Developmental Toxicity And Potential Mechanism Of Letrozole In Juvenile SD Rats

ObjectiveEstrogen is essential for heart,reproduction,brain,and bone metabolism through the action of aromatase enzymes.Letrozole（LTZ）has been used in recent years to treat pediatric endocrine disorders such as short stature and precocious puberty because it blocks aromatase and reduces estrogen synthesis.However,the effects of letrozole on cognitive,reproduction function,and bone metabolism in children are unknown,and studies of developmental toxicity in juvenile animals are limited.Therefore,in this study,learning and memory ability,bone mineral density,bone metabolic indexes,sex hormones,expressions of type III receptor tyrosine kinase（C-Kit）in ovarian and testicular cell were observed to investigate the effects of letrozole on cognitive function,bone development,reproductive function and potential toxic mechanisms in juvenile SD rats.MethodsIn this study,juvenile SD rats were used and letrozole was used as the study reagent,and first went through two four-week dose range exploration tests to observe clinical signs,body weight,bone metabolism,sex hormones,and pathological changes in the brain,femur,uterus,ovaries,testes and epididymis to find the appropriate dose.Then,a six-week dosing and four-week recovery developmental toxicity test was performed by passive avoidance,Dual-energy X-ray Absorptiometry（DXA）,ELISA,immunohistochemistry,liquid chromatography-tandem mass Spectrometry（LC-MS/MS）techniques to assess the changes on learning memory capacity,bone mineral density,bone metabolism,sex hormones,ovarian and testicular cell type III receptor tyrosine kinase（C-Kit）expression,and plasma letrozole drug concentrations to explore the effects of letrozole on reproductive function,bone development and cognitive function in juvenile rats,as well as the potential mechanism of toxic effects.To provide a basis for safer and more effective use of letrozole in clinical pediatrics.Results1 Dose-range exploration experiment of letrozole for four weeks in juvenile SD ratsAfter 28 days of administration,compared with the control group,increased ovaries weights and reduced uterine weights and its organ coefficients were noted at all dose groups（P≤0.05 or P<0.01）,and the ovaries showed moderate increase in atretic follicles and the uterus showed mild uterine atrophy in each dose group.2 Preliminary exploration of developmental toxicity of letrozole in juvenile SD ratsCompared with the control group,reduced uterine weights and its organ coefficients were noted in medium and high-dose group in females（P≤0.05）;increased ovarian atretic follicles and uterine atrophy were noted at all dose groups,and 2 cases of slight unilateral epididymal changes were noted in low and medium-dose group.Reduced ovarian estradiol（E2）contents were noted in medium and high-dose group in female juvenile rats（P≤0.05）,and increased testosterone（T）contents were noted in high-dose group in male juvenile rats（P≤0.05）.3 Developmental toxicity and potential mechanisms of letrozole in juvenile SD ratsAfter 42 days of administration and 28 days of recovery,compared with the control group,increased body weight and femur lengths were noted in medium and high-dose group,and decreased uterine visceral brain coefficient was noted in medium and high-dose group,increased heart,liver,and kidney visceral brain coefficients were noted at all dose groups,vaginal atrophy and renal tubular degeneration were noted at all dose groups,inflammatory cell infiltration in the heart was noted in high-dose group,increased uterine atrophy,ovarian atretic follicles and increased femoral hematopoietic cells were noted in medium and high-dose group in female juvenile rats（P≤0.05 or P<0.01）.For male juvenile rats,decreased body weight,right femur lengths and weights were noted in high-dose group,testis in medium-dose group and epididymis,heart,spleen,kidney,lung in high-dose group visceral brain coefficients were reduced,increased liver visceral brain coefficients were noted in medium-dose group,increased renal tubular pattern was noted in low-dose group,increased femoral hematopoietic cells were noted in medium and high-dose group（P≤0.05 or P<0.01）.After 28 days of recovery,increased renal and ovarian brain coefficients were noted in high-dose group in female rats,and spleen brain coefficients decreased were noted in medium and high-dose group in male rats（P≤0.05or P<0.01）.Compared with the control group,increased T levels in serum and ovarian tissue were noted in high-dose group,decreased E2 levels of ovarian tissue were noted in medium and high-dose group,and the expression of C-Kit in the ovaries were decreased were noted at medium and high-dose group in females（P≤0.05）;For males,increased serum T levels were noted in medium and high-dose group（P≤0.05 or P<0.01）.Compared with the control group,reduced serum prepropeptide of type I Procollagen（PINP）levels were noted in high-dose group（P≤0.05）,reduced bone mineral density of the right femurs were noted in low and high-dose group（P≤0.05）,and increased Areas of the right femur were noted in low-dose group in females（P≤0.05）.For males,increased serum C-terminal Telopeptides of typeⅠcollagen（CTX-Ⅰ）levels were noted in high-dose group on D42（P≤0.05）.On D70,decreased right femoral Areas were noted in high-dose group（P≤0.05）,and increased Bone Mineral Content（BMC）of the right femurs were noted in low-dose group（P<0.01）.At D1 and D42,no significant differences were observed between the exposure of female rats and that of male rats at equivalent doses.After D1 and D42 administration,the increase in exposure（AUC and Cmax）was accompanied by an increase in the range of0.1 to 100 mg/kg/day of letrozole doses,but this was less than the proportional increase in dose.After 42 days of administration,drug accumulation was present in 1.0 mg/kg/day letrozole in females and 100 mg/kg/day letrozole in males(based on AUC_0-24).Conclusions1.The dose range of letrozole was initially determined by four-week dose exploration and preliminary developmental toxicity exploration experiments,and it affected the reproductive function of female juvenile rats.2.Six-week dosing and four-week recovery developmental toxicity tests showed that 1/10mg/kg/day letrozole had severely impaired growth,reproductive function and bone development in females;and 100 mg/kg/day letrozole had severely impaired growth and bone development in males.3.Letrozole reduced E2 levels and diminished ovarian oocyte C-Kit expression,resulting in an increase in ovarian atretic follicles,which in turn affected ovarian development.