (G2), and ASE protective (G3-5)and therapeutic (G6-8) groups.

This means drug and ASE were able to hinder a considerable amount of charge carriers causing them to lag forming another relaxation time and/or creating other compounds through their interaction with each other or with liver cells. These newly created compounds produced the second peak appearing in the electric modulus relaxation.

Conclusion

This work concluded that amoxicillin has a toxic impact on liver tissue as shown in histopathological examination. The role of ASE as a therapeutic agent was relatively more positively effective than the protective role, especially at 200 and 300 mg/kg.

Dielectric and thermodynamic data illustrated that amoxicillin consumption, 90 mg/kg/day for 12 days, caused a significant increase in relaxation time, while the intermediate protective (200 mg/kg) and in therapeutic doses of ASE the lower and higher doses receded its values near control. Amoxicillin drug had little effect on permittivity and free energy change. Lower and intermediate therapeutic doses of ASE had results close to control. In contrast, amoxicillin treatment caused high disturbance for entropy changes, while using protective ASE decreased this disturbance. For the internal energy change, amoxicillin raised it significantly. A lower protective dose and the highest one of therapeutic ASE helped to reduce amoxicillin effects.

In addition, the results of electric modulus relaxation peak of liver showed that ASE interacts with amoxicillin and the interaction had its maximum effect at a high ASE protective dose, which along with amoxicillin, causing the relaxation peak to split up into two relaxations.

Finally, Histopathological analysis and physical measurements proved that ASE has a more positive effect in case of therapy and it should not be used in high doses with drugs for the probability of inducing undesirable interactions.

Author contributions

Azhar M. Elwan, Methodology, histology, biophysical measurements, data analysis, writing and reviewing.

Ibrahim M Farag, Animal care.

Mohamed M.M. Elnasharty, Methodology, histology, biophysical measurements, data analysis, writing and reviewing.

Funding

Authors declare that they have not received any funds for this work.

Conflict of interest statement. Authors declare that they have no competing interests about the paper.

Data availability

Data will be available upon reasonable request.