In-vivo Mutagenicity of Protein-modified Enterococcus faecalis

Lijun DING Shasha ZHAO

Abstract The mutagenicity of the protein modified Enterococcus faecalis was evaluated by a mouse bone marrow micronucleus test and a mouse sperm abnormality test. The test substance was designed with three dose groups （1， 2 and 5 g/kg·bw） and intragastrically administrated， with cyclophosphamide as a positive control and normal saline as a normal control. The micronucleus rate and sperm abnormality rate were measured. The results showed that the micronucleus rates and sperm abnormality rates in the different dose groups were not significantly different from those in normal control group （P>0.05）， while the positive control group was significantly higher than the normal control group （P<0.01）. The conclusion is that the protein modified E. faecalis was tested to be negative in both the mouse bone marrow micronucleus test and mouse sperm abnormality test， suggesting that it has no mutagenicity in vivo.

Key words Protein modified E. faecalis; Mouse bone marrow micronucleus test; Mouse sperm abnormality test

Enterococcus faecalis， also known as Streptococcus faecalis， belongs to group D Streptococcus and is one of the major floras in the intestinal tract of human and animals[1]. It can form a biofilm attaching to the intestinal mucosa of animals intestines， to grow， develop and proliferate. E. faecalis is one of the strains permitted to be added into feeds by Feed Additive Species Catalogue （2013）. It can soften the fiber in feeds and improve the conversion rate of feeds. It was found that the addition of E. faecalis and cellulase to corn straw silage can significantly improve the color， odor and texture of the silage. Specifically， the addition of E. faecalis and cellulase to corn straw silage increased the dry matter loss rate by 8%， reduced the mass ratio of ammonia nitrogen to total nitrogen by 33%， and reduced the mass fraction of butyric acid by 82%， thereby significantly improving feed quality[2]. The addition of  E. faecalis  to the diet has the effects of improving animal performance， improving nutrient metabolism and improving immune function[3-5]. E. faecalis can produce a variety of antibacterial substances， which have a good inhibitory effect on pathogenic bacteria such as Salmonella， Escherichia coli and Staphylococcus aureus[6-8]. The research and application of E. faecalis as a probiotic in livestock and poultry is also increasing. In this study， the mutagenic effect of protein modified E. faecalis on mice was investigated by mouse bone marrow micronucleus test and mouse sperm abnormality test， so as to provide a scientific basis for safety evaluation of E. faecalis.

Materials and Methods

Materials

Test substance， experimental animals and strains

The test substance was protein modified E. faecalis 5×109 CFU/g （lot number： 20141206）， provided by some biotechnology company in Jiangsu Province. It was prepared to water solutions with desired concentrations before use.

50 ICR mice， half male and half female， with a body weight of （22±2） g， and 50 TCR mice （♂） with a body weight of  （28±2） g ， were purchased from the Comparative Medicine Center， Yangzhou University， under animal production license number： SCXK （Su） 2012 0004， use license number： SYXK （Su） 2012 0029. The animals were fed with 60 Co irradiated feed， and raised at （24±2） °C and humidity of （60%±20%）. They ate food and drink water freely， and the drinking water was tap water that meets urban drinking water standards.

Preparation of phosphate buffer （pH 6.47）： A certain  amount  of Na 2HPO 4·12H 2O （23.88 g） was dissolved in 1 000 ml of distilled water， obtaining solution A; and a certain amount of KH 2PO 4 （9.08 g） was dissolved in 1 000 ml of distilled water， obtaining solution B. 30 ml of solution A and 70 ml of solution B were mixed， giving the phosphate buffer with a pH of 6.47.

Giemsa dye solution： A certain amount of Giemsa raw powder （1.5 g） was add with 50 ml of glycerin， and placed in an incubator at 60 ℃， to allow dissolution within about 3 h. The mixture was then taken out and added with 50 ml of methanol to prepare a mother liquor. Before use， the mother liquor and the phosphate buffer （pH 6.47） were mixed into a working solution at a ratio of 1∶10.

Methods

Acute toxicity test

In the pre test， no mice died when the dose was as high as 10 g/kg·bw， indicating that the test substance had less toxicity， and a maximum dose test was carried out according to the acute toxicity operation procedure. Twenty mice， half male and half female， were intragastrically administered twice within 24 h， and the total dose was 10 g/kg·bw. Before the  intragastric  administration， they were fasted for 12 h with the  permission  of drinking water freely. After the intragastric administration， they were fasted for 1 h; and the mice were observed for  7-14 d ， during which symptoms of poisoning or death were  recorded.

Mouse bone marrow micronucleus test

Exposure to toxicant

The three dose groups of the test substance were 5， 2 and 1 g/kg·bw， respectively. A normal control and cyclophosphamide positive control group were also set. The normal control group was orally administered with normal saline， and the positive control was given cyclophosphamide at  80 mg/kg  once intraperitoneally. Intragastric administration was performed with the different concentrations of solutions at equal volume （0.2 ml/10 g·bw）. The mice were subjected to  exposure  to the toxicant twice with an interval of 24 h. Each group included 10 mice， half male and half female， which were killed by cervical spine dislocation 6 h after the second intragastric administration.

Preparation of slide and dyeing

The femurs at both sides of mice were taken， and the two ends were cut off. The bone marrow cavity was washed with 0.05 ml of calf serum， and the marrow was spread on a glass slide， and dried.

The smears were fixed with methanol for 5-10 min， dried， and stained with Giemsa dye solution for 20 min. Microscopic  examination  was performed after flushing with distilled water and drying.

Microscopic examination and micronucleus cell counting

Under oil lens， polychromatic erythrocytes （PCEs） were gray blue， and mature normochromatic erythrocytes （NCEs） were pink. The number of cells having micronuclei in 1 000 PCEs of each mouse was counted， and the micronucleus rate was calculated according to Micronucleus rate = Total number of PCEs with  micronuclei/Total  number of examined PCEs * 100%. The micronucleus rate was expressed in ‰. In addition， the numbers of PCEs and NCEs in 200 erythrocytes were counted， and the value of PCE/NCE was obtained.

Judgment of results

If the statistical results of the micronucleus rate show significantly differences （P<0.05） and dose response relationship， the test substance is tested to be negative.

The ratio of PCE/NCE should be in the range of 0.6 to 1.2. If the ratio is less than 0.1， it means that the formation of PCE is severely inhibited; and if the ratio is less than 0.05， it means that the dose of the test substance is too large， and the test result is not reliable.

The PCE/NCE ratio should be in the range of 0.6-1.2. If the ratio is smaller than 0.1， PCEs are inhibited seriously. If the ratio is smaller than 0.05， the dose of the test substance is too high， and the test results are not reliable.

Mouse sperm abnormality test

Exposure to toxicant

ICR male mice， 10 in each group， were exposed once every day for five consecutive days. The test substance was intragastrically administered to mice at 0.2 ml/10 g·bw， and the doses were 5， 2 and 1 g/kg·bw， respectively. Furthermore， intraperitoneal injection of cyclophosphamide at a dose of  40 mg/kg·bw  was used as a positive control， and normal saline was administered as a negative control group.

Observation

The mice were killed 35 d after the first administration of the test substance by cervical spine dislocation. A sperm suspension was prepared with epididymides at two sides， followed by preparation of smears which were dried and subjected to dyeing with 2% eosin and microscopic examination. 1 000 sperms of each mouse were observed for the calculation of abnormal sperm number and abnormal sperm rate and the analysis of abnormality types.

Judgment of results

When a repeatable dose response relationship occurs， the test result can be judged to be positive. That is to say if the sperm abnormality rates of at least two adjacent dose groups are significantly higher than that of the negative control group （P<0.01）， or are 2 times or more than 2 times of the negative control group， and the results are repeatable， the test could be considered as positive. Otherwise， the test is negative.

Data statistics

Data statistics was performed using SPSS 12.0， and comparison of significance was performed by χ2 analysis.

Results

Mouse acute toxicity test

No mice died when the dose reached 10 g/kg·bw. Therefore， the test substance has an oral LD  50 >10 g/kg·bw to mice.

According to the Exogenous Chemical Acute Toxicity Grading Standard of WHO， LD  50 >5 000 mg/kg·bw is the actual non toxic level， and the tested protein modified E. faecalis is an actual non toxic substance.

Mouse bone marrow micronucleus test

The micronucleus rate and PCE/NCE ratio are shown in Table 1. The PCE/NCE ratio of each test group was within the normal range， indicating that the test method was feasible. The difference between the positive control group and the normal control group was extremely significant （P<0.01）. The micronucleus rate of each experimental group was significantly different from that of the positive control group （P<0.01）， but had no significant difference compared with the normal control group （P> 0.05 ）， and there was no dose response relation in micronucleus rate between various experimental groups.

Mouse sperm abnormality test

The test results of the sperm abnormality test on the test subject mice are shown in Table 2 and Table 3. The sperm abnormality rate of the positive control group was significantly higher than that of the negative control group （P<0.01）. The sperm abnormality rates of the three test dose groups had no significant differences from the negative control group （P>0.05）. The sperm abnormality types of the test substance were similar to those of the positive control group， mainly including no hook， banana shape， amorphous shape and fat head， accounting for more than 90%， and other types accounted for a small proportion.

The test substance was tested to be negative in the sperm abnormality， indicating that the protein modified E. faecalis has no obvious mutagenicity to the germ cells of mice.

Conclusions and Discussion

The test substance was tested to be negative in both the mouse bone marrow micronucleus test and the mouse sperm abnormality test， indicating that the test substance has no obvious mutagenic effect on somatic cells and germ cells in vivo.

The Salmonella typhimurium mutation test carried out by the research group of this study also showed a negative result （which is published in another paper）. Therefore， the test substance was negative in the in vitro Ames test， in vivo mouse bone marrow micronucleus test and mouse sperm abnormality test， indicating that the protein modified E. faecalis has no mutagenic effect.

Because the LD  50  of the test substance was greater than  10 g/kg·bw ， the doses in the mouse bone marrow micronucleus test and the mouse sperm abnormality test were 5， 2 and 1 g/kg·bw， respectively， which were set according to 1/2 LD  50 ， 1/5 LD  50  and 1/10 LD  50 . Although the test doses were relatively large， the results of the mouse bone marrow micronucleus test and the mouse sperm abnormality test were still negative.

Agricultural Biotechnology2019

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