Toxicological Evaluation of Kaempferol and Linearolactone as Treatments for Amoebic Liver Abscess Development in Mesocricetus auratus
Abstract
:1. Introduction
2. Results
2.1. Cytotoxic Effect of the Active Principles KP and LL in Cell Lines
2.2. Treatment with KP or LL Selectively Induced Cell Death and Morphological Alterations in Trophozoites but Not in Normal Cells
2.3. The Effect of Treatment with KP or LL in the M. auratus ALA Model
2.4. Paraclinical Analysis of Post-Treatment Liver and Kidney Function
2.4.1. Evaluation of the Hepatic Function
2.4.2. Evaluation of the Renal Function
2.4.3. Determination of the Liver Injury Ratios
2.4.4. Determination of the ALA Prognosis Ratios
2.4.5. Determination of the Drugs Toxicity Ratios
2.4.6. Determination of the Kidney Damage Ratios
2.4.7. Evaluation of the Hematic Biometry
2.4.8. Determination of the Hematic Ratios
2.5. Treatment with LL or KP Effectively Inhibited the Development of ALA in M. auratus
2.6. Toxicological Evaluation of Hamsters Treated with KP or LL
2.7. In Silico Analyses to Determine the Toxicity of KP and LL
3. Discussion
4. Materials and Methods
4.1. Chemical Compounds
4.2. Cell Cultures
4.3. Cytotoxicity Assays in Normal Cell Lines by Formazan Salts
4.4. Cell Death Determination by Flow Cytometry
4.5. Cell Morphology by Confocal Microscopy
4.6. Ultrastructural Morphology Analysis with TEM
4.7. Hamster Model for ALA
4.8. ALA Induction and the Antiparasitic Protocol
4.9. MRI
4.10. Paraclinical Analysis
4.11. Histopathological Analysis
4.12. In Silico Analyses
4.13. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
References
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In Vitro Studies: IC50, SI and LD50 | ||||
---|---|---|---|---|
Samples | Vh | MTZ | KP | LL |
IC50 HM-1 | Innocuous | 0.16 ++ * | 31.6 + * | <7.8 + * |
IC50 CHO-K1 | Innocuous | 500 - * | 70 +/- * | 300 - * |
IC50 BEAS-2B | Innocuous | 400 - * | 100 +/- * | 200 - * |
SI | ND | 3461 ++ | 2.7 +/- | >32 + |
LD50 (Theoretical) | ND | 1025.64 | 551.75 | 824.20 |
In Vivo Studies: Acute Toxicity and ALA Characteristics | ||||
---|---|---|---|---|
Treatments | Vh | MTZ | KP | LL |
Hamster weight (g) | 91.9 ± 2.3 | 95.4 ± 5.1 | 93.5 ± 6 | 93.4 ± 7.2 |
Toxicity signs | None | None | None | None |
Survival rate (%) | 100 | 100 | 100 | 100 |
ALA Weight (g) | 1.23 ± 0.15 | 0.33 ± 0.32 * | 0.86 ± 0.15 * | 0.40 ± 0.20 * |
ALA Volume (mm3) | 2.257 ± 523.7 | 528 ± 183.5 * | 738 ± 150 * | 562.3 ± 23.3 * |
MRI (T1/T2) | Intermediate intensity/ hyperintense | Intermediate intensity/ hypointense | Intermediate intensity/ hypointense | Intermediate intensity/ hypointense |
Leukocyte infiltrate | ++ | − | +/− | +/− |
Fibrosis | + | −− | −− | −− |
Liquid | ++ | − | +/− | − |
Edges | Irregular and poorly defined | Regular and well defined | Irregular and poorly defined | Regular and well defined |
Morphology | Loculated ovoid | Round | Ovoid | Loculated ovoid |
Treatments (%) Hematological Parameters | Disease-Free | Vh | MTZ | KP | LL | Reference Range (Mean) |
---|---|---|---|---|---|---|
Lymphocytes | 42 ± 4 | 35 ± 13 | 50 ± 13 | 39 ± 12 | 44 ± 21 | 40–85 (63) |
Monocytes | 0 | 0 | 0 | 0 | 0 | 1–6 (3) |
Eosinophils | 0 | 0 | 0 | 0 | 0 | 1–2 |
Basophils | 0 | 0 | 0 | 0 | 0 | 0–5 (2) |
Segmented neutrophils | 54 ± 2 | 64 ± 14 | 51 ± 13 | 60 ± 11 | 56 ± 21 | 25–55 (40) |
Band neutrophils | 4 ± 2 | 2 ± 1 | 0 | 2 ± 1 | 0 | 5–13 (9) |
Immature forms | 0 | 0 | 0 | 0 | 0 | 0 |
Organs | Disease-Free | Vh | MTZ | KP | LL |
---|---|---|---|---|---|
Liver | 6.7 ± 0.07/4.04 ± 0.05 | 8.28 ± 1.1 */4.7 ± 0.5 * | 5.76 ± 1.4/4 ± 0.15 | 5.9 ± 0.8/3.88 ± 0.11 | 5.5 ± 0.9/3.94 ± 0.16 |
Heart | 0.4 ± 0.0/0.8 ± 0.09 | 0.38 ± 0.05/1.06 ± 0.09 | 0.38 ± 0.1/1.08 ± 0.04 | 0.38 ± 0.1/1.06 ± 0.08 | 0.38 ± 0.08/1.1 ± 0.07 |
Kidneys | 0.65 ± 0.07/1.2 ± 0.14 | 0.62 ± 0.03/1.2 ± 0.07 | 0.52 ± 0.0/1.3 ± 0.08 | 0.49 ± 0.04/1.2 ± 0.1 | 0.49 ± 0.07/1.1 ± 0.08 |
Lungs | 0.67 ± 0.0/2.8 ± 0.2 | 0.7 ± 0.05/2.6 ± 0.2 | 0.68 ± 0.05/2.5 ± 0.2 | 0.74 ± 0.06/2.5 ± 0.05 | 0.74 ± 0.06/2.4 ± 0.1 |
Spleen | 0.2 ± 0.0/3.4 ± 0.38 | 0.2 ± 0.0/3.30 ± 0.4 | 0.2 ± 0.05/3.3 ± 0.3 | 0.36 ± 0.05/3.7 ± 0.1 | 0.26 ± 0.05/3.4 ± 0.4 |
Samples | MTZ | KP | LL |
---|---|---|---|
SwissADME©: Physicochemical Properties | |||
Density: | 1.5 ± 0.1 g/cm3 | 1.7 ± 0.1 g/cm3 | 1.3 ± 0.1 g/cm3 |
Refraction index: | 1.612 | 1.785 | 1.612 |
Polarizability: | 16.2 ± 0.5 × 10−24 cm3 | 28.3 ± 0.5 × 10−24 cm3 | 35.0 ± 0.5 × 10−24 cm3 |
Surface tension: | 60.5 ± 7.0 dyne/cm | 98.9 ± 3.0 dyne/cm | 54.6 ± 5.0 dyne/cm |
Number heavy atoms: | 12 | 21 | 25 |
Number of bonds: | 12 | 23 | 29 |
Number of rings: | 1 | 3 | 5 |
Number aromatic heavy atoms: | 5 | 16 | 5 |
Fraction Csp3: | 0.50 | 0.00 | 0.50 |
Number rotatable bonds: | 3 | 1 | 1 |
Total charge: | 0.0 | 0.0 | 0.0 |
Molar refractivity: | 43.25 Å | 76.01 Å | 88.42 Å |
SwissADME©: Pharmacokinetics/Molinspiration©: Bioactivity score | |||
GI absorption: | High | High | High |
P-gp substrate: | No | No | No |
Log Kp: | −7.36 cm/s | −6.70 cm/s | −6.37 cm/s |
GPCR ligand: | −1.09 | −0.10 | 0.65 |
Ion channel modulator: | −0.87 | −0.21 | 0.16 |
Kinase inhibitor: | −0.59 | 0.21 | −0.13 |
Nuclear receptor ligand: | −1.74 | 0.32 | 0.66 |
Protease inhibitor: | −1.68 | −0.27 | 0.04 |
Enzyme inhibitor: | −0.32 | 0.26 | 0.47 |
SwissADME©: Medicinal Chemistry | |||
PAINS: | 0 alert | 1 alert (catechol A) | 0 alert |
Brenk: | 2 alerts (nitro group) | 1 alert (catechol A) | 1 alert (>2 esters) |
Leadlikeness: | No (M.W. < 250) | Yes | Yes |
Synthetic accessibility: | 2.30 | 3.14 | 5.56 |
T.E.S.T.© and LAZAR©: Toxicological properties | |||
LD50 Fathead minnow (96 h): | 424.1 mg/L | 1.28 mg/L | ND |
LD50 Daphnia magna (48 h): | 39.14 mg/L | 3.62 mg/L | ND |
IGC50 T. pyriformis (48 h): | 270.22 | 10.54 mg/L | ND |
LD50 Rat (Oral): | 2444 | 2018 mg/kg | ND |
Bioconcentration factor: | 1.914 | 8.032 | ND |
Developmental toxicity: | Not | Yes | ND |
AMES mutagenicity: | Yes (p = 0.67) | Yes (p = 0.42) | ND |
Carcinogenicity (rodents): | ND | No (p = 0.43) | ND |
Adverse effects (rat): | ND | 1320 mg/kg/day | ND |
Estrogen Receptor RBA: | 5.089 × 10−4 | 0.004 | ND |
Estrogen Receptor Binding: | Yes | Yes | ND |
Groups | n | Treatment | Administration Route | Doses/Time |
---|---|---|---|---|
I (normal control, without ALA) | 5 | Disease-free | i.p. | N/A |
II (negative control, non-treatment) | 5 | Vh (1× PBS) | i.p. | 200 µL/day |
III (positive control, antiamoebic drug) | 5 | MTZ | i.p. | 10 mg/kg body weight/day |
IV (sample) | 5 | KP | i.p. | 5 mg/kg body weight/day |
V (sample) | 5 | LL | i.p. | 10 mg/kg body weight/day |
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Varela-Rodríguez, L.; Calzada, F.; Velázquez-Domínguez, J.A.; Hernández-Ramírez, V.I.; Varela-Rodríguez, H.; Bautista, E.; Herrera-Martínez, M.; Pichardo-Hernández, D.L.; Castellanos-Mijangos, R.D.; Chávez-Munguía, B.; et al. Toxicological Evaluation of Kaempferol and Linearolactone as Treatments for Amoebic Liver Abscess Development in Mesocricetus auratus. Int. J. Mol. Sci. 2024, 25, 10633. https://doi.org/10.3390/ijms251910633
Varela-Rodríguez L, Calzada F, Velázquez-Domínguez JA, Hernández-Ramírez VI, Varela-Rodríguez H, Bautista E, Herrera-Martínez M, Pichardo-Hernández DL, Castellanos-Mijangos RD, Chávez-Munguía B, et al. Toxicological Evaluation of Kaempferol and Linearolactone as Treatments for Amoebic Liver Abscess Development in Mesocricetus auratus. International Journal of Molecular Sciences. 2024; 25(19):10633. https://doi.org/10.3390/ijms251910633
Chicago/Turabian StyleVarela-Rodríguez, Luis, Fernando Calzada, José Antonio Velázquez-Domínguez, Verónica Ivonne Hernández-Ramírez, Hugo Varela-Rodríguez, Elihú Bautista, Mayra Herrera-Martínez, Diana Laura Pichardo-Hernández, Rodrigo Daniel Castellanos-Mijangos, Bibiana Chávez-Munguía, and et al. 2024. "Toxicological Evaluation of Kaempferol and Linearolactone as Treatments for Amoebic Liver Abscess Development in Mesocricetus auratus" International Journal of Molecular Sciences 25, no. 19: 10633. https://doi.org/10.3390/ijms251910633