Toxicological Evaluation of Kaempferol and Linearolactone as Treatments for Amoebic Liver Abscess Development in Mesocricetus auratus

Abstract

Several studies with kaempferol (KP) and linearolactone (LL) have demonstrated their antiparasitic activity. However, the toxicity of these treatments is unknown. Therefore, this study aimed to evaluate the possible toxicological effects of intraperitoneal (i.p.) administration of KP or LL on the amoebic liver abscess model (ALA) in Mesocricetus auratus. An ALA was induced in male hamsters with 1.5 × 10⁵Entamoeba histolytica (E. histolytica) trophozoites inoculated in the left hepatic lobe. The lesion evolved for 4 days, and then KP (5 mg/kg body weight/day) or LL (10 mg/kg body weight/day) was administered for 4 consecutive days. Then, magnetic resonance imaging (MRI), paraclinical analyses, and necropsy for histopathological evaluation were performed. There was similar ALA inhibition by KP (19.42%), LL (28.16%), and metronidazole, the antiamoebic control (20.87%) (p ≤ 0.05, analysis of variance [ANOVA]). There were hepatic and renal biochemical alterations in all treatment groups, mainly for KP (aspartate aminotransferase: 347.5 ± 37.5 U/L; blood urea nitrogen: 19.4 ± 1.9 g/dL; p ≤ 0.05, ANOVA). Lesions found in the organs were directly linked to the pathology. In conclusion, KP and LL decreased ALA development and exerted fewer toxicological effects compared with metronidazole. Therefore, both compounds exhibit therapeutic potential as an alternative treatment of amoebiasis caused by E. histolytica. However, additional clinical studies in different contexts are required to reaffirm this assertion.

Keywords: Entamoeba histolytica; Mesocricetus auratus; amoebic liver abscess; kaempferol; linearolactone; toxicity.

Figure 1

The cytotoxic effects of kaempferol (KP) and linearolactone (LL) on the CHOK-1 and BEAS2B cell lines. The half-maximal inhibitory concentrations (IC₅₀) of KP and LL in the CHO-K1 (A) and BEAS-2B (B) cell lines were determined by dose-response viability curves after 24 h using the MTT assay. The results are presented as the mean ± standard deviation of three biological replicates (n = 3, in triplicate). (*) p ≤ 0.05 vs. vehicle (1× phosphate-buffered saline, negative control) by analysis of variance. Metronidazole (MTZ) was used as a positive control (antiparasitic reference).

Figure 2

Paraclinical analyses of blood and serum from the five groups of hamsters. Nutritional parameters (A), biochemical parameters (B), ratios (C), and hematological parameters (D) were evaluated (for details, see Table S1). The results are presented as the mean ± standard deviation (A,B,D) or the median and range (C) of two biological replicates (n = 5). * p ≤ 0.05 vs. the control group by analysis of variance (red for the disease-free group, green for the Vh group, and yellow for both groups). Metronidazole (MTZ) was used as a positive control, vehicle (Vh, 1× phosphate-buffered saline, no treatment) was used as a negative control, and disease-free (without ALA) was used as a normal control. ALP, alkaline phosphatase; ALT, alanine aminotransferase (GPT); AST, aspartate aminotransferase (GOT); GGT, gamma glutamyl-transpeptidase; TB, total bilirubin; AL, albumin; BUN, blood urea nitrogen; CR, creatinine; DB, direct bilirubin; Hb, hemoglobin (g/dL); HCT, hematocrit (%); PCT, plateletcrit (%); RBC, red blood cells (×10⁶/mm³); WBC, white blood cells (×100/mm³); PLT, platelets (×10⁶/μL).

Figure 3

Histopathological analysis of the organs collected from the five groups of hamsters. The photographs show the anatomical morphology of the kidneys, heart, spleen, and lungs collected from the hamsters (A). Representative liver and kidney sections stained with hematoxylin and eosin are shown at 10× and 40× magnification (B). Digital zoom with 40× magnification of liver sections stained with hematoxylin and eosin clearly shows the following tissue characteristics: hepatocytes (H), sinusoids (S), Kupffer cells (K), lymphocytes (L), focal intrahepatic cholestasis (gray arrow), bile canaliculi (black arrow), and lipid microvesicles (white arrow) (C). The representative results from two biological replicates (n = 5) are shown. KP, kaempferol; LL, linearolactone. Metronidazole (MTZ) was used as a positive control, vehicle (Vh, 1× phosphate-buffered saline) was used as a negative control, and the disease-free status was used as a normal control.

Figure 4

Predictions of the pharmacokinetics, toxicity, and molecular targets of KP and LL based on their physicochemical properties. The molecular properties and chemical structures of (A) KP and (B) LL were used to predict similarities with biologically active drugs through the Molsoft© drug similarity model and their potential target pharmacophores using SIB© SwissTarget prediction. (C) The toxicological activity of both active principles, similarly to antipara-sitic drugs in humans, was predicted through inhibitory effects on the enzyme complex associated with CYP using a predictive machine learning model based on specific fingerprints called MCF with SuperCYPsPred of Structural Bioinformatics©. (D) Other toxicity characteristics, such as BBB permeability, HIA, and PGP affinity, were predicted by the SIB© SwissADME boiled egg permeation method. The results were compared with metronidazole (MTZ), an amoebicidal compound used to treat diverse parasitic diseases (see Supplementary Materials). Abbreviations used: KP, kaempferol; LL, linearolactone; MCF, most common features; BBB, blood-brain barrier; HIA, health impact assessment; PGP, P-glycoprotein; CYP, cytochrome-P450 system; HBA, hydrogen bond acceptors; HBD, hydrogen bond donors; log P, partition coefficient; log S, aqueous solubility coefficient; pKa, acid dissociation coefficient; PSA, polar surface area; LIPO, lipophilicity; POLAR, polarity; INSOLU, insolubility; INSATU, insaturation; FLEX, flexibility, NOX4, NADPH oxidase IV; OPRK, Kappa Opioid receptor.

Figure 5

Timeline of the amoebic liver abscess model (A) and antiparasitic activity protocol (B) implemented for KP and LL. The antiprotozoal activity of kaempferol (KP) and linearolactone (LL) was determined in Entamoeba histolytica trophozoites xenotransplanted into the livers of male Mesocricetus auratus (Syrian hamsters), according to the protocol proposed by Tsutsumi et al. [18] and the Official Mexican Regulations (NOM-062-ZOO-1999) [72]. Figure created in BioRender by Talamas-Rohana, P. (accessed on 14 September 2024).