环磷酰胺对新西兰兔胚胎-胎仔发育的毒性作用研究

Effects of cyclophosphamide on embryo-fetal developmental toxicity test with New Zealand rabbits

目的探讨环磷酰胺用作兔胚胎一胎仔发育毒性实验阳性对照药物的可行性及最佳用药方案，为顺利开展规范化生殖毒性试验提供依据。方法实验用普通级新西兰兔，以交配成功日为妊娠第0天（GD0），采用计算机完全随机区组法将妊娠兔分为4组，包括环磷酰胺A组（14只，GD6～20环磷酰胺18mg／kg，10：／a灌胃）、B组（11只，GD10—13环磷酰胺25mg／kg，1次／d皮下注射）、C组（12只，GD6～18环磷酰胺15mg／kg，1次／d肌内注射）和溶媒对照组（12只，GD6—18生理盐水2ml／kg，1次／d灌胃）。观察用药期间动物的一般情况，分别于GD0、3、6、10、13、15、18、20、24、28对各组妊娠兔称重并计算宫外增重，于GD5和GD28取兔血清测定睾酮、黄体酮和雌二醇水平。GD28处死各组兔，取胎仔，记录黄体数、着床数、活胎率、吸收胎率、死胎率；检查胎仔，计算外观、内脏和骨骼畸形发生率。结果环磷酰胺A组兔GD10和GD15、环磷酰胺B和C组兔GDl5体重均明显低于溶媒对照组（P〈0．05或P〈0．01）；环磷酰胺A组宫外增重明显低于溶媒对照组[（-0．013±0．163）kg比（0．208±0．194）kg，P〈0．01]。GD28与GD5的激素水平差值比较显示，环磷酰胺A组激素水平变化明显，与溶媒对照组相比，雌二醇和睾酮变化幅度差异有统计学意义（P〈0．01或P〈0．05），黄体酮变化幅度差异无统计学意义（P〉0．05）；环磷酰胺B组及C组激素变化幅度与溶媒对照组相比，均无统计学意义（P〉0．05）。大体解剖未见母兔有明显异常。环磷酰胺A组、B组活胎率（65．1％、19．2％）均明显低于溶媒对照组（93．1％）（均P〈0．01），吸收胎率（17．4％、53．9％）及死胎率（17．4％、26．9％）均明显高于溶媒对照组（4．2％、2．8％）（均P〈0．01）。环磷酰胺A组、B组、C组胎仔外观畸形发生率（60．6％、93．3％、16．1％）、内脏畸形发生率（62．9％、93．3％、25．9％）及骨骼畸形发生率（91．4％、100．0％、61．7％）均明显高于溶媒对照组（0、0、3．0％）（均P〈0．01）。结论环磷酰胺用作新西兰兔胚胎-胎仔发育毒性实验阳性对照药物是可行的，其最佳用药方案为18m∥kg，10c／a灌胃，GD6～20连续用药15d。

Objective To explore the feasibility and the best application regimen of cyclophosphamide as the positive control drug of embryo-fetal developmental toxicity test with rabbits and provide evidences for smoothly developing standardized reproductive toxicity tests. Methods Common New Zealand rabbits were used for the experiment and the date of mating success was day 0 of pregnancy （GD 0）. Pregnant rabbits were divided into 4 groups including group A using cyclophosphamide （14 rabbits, given intragastric cyclophosphamide 18 mg/kg once daily from GD 6 to 20 ）, group B using cyclophosphamide （ 11 rabbits, given subcutaneous injection of cyclophosphamide 25 mg/kg once daily from GD 10 to 13 ）, group C using cyclophosphamide （12 rabbits, given intramuscular injection of cyclophosphamide 15 mg/kg once daily from GD 6 to 18, and solvent control group （12 rabbits, given intragastric normal saline 2 ml/kg once daily from GD 6 to 18 ）. The general situation of the animals during drug use was observed. The pregnant rabbits were weighed in every group at GD 0, 3,6, 10, 13, 15, 18, 20, 24, and 28 respectively and extranterine weight gain was calculated. Serum of rabbits was taken at GD 5 and 28 respectively and the levels of testosterone, progesterone, and estradiol were tested. At GD 28, the rabbits were killed and fetal rabbits were taken. Corpora lutea count, implantation count, live birth rate, absorbed embryo rate, and stillbirth rate were recorded. Fetal rabbits were examined and aberration rates of appearance, internal organs, and bones were calculated. Results Body weight of rabbits in group A at GD 10 and 15 and body weight of rabbits in group B and C at GD 15 were all markedly lower than that in solvent control group （P 〈 0. 05 or P 〈 0. O1 ）. Extrauterine weight gain in group A with use of cyclophosphamide was significantly lower than that in solvent control group [ （ - 0. 013 _＋ O. 163 ） kg vs. （0. 208 _＋0. 194）kg, P 〈0.01 ]. The differences of hormone levels at GD 28 and 5 showed the hormone levels changed markedly in group A while using cyclophosphamide and, compared with solvent control group, there were statistically significant difference in change range of estradiol and testosterone （P 〈 0.01 or P 〈 0.05 ） and there was no significant difference in change range of progesterone （ P 〉 0.05 ）. There was no statistical significance in change range of hormone between group B, C using cyclophosphamide and solvent control group. Gross anatomy showed that there was no markedly abnormal manifestation of female rabbits. Live birth rate in group A and B using cyclophosphamide （65. 1%, 19.2% ） were markedly lower than that in solvent control group （93.1%） （all P 〈 0.01 ）. Absorbed embryo rates （ 17.4%, 53.9% ） and stillbirth rates （ 17.4% , 26.9% ） in group A, B using cyclophosphamide were markedly higher than that in solvent control group （4.2%, 2.8% ） （ all P 〈 0.01 ）. Aberration rates of appearance （60.6%, 93.3%, 16. 1%）, internal organs （62.9%, 93. 3%, 25.9%）, and bones （91.4%, 100.0% , 61.7% ） were markedly higher than that in solvent control group （0, 0, 3.0% ） （all P 〈 0.01 ）. Conclusion It was feasible that using cyclophosphamide as the positive control drug of embryo- fetal developmental toxicity test with New Zealand rabbit and the best regimen was intragastric cyclophosphamide 18 mg/kg once daily from GD 6 to 20 for 15 days continuously.