Colchicine causes prenatal cell toxicity and increases tetraploid risk
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Colchicine is a clinical medicine used for relief from gout and familial Mediterranean fever. Because of its toxic effects, intravenous injection of colchicine has been banned, but it is still widely administered orally. We assayed the toxic effects of colchicine in cultured primary chorionic villus cells and amniotic fluid cells to interpret its influence on the placenta and foetus.
Bright field record and cell count kit 8 were used to value cell viability. Flow cytometer was used to identify cells markers, cell cycle and cell apoptosis. G-banding was used for karyotype analysis for sample genetic and drug effect evaluation.
Chorionic villus cells and amniotic fluid cells were characterized as mesenchymal cells that share most cell surface markers and have a similar response to colchicine. Colchicine did not induce a decline in cell viability at low concentrations but suppressed cell proliferation by arresting the cell cycle in the G2/M phase and increased the risk of tetraploid generation in a small subset of cases.
Our study revealed the results of a colchicine-induced toxicity test in prenatal cells and determined the anti-mitotic biologically functional dose and manner of administration that might reduce the risk of tetraploid generation.
KeywordsColchicine Chorionic villus cells Amniotic fluid cells Cell cycle arrest Tetraploid generation
Amniotic fluid cells
Cell Counting Kit-8
Cluster of Differentiation
Chorionic villus cells
Cytochrome P450 proteins
Food and Drug Administration
familial Mediterranean fever
Colchicine is a natural product extracted from the autumn crocus (Colchicum autumnale, the name is derives from the Greek word) plant, which is a taxonomic species within the family Colchicaceae. Owing to its biological activity, colchicine has been used in medical care for centuries. There are two main medical uses of colchicine: first, colchicine was regarded as an anti-inflammatory agent used in treating familial Mediterranean fever (FMF), Behcet’s disease and gout; second, colchicine was used as a cardioprotective agent to treat acute and recurrent pericarditis and other cardiovascular-associated abnormalities . In 2009, the United States Food and Drug Administration (FDA) approved colchicine for the treatment of gout and FMF. Based on its pharmacological effects, some clinical trials are in progress and, for some, the results have recently been published, such as those on stroke prevention  and enhanced graft survival , creating new means for treating disease and promoting wide colchicine medicinal usage. Colchicine is one of the oldest drugs still used today.
Side effects of colchicine accompany its medical usage. At recommended doses, 10% of the patients show some gastrointestinal distress syndromes, such as nausea, vomiting, and particularly diarrhoea. With overdose, it is highly toxic, even fatal . Considering the side effects, the FDA withdrew approval for intravenous colchicine and provided guidelines for reducing the recommended dose and for concomitant use of CYP3A4 and P-glycoprotein inhibitors , which target the toxic effects caused by pharmacological use of colchicine.
Most toxicological research on colchicine focuses on treating patient but seldomly focuses on pregnant women or their foetuses. Chorionic villus tissue is a constituent of the placenta, and chorionic villus cells (CVCs) are mesenchymal cells derived from the chorionic villus, from which biopsy samples are taken at the end of the first trimester. Amniotic fluid contains foetal membrane cells and foetal cells, and amniotic fluid cells (AFCs) are mesenchymal cells derived from the amniotic fluid and are extracted during the second trimester. Primary cultures of CVCs and AFCs to obtain chromosomes for G-banding are widely used in prenatal genetic diagnoses. CVCs and AFCs also have with the same biochemical and genetic characteristics as the placenta and foetus, which are in contact with the amniotic fluid. For this study, we used cultured primary chorionic villus cells (CVCs) and amniotic fluid cells (AFCs) to evaluate the toxic effects of colchicine on the placenta and foetus.
This study was approved by the Ethics Committee of The Third Affiliated Hospital of Guangzhou Medical University (Approval number: 2017–001). Six pregnant patients signed informed consent and were told that the diagnostic sample would be used for research. Four pregnant women at 18–21 weeks gestation were undergoing amniocentesis, and two at 12 weeks gestation were undergoing chorionic villus sampling. After the cytogenetic diagnoses were completed, the four AFC lines and two CVC lines were used in this study.
The clinical samples were 8 ml amniotic fluid or 100 g chorionic villus tissue for each patient. For primary culture, amniotic fluid was centrifuged in 300G and chorionic villus tissue was cut into approximately 1 mm2 tissue masses. Then, cell precipitates (or the tissue masses) were resuspended in 4 ml amnioMAX-C100 medium (Cat#12558011, Invitrogen Life Technologies, Carlsbad, CA, USA) and seeded into two 25-cm2 culture flasks where they were maintained for 7–9 days at 37 °C in 5% CO2. Cells were detached by 0.05% trypsin (Cat#25300054, GIBCO, Invitrogen China Limited, Shanghai, China) for subculture. Clinical cytogenetic diagnosis was performed on passage 1. AFCs and CVCs were subcultured in complete MEM medium (Cat#12571071, GIBCO) supplemented with 20% foetal calf serum (Cat#10100, GIBCO) and 100 U/ml of penicillin and streptomycin (Cat#10378016, Invitrogen). Cells from passages 3–5 were used for the toxicity assays of colchicine (Cat# S2284, Selleck Chemicals, Houston, TX, USA). Colchicine was dissolved in MEM medium, for dose-dependent, cells were treated by 0, 0.15, 0.3, 0.6, 1.2 and 2.4 μg/ml colchicine for 3 h; for time-dependent, cells were treated by 0.15 μg/ml colchicine for 0, 12, 24, 48 and 72 h. As colchicine diluted in MEM, untreated group was control.
The P1 subcultured cells were treated with 0.15 μg/ml colchicine for 3 h, detached by 0.05% trypsin, subjected to hypotonic treatment with 0.02% sodium citrate and 2% potassium chloride and fixed in a mixture of methanol and acetic acid in 3:1 (volume to volume). The fix cells were dripped on cold slides and stained with Giemsa for metaphase chromosome analysis that was conducted with an Ikaros system (Carl Zeiss AG, Oberkochen, Germany).
Cell viability assay
A total of 5000 cells were counted in each well of 96-well plates, with triplicate sets for each group. After treatment with different drug concentrations at specific time points, the cell viability was assessed using a commercial CCK-8 (Cell Counting Kit-8) assay (Cat#C0039, Beyotime Biotechnology, Shanghai, China).
Cell surface marker assays
The cells were detached by trypsin and stained by the following fluorescence-conjugated antibodies: CD14-APC, CD34-PE, CD45-FITC, CD105-FITC, CD29-PE, CD44-FITC and CD73-APC. The population analysis was conducted on an Attune NxT flow cytometer (Invitrogen).
Cell cycle assay
Drug-treated cells were detached by trypsin and fixed in 70% ethanol overnight. The fixed cells were assayed using a cell cycle and apoptosis analysis kit (Cat#C1052, Beyotime Biotechnology) on a flow cytometer. The fluorescence intensity was used to distinguish different phases of the cell cycle.
Cell apoptosis assay
1 × 106 cells were seeded in to a 100 cm2 dish overnight for attachment. Drug treatment was accessed by change medium supplemented with 0.15 μg/ml colchicine. After drug treatment, the cells were stained with an annexin V-FITC apoptosis detection kit (Cat#C1062M, Beyotime Biotechnology) and counted by a flow cytometer for analysis. The cells in early apoptosis were the annexin V-positive and PI (propidium iodide)-negative cells, and the cells in late apoptosis were the annexin V- and PI-positive cells.
Statistical analysis of diploid and tetraploid cells in the prenatal samples
Tetraploid VS. Diploid
0.075 μM Colchicine (P Value)
0.15 μM Colchicine (P Value)
2 VS. 20
0 VS. 15 (>0.05)
0 VS. 19 (>0.05)
10 VS. 89
3 VS. 49 (>0.05)
8 VS. 65 (>0.05)
0 VS. 15
4 VS. 22 (>0.05)
2 VS. 25 (>0.05)
2 VS. 25
3 VS. 29 (>0.05)
10 VS. 19 (0.014)
1 VS. 78
3 VS. 87 (>0.05)
0 VS. 90 (>0.05)
3 VS. 80
5 VS. 97 (>0.05)
2 VS. 61 (>0.05)
Isolation, culture and characteristics of prenatal cells
Colchicine affects cell viability in a time- and dose-dependent manner
Colchicine reduces cell proliferation ability
Colchicine causes cell cycle arrest and induces polyploids
Pregnant women exhibit specific and substantially different anatomical and physiological characteristics, resulting in the necessity to evaluate drug pharmacokinetics, functional mechanisms and toxic effects comprehensively. On one hand, the drug must benefit the individual; on the other side, attention must be paid to minimize any side effects not only in the mother but also in the foetus . Colchicine is a clinical medicine that has been in continuous use, and epidemiological studies have suggested that clinical colchicine usage is not associated with foetal malformations or miscarriage, but its anti-mitotic properties may cause pre-term birth, short gestational age and low birthweight . In this study, we used primary prenatal cells to evaluate the cell toxicity induced by colchicine and to uncover the cell biology of the colchicine-induced toxicity in the placenta and foetus.
The CVC and AFC models can be used to evaluate drug cell toxicity to the placenta and foetus. Primary cultures of CVCs and AFCs are widely used in prenatal diagnosis, especially as the foundation of cytogenetic determination. The AFCs and CVCs were harvested at different gestational ages and represent different tissues. Chorionic villus tissue was acquired by biopsy of the placenta at 11–12 weeks of gestation, which was an important period of its growth and maturity . The CVCs were acquired from and represent the placenta. Amniotic fluid was extracted at 18–21 weeks gestation, at which time, the primary circulatory system of the foetus is established . The amniotic fluid is the urine of the foetus, being mainly composed of water but also containing foetal faeces and exfoliated cells of the body and amniotic membranes. The acquired AFCs were regarded as foetal cells. In clinical diagnosis, the CVC genetic report reflects the placenta, and analysis of the AFCs was used to diagnose the foetus, especially for mosaicisms . We used clinically obtained CVCs and AFCs to test colchicine-induced toxicity, partly to determine the influence on the placenta and foetus body. The morphology of passaged CVCs and AFCs differed, with CVCs being slender with clear boundaries, and the AFCs being flat; both were homogeneous as determined by bright field observation. Actually, CVCs  and AFCs  were mesenchymal stromal cells (MSCs), which represent a part bio-function of their sources. Here, we check the surface biomarkers of MSCs . The AFCs shared most of their cell surface markers with the CVCs: both were positive for CD29, CD44 and CD73, and both were negative for CD14, CD34 and CD45; however, CD105 was expressed differently in the two cell lines, and long-term culture increased the CD105 ratio . In our study, although AFCs and CVCs showed the same trend in terms of induced cell toxicity, the CVCs were more sensitive to colchicine in terms of cell viability and proliferation, showing more changes than AFCs, which suggests that colchicine affects the placenta much more than the foetus body, findings that are consistent with the epidemiology studies .
To precisely interpret the colchicine-induced toxicity of the placenta and foetus, we need a complex biological model and the ability to monitor the system. The micro-environment is complex and includes multiple types of cells and a complicated extracellular matrix that changes quickly, a situation quite different from our cell model. On the other hand, the concentration of colchicine in our study was higher than the peak plasma concentrations ; however, despite the low concentrations, the effect over time might be greater, as the exposure to colchicine would be maintained throughout the entire pregnancy.
It is necessary to evaluate colchicine toxicity in the placenta and foetus. Colchicine is the first-line therapy for gout and FMF due to its anti-inflammatory properties . Three proteins preferentially bind to colchicine: tubulin, CYP3A4 and P-glycoprotein. The anti-mitotic properties of colchicine that accelerate cells proliferation derive from tubulin binding, the metabolism of colchicine depends on CYP3A4 and P-glycoprotein in the liver, and colchicine is excreted via P-glycoprotein in the kidney . Pregnant women lack an adequate response to colchicine because of an increase in hepatic CYP3A4 enzyme activity . The increase in the dose administered to pregnant women could result in side effects in the foetus, as placenta cells express P-glycoprotein as an important transporter for substance exchange between the mother and foetus .
Our prenatal cell toxicity data may reflect the influence of colchicine on the placenta and foetus, but it did not completely mimic the toxic reaction in vivo. For clinical cytogenetic diagnosis, 0.15 μg/ml of colchicine was used to arrest rapidly proliferating cells in metaphase . In our study, prenatal cell viability was not affected in 12 h by 0.15 μg/ml colchicine treatment, but the ratio of late apoptotic cells (annexin V- and PI-double positive cells) was significantly increased; the continuous treatment over 24 h resulted in a decline in cell viability but not an increase in apoptotic cells. Furthermore, treatment with 0.15 μg/ml of colchicine for 3 h did not affect cell viability but limited cell proliferation in the continuous cell culture. Another biological phenomenon of colchicine resulted in an increase in the ratio of mitotic tetraploid cells, which is common in plants . Although the total number of tetraploids was low and they were not found in all the samples, it is necessary to take notice of the risk for developing genome abnormalities. Based on these findings, we conclude that the biological function of colchicine was arrest of the cell cycle but not induction of cell death.
In summary, we used primary cells derived from chorionic villus and amniotic fluid to assay colchicine-induced toxicity. Colchicine did not induce a decline in cell viability at low concentrations, but chronic exposure triggered suppressed cell proliferation by arresting the cell cycle in the G2/M phase and increased the risk of tetraploid generation. Our study reflects the influence of colchicine-induced toxicity in the placenta and foetus, but additional studies are needed on a more representative biological model that can be precisely monitored for interpretation of the relevant biological mechanisms.
DW and XS carried out study design. YX, MY, QP and YL performed the experiments. YX and MY did statistical analysis. DW wrote the paper. All authors read and approved the final manuscript.
This work was supported by National Natural Science Foundation of China (81401205 and 31872800), Clinical Innovation Research Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory (2018GZR0201002) and Guangzhou City Science and Technology Key Topics Project (201803040009 and 201904020025). The funding source and the sponsor did not participate in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Ethics approval and consent to participate
This study was approved by the Ethics Committee of The Third Affiliated Hospital of Guangzhou Medical University (Approval number: 2017–001). As there is no direct interaction with the study participants, informed consent was written to notice the diagnostic sample would be used for research.
Consent for publication
The authors declare that they have no competing interests.
- 3.Choi MY, Wee YM, Kim YH, Shin S, Yoo SE, Han DJ: Novel colchicine derivatives enhance graft survival after transplantation via suppression of T-cell differentiation and activity. Journal of cellular biochemistry 2019.Google Scholar
- 12.Alessio N, Pipino C, Mandatori D, Di Tomo P, Ferone A, Marchiso M, Melone MAB, Peluso G, Pandolfi A, Galderisi U. Mesenchymal stromal cells from amniotic fluid are less prone to senescence compared to those obtained from bone marrow: an in vitro study. J Cell Physiol. 2018;233(11):8996–9006.CrossRefGoogle Scholar
- 13.Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315–7.CrossRefGoogle Scholar
- 18.Aweeka FT, Hu C, Huang L, Best BM, Stek A, Lizak P, Burchett SK, Read JS, Watts H, Mirochnick M, et al. Alteration in cytochrome P450 3A4 activity as measured by a urine cortisol assay in HIV-1-infected pregnant women and relationship to antiretroviral pharmacokinetics. HIV medicine. 2015;16(3):176–83.CrossRefGoogle Scholar
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