Toxicity effects of paclitaxel exposure on Caenorhabditis elegans

Jia-Li Ruan, Qi-Chun Huang

Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, China

Keywords:Paclitaxel Caenorhabditis elegans Neurotoxicity Apoptosis

ABSTRACT Objective: In order to understand the neurotoxic mechanism of paclitaxel, the motility and apoptosis of the animal model Caenorhabditis elegans were studied. Methods: A paclitaxel dilution (0.4, 4, 40, 400 mg/L) was prepared, and a K-medium solution was used as a blank control group (0 mg/L).Taking Caenorhabditis elegans as an experimental animal model, the effects of paclitaxel at different dose concentrations on the movement behavior of Caenorhabditis elegans and changes in apoptosis levels were evaluated to evaluate the toxicity of Paclitaxel to Caenorhabditis elegans. Results: After acute exposure, compared with the solution control group, the exercise capacity of the nematodes in the experimental group (0.4, 4, 40, 400 mg/L) was suppressed.Among them, only 0.4 mg/L in the experimental group showed a decrease in the head swing frequency and body bending frequency (P ＜0.05), while the other experimental groups had a significant decrease in their motor capacity (P ＜0.01), and it showed a dose-response relationship with the concentration of exposure.Compared with the solution control group, the apoptosis level of nematodes in the 400 mg/L experimental group was significantly increased after AO staining (P ＜0.01). Conclusion: Paclitaxel solution has neurotoxic effects on Caenorhabditis elegans. Neurotoxicity is related to the dose concentration of paclitaxel. Apoptosis may be one of the mechanisms that cause nematode neurotoxicity.

1. Introduction

According to statistical reports,the incidence of malignant tumors in China has increased by 3.9% year-on-year in the past decade,and the cumulative rate (0-74 years old) is 21.44%. At present, the number of cancer patients is huge [1];therefore, the clinical dosage of anti-tumor drugs related to it is large and the usage is diverse.Paclitaxel (PTX) is an anti-microtubule cytotoxic inhibitor, which is extracted from the bark of Taxus chinensis or obtained semi-synthetically;The mechanism of action of paclitaxel is not to directly kill cancer cells,but to stabilize microtubules during cell division and cause cancer cells to stop growing.It is a commonly used broad-spectrum antitumor drug[2～4];At present, paclitaxel has become the first-line treatment for ovarian cancer, lung cancer, breast cancer, lymphoma, etc[5].With the expansion of the number of clinical medicines and cancer types, studies on the higher side-effects caused by the use of paclitaxel have been reported frequently, but the mechanism of toxic and side effects is still not completely clear[6, 7].Caenorhabditis elegans is one of the most widely used animal models at present, it has been more commonly used in environmental and pharmaceutical toxicology research.In the late 1970s,the mechanism of the apoptosis pathway of Caenorhabditis elegans was completely interpreted by Brenner for the first time[8].As the life of C. elegans research continues to deepen, its neural lineage and nervous system circuit diagram are also fully described[9].Caenorhabditis elegans has a basically transparent pathway mechanism and its high degree of homology and conservatism. This particularity makes the nematode with small size, easy to reproduce, and short growth cycle an ideal model for analyzing neurotoxic mechanisms[10].Considering that the toxicity of paclitaxel to the human body is related to the dosage of the drug, this study analyzed different concentrations of paclitaxel solutions as experimental variables, and observed the effects of paclitaxel infiltrating Caenorhabditis elegans on motor behavior and the level of apoptosis. Use the experimental results to analyze the correlation between paclitaxel dose concentration and toxic and side effects, and provide theoretical basis and experimental models for clinical medication.

2. Materials and Methods

2.1 Laboratory animals and reagents

The wild-type Caenorhabditis elegans strain N2 and the nematode food E. coli OP50 were all donated by Mr. Liang Bin, Kunming Institute of Zoology, Chinese Academy of Sciences.C.elegans grows on NGM medium (peptone 0.625g,agar powder 4.25g,NaCl 0.75g) coated with food E. coli OP50 in a sterile constant temperature incubator at 22℃.Paclitaxel injection (5ml: 30mg) was purchased from Yangzijiang Pharmaceutical Group Co., Ltd .; acridine orange stain was purchased from Beijing Solabao Co., Ltd .; yeast powder and tryptone were purchased from OXOID company; agar powder was purchased from Biosharp company, peptone Purchased from Beijing Aoboxing Biotechnology Co., Ltd., sodium chloride, sodium hydroxide, potassium chloride, sodium hypochlorite, cholesterol, etc. are all made in China.

2.2 Experimental instruments and equipment

XTL-3400E type continuous zoom stereo microscope (Guangxi Wuzhou Aoka Optical Instrument Co., Ltd.);fluorescence inverted microscope (Olympus, Japan);DRP-9272 electric heating constant temperature incubator (Shanghai Senxin Experimental Instrument Co., Ltd.);Avanti J- 26 XP high-efficiency centrifuge (American Beckman coulter company);autoclave (American Thermo company).

2.3 Exposure test

2.3.1 Synchronization experiment of C.elegans

According to the literature and the previous method of the research group[11] ,The nematode culture solution K-medium solution (KCl 1.18g, NaCl 1.5g) is prepared.The NGM medium containing a large number of adults at the peak of reproduction was selected. Adult nematodes were rinsed off and collected in 15ml centrifuge tubes. After centrifugation at 1500 rpm, the upper layer solution was aspirated to leave only 3.5 ml of cloudy solution. 1.5ml of lysis solution (5 M / L NaOH: 5% NaClO = 1: 2, now used) is added to the nematode turbid solution and shaken vigorously for 6 ～ 8min. After all adult worms are lysed, centrifuge the turbid liquid at 1000 rpm. The supernatant was removed to collect the egg deposits and washed repeatedly with the culture medium 3 times. The eggs were collected and incubated overnight in the nematode culture medium without food. The growth and development of the resulting nematodes all stagnate in the L1 phase. The L1 phase nematodes were transferred to NGM culture dishes coated with fresh food and cultured for 36-48 hours. Synchronized L4 phase nematodes were available for experiments.

2.3.2 The concentration setting of Paclitaxel solution

Based on the experimental results of the half-lethal rate of paclitaxel in the early stage of the research group, we set the concentration gradient of 0.1%, 1%, and 10% for 24h LC50, and set the final concentration of paclitaxel exposure solution to 0.4, 4, 40, 400 mg / L . K-medium solution was used for solution dilution preparation, and a solution control group (0 mg / L) was also provided. Six-well sterilized petri dishes were used in the exposure experiment. 4ml of different concentrations of exposure solution were added to each well, and three parallels were set for each concentration group. The synchronized L4 Caenorhabditis elegans was inoculated into each petri dish, and was acutely exposed for 24h in a dark environment at at 20℃±2℃. After the exposure, the nematode motility and apoptosis levels were measured.

2.4 Movement behavior changes of C.elegans

2.4.1 Measurement of head swing frequency

The NGM medium without the addition of E. coli OP50 with 100µL of K-medium in the center was placed in advance. Fifteen nematodes at the end of exposure were randomly selected in each group concentration, and they were placed in K-medium for free movement for 1 min. The nematode swings from one side to the other side and back again is defined as one head swing. The number of head swings of the nematode in 20s was recorded as the head swing frequency index.

2.4.2 Measurement of body bending frequency

NGM medium without food was prepared in advance. Fifteen nematodes at the end of exposure were randomly selected in each group concentration, and they were placed on the dish and moved freely for 1 min. The movement of one wavelength in the direction of the long axis of the nematode body is defined as one body bend. The number of times the nematode's body was bent within 20 s was recorded as an indicator of body bending frequency.

2.4.3 Measurement of Pharyngeal pump frequency

NGM Petri dishes with OP50 added were prepared in advance. Fifteen nematodes at the end of exposure were randomly selected in each group concentration, and they were placed on the dish for free for 1 min. One beating of the pharyngeal pump during the nematode feeding process was determined as the frequency of one pharyngeal pump. The frequency of the nematode pharyngeal pump beating within 20s was recorded as an index of pharyngeal pump frequency.

2.5 Determination of apoptosis in C.elegans

Groups of C. elegans were collected with K-medium solution after the exposure. The nematodes that had been washed three times were moved to a 24-well sterile culture plate containing 1 ml of acridine orange (25µg / mL) and incubated for 2 h at 20℃[12].A glass slide with a 2% agarose pad in the center was placed in advance. The nematodes that had been exposed to acridine orange were collected and washed three times, anesthetized with levamisole solution and placed in the center of the agarose pad. The slides with nematodes were placed under a fluorescent inverted microscope to take pictures (excitation wavelength 488 nm, blocking wavelength 515 nm), and the apoptosis levels of various parts of the nematode were observed under the microscope. The average relative fluorescence intensity value indicates the level of apoptosis.

2.6 Statistical methods

The measurement data is expressed as mean±standard deviation (±s). SPSS 22.0 was used for single-factor analysis of variance for comparison between groups. When pairwise comparisons were made, the LSD method was used for all variances, and Tamhane's T2 method for uneven variances. Graphpad Prism 7.0 software was used for plotting, and P ＜0.05 and P ＜0.01 judged the data to be different and significantly different, respectively. All experiments were repeated three times in parallel.

3. Result

3.1 Changes in appearance of Paclitaxel on C.elegans

After 24 hours of acute administration of Caenorhabditis elegans, compared with the blank group, the nematodes in each experimental group curled up significantly and did irregular non-sinusoidal exercise. In the high-concentration group (40, 400 mg / L), the nematode's ability to exercise after being touched by the silver needle was obviously not stressful. The amplitude and frequency of C.elegans' body swings decreased significantly, turning over slowly and the time prolonged significantly. Their athletic ability changed significantly.

3.2 Effects of Paclitaxel on the Movement Behavior of Caenorhabditis elegans

Each experimental group and control group were compared with each other. The movement ability of C.elegans (including head swing frequency, body bending frequency, pharyngeal pump beat frequency) had statistically significant changes in the infiltration changes of different doses of paclitaxel solution (P ＜0.05). The 0.4 mg / L experimental group's head swing frequency and body swing frequency (P ＜0.05), while the other experimental groups' behavioral ability decreased significantly (P ＜0.01). Experimental data also shows that the toxic effect of paclitaxel on C. elegans has a certain concentration dependence, that is, the cytotoxic effect of paclitaxel on C. elegans shows an upward trend with increasing concentration dose, and there is a dose-effect relationship. ( Table 1)

Tab1 Effects of Paclitaxel on Head Swing Frequency, Body Bending Frequency, and Pharyngeal Pump Frequency of nematodes in each group（n=15，±s）

Tab1 Effects of Paclitaxel on Head Swing Frequency, Body Bending Frequency, and Pharyngeal Pump Frequency of nematodes in each group（n=15，±s）

Note: When comparing each experimental group with the control group, * p ＜0.05, ** p ＜0.01.

Pharyngeal pump beat frequency （times/20s） Control（0 mg/L） 138.67±4.01 16.73±1.49 86.60±5.03 0.4 mg/L 128.73±9.22* 13.67±0.98* 81.33±5.33**4 mg/L 108.73±7.11** 13.27±1.34** 71.60±5.19**40 mg/L 103.60±8.59** 12.67±1.35** 74.20±2.57**400 mg/L 103.40±6.31** 11.00±1.56** 71.93±5.20** F 74.447 36.590 28.470 P 0.000 0.000 0.000 Group Head swing frequency （times/1min） Body bending frequency （times/20s）

3.3 Effect of paclitaxel on the apoptosis of C.elegans

Normal cells stained green with acridine orange AO. The apoptotic cells appear bright green or yellow under the microscope.As shown in Figure 1, the nematode body can be seen as bright green in the experimental group with a paclitaxel concentration of 400 mg / L. The apoptosis of C.elegans is more serious ( Figure 1).Compared with the control group, there was no significant difference in the apoptosis levels of C. elegans cells in the experimental group from 0.4 mg / L to 40 mg / L (p＞ 0.05). When the acute exposure concentration of paclitaxel was 400 mg / L, the apoptosis level of C. elegans increased significantly (p ＜0.01), which was consistent with the nematode status observed under the microscope. The level of apoptosis of C. elegans in the experimental group increased by 23% compared with the control group. ( Table 2)

Figure 1 Apoptosis of nematode cells in each group after AO staining

Table2 Effect of Paclitaxel on the relative fluorescence level intensity of Apoptosis in nematode cells of each group（n=43，±s）

Table2 Effect of Paclitaxel on the relative fluorescence level intensity of Apoptosis in nematode cells of each group（n=43，±s）

Note: When comparing each experimental group with the control group, * p ＜0.05, ** p ＜0.01.

Group Relative fluorescence intensity value Control（0 mg/L） 91.00±14.47 0.4 mg/L 85.63±10.16 4 mg/L 87.40±13.02 40 mg/L 100.86±21.62 400 mg/L 111.98±17.88** F 20.476 P 0.000

4. Discussion

Caenorhabditis elegans is extremely susceptible to changes in the external environment. They can obtain various nutritional mutants or gene mutants by changing the medium or adding mutagen (such as ethyl methanesulphonate, EMS)[13].C.elegans has a simple nervous system structure and transparent cell lineage[14]；Changes in nematode behavior are responses to changes in nervous system function.C.elegans' head swing, body bending, and pharyngeal pump frequency are all commonly used indicators to detect changes in the basic functions of the nervous system, and are now widely used in the analysis of neurotoxicity and clinical drug toxicity.This study suggests that compared with the blank group, the exposure of paclitaxel solution will have a certain effect on the nematode's ability to exercise. The nematode's ability to exercise is weakened, and this behavioral response to paclitaxel concentration reflects a decline in nervous system function. With the increase of paclitaxel concentration, the frequency of head swing, body bending ability and frequency of pharyngeal pump all decreased progressively.

Caenorhabditis elegans conducts a series of physiological behaviors through exercise behaviors, such as seeking benefit and avoiding harm, and feeding. Motor ability is controlled by a variety of neurons, and motor neurons are mainly affected by environmental factors and genetic factors.A large number of studies[15～17]have proved that the use of paclitaxel will produce obvious neurotoxic effects. The induced neurotoxicity will promote abnormal apoptosis of neurons, which will lead to the defects of basic ability of learning, memory and exercise. The incidence of neurotoxicity in the high concentration group was higher than that in the low concentration group.In the 0.4 mg / L experimental group, the nematode head swing frequency and body bending ability were not significantly reduced, which may be just the nematode's reduced body response to maintain normal body function but the continuous increase in paclitaxel concentration makes the nematode irresistible and unable to repair itself resulting in damage to the nervous system.The change in the frequency of nematode pharyngeal pump is related to the nematode feeding ability.The nerves or muscles associated with nematode feeding are affected by foreign chemicals. They avoids the damage caused by toxic substances to the body by reducing food intake, which indirectly suggests the neurotoxicity of paclitaxel to Caenorhabditis elegans.The decrease in pharyngeal pump frequency may be due to paclitaxel causing irreparable damage to the pharyngeal nerves or muscles of Caenorhabditis elegans.This change causes nematode obstacles in feeding.Studies have reported that the adverse reaction rate of paclitaxel is related to the dose used, which is a dose-dependent drug toxicity[18].In this experiment, the nematode exercise capacity decreased with the increase of paclitaxel concentration, which showed a significant dose-effect relationship. This experimental result once again verified this relationship.

The biological significance and mechanism of apoptosis was first discovered through nematode research.Different from the death mode of cell necrosis, apoptosis is the behavior of normal cells that are highly regulated by external stimuli and actively die[19].This is a way for the body to maintain its balance[20].C. elegans cell apoptosis is also a sensitive indicator of exogenous chemical exposure[21].The premature apoptosis of normal cells will significantly affect the normal growth and development of the organism.There are four mechanisms of action of paclitaxel that are generally accepted by current research.Apoptosis is one of these four types, which promotes the expression of the apoptosis protein Caspaese-3 and leads to apoptosis[22].In this experiment, when the exposure of paclitaxel reached 400 mg / L, the level of apoptosis in C. elegans significantly increased (P ＜0.05), and a significant decrease in nematode movement ability in the same group was observed. This may help to infer that the toxic effect of paclitaxel on C. elegans may be related to apoptosis.In clinical use, the anti-tumor effect of paclitaxel increases with increasing paclitaxel concentration and cumulative dose. Similarly, its incidence of toxic and side reactions is also increasing;At the same time, the role of its apoptosis mechanism in drug efficacy and toxic and side effects was confirmed.

In this experiment, the exercise ability and apoptosis level of Caenorhabditis elegans were used to evaluate the toxic effects of different doses of paclitaxel.The experimental results confirmed that paclitaxel has a significant neurotoxic effect on nematodes, and there is a dose-effect relationship.Under high concentration environment, the degree of apoptosis of C. elegans was significantly enhanced.This progression is consistent with the increase in the dose of clinical patients or the increase in cumulative dose leading to more adverse reactions, which can evaluate the toxic and side effects of different doses of paclitaxel more accurately and quickly.It also provides a new basis for elucidating the possibility of paclitaxel's neurotoxicity and even the mechanism of toxicity. Due to the species differences between humans and model animals, the mechanism of toxic and side effects associated with the clinical use of paclitaxel still needs further study.