盐酸克伦特罗对成年大鼠睾丸类固醇激素合成急性调节蛋白（StAR）基因mRNA表达的影响
|
摘要
目的
研究β2肾上腺素受体激动剂盐酸克伦特罗(Clenbuterol hydrochloride, CLB)急性和亚急性染毒对成年大鼠睾丸类固醇激素合成急性调节蛋白(StAR)基因mRNA表达的影响,在分子水平探讨盐酸克伦特罗对睾丸遗传物质影响的相关机制,以期为进一步研究β2肾上腺素受体激动剂类药物对雄(男)性的生殖系统的影响及其环境危险度评价提供初步实验依据。
材料与方法
1.实验动物及试剂
CLB急性和亚急性染毒实验的动物均选取9-10周龄的SPF级的成年雄性Sprague-Dawley (SD)大鼠,体重200-220 g。所有大鼠均分笼饲养于标准动物房一周后用于实验,每日光照12 h,全价颗粒饲料喂养,自由摄食。每天固定时间染毒,染毒前禁食,染毒后喂食。每个剂量组的CLB均溶于0.9%的生理盐水至1 mL。
2.动物分组及染毒方式
70只大鼠随机均分为14组,每组5只动物。用于CLB急性染毒实验共6组。按照CLB给药浓度分别设为：对照组A和A’(0 mg k-1bw)、低剂量组B和B’(20 mg kg-1bw)和高剂量组C和C’(40 mg kg-1bw)。CLB均通过灌胃方式对大鼠给药一次,对照组A,A’以等量生理盐水(1 mL)代替CLB进行灌胃。字母A、B和C表示CLB急性染毒后停药一天组,字母加上标“’”表示CLB急性染毒后停药七天组。
用于CLB亚急性染毒实验的大鼠共8组,每组5只动物。按照CLB浓度分别设为：对照组F和F’(0 mg kg-1bw day-1)、低剂量组G和G’(0.4 mg kg-1bw day-1)、中剂量组H和H’(2.0 mg kg-1 bw day-1)、高剂量组I和I’(18.5 mg kg-1bw day-1)。CLB均通过灌胃方式对大鼠连续给药十四天,而对照组F和F’以等量生理盐水(1 mL)代替CLB进行灌胃。字母F、G、H和I表示CLB亚急性染毒后停药一天组,字母加上标“’”表示CLB亚急性染毒后停药七天组。
检测方法
分别记录各组大鼠在CLB给药前和处死前的体重；记录各组大鼠睾丸重量并计算其睾丸脏器系数；应用半定量反转录聚合酶链式反应(RT-PCR)检测睾丸组织中的类固醇激素合成急性调节蛋白(StAR)基因mRNA的表达变化。所有实验数据均采用分析统计软件SPSS 13.0进行单因素方差分析,组间比较采用t检验,P<0.05具有统计学意义。所有实验数据以均数±标准差((?)±s)表示。
结果
1.CLB急性、亚急性染毒大鼠体重的变化
与对照组相比,CLB急性染毒实验B、C组(停药一天)大鼠体重变化无明显差异(P>0.05),无统计学意义,但B’、C’组(停药七天)大鼠体重增加显著(P<0.05),有统计学意义；CLB亚急性染毒实验各实验组大鼠体重与对照组相比均明显增加,其中G,G’增加明显(P<0.05)有统计学意义,而H,H’,I和I’组体重增加极为显著(P<0.01),有统计学意义。
2.CLB急性、亚急性染毒实验大鼠睾丸重量及睾丸脏器系数的变化
CLB急性、亚急性染毒实验各组大鼠睾丸重量与对照组相比差异不显著(P>0.05),无统计学意义；CLB急性、亚急性染毒实验各组大鼠睾丸重量与体重的比值(睾丸脏器系数)与对照组相比有所降低,但差异不显著具(P>0.05),无统计学意义。3.CLB急性染毒实验大鼠睾丸StAR基因mRNA表达的变化
CLB急性染毒停药一天组B、C的StAR mRNA表达与对照组相比均显著升高(P＜0.05),有统计学意义；CLB急性染毒停药七天组B’,C’的StAR mRNA表达与对照组相比均略有升高,但差异不显著(P>0.05),不具统计学意义。
4.CLB亚急性染毒实验大鼠睾丸StAR基因mRNA表达的变化
CLB亚急性染毒实验停药一天的G、H、I三组的StAR mRNA表达水平与对照组相比均降低,降低程度随CLB的给药剂量增加而加大,其中I组降低显著(P<0.05),具有统计学意义；CLB亚急性染毒实验停药七天组G’和H’的StAR mRNA表达水平与对照组相比均降低,但差异不显著(P>0.05),不具统计学意义。但与相对应的G、H组相比其StARmRNA的表达水平随CLB给药剂量的降低而升高。I和I’组StAR mRNA表达水平与对照组相比呈现显著性降低(P<0.05),具有统计学意义。
结论
1.CLB急性染毒(停药七天)20 mg kg-1 bw和40 mg kg-1 bw均能使大鼠体重快速增加,但其体重增长速度没有呈现剂量效应。说明CLB能对大鼠体重产生影响的单次给药的最高剂量可能为20 mg kg-1 bw或者此剂量以下。CLB亚急性染毒实验(14 d)各实验组大鼠体重均显著增加,且随CLB给药剂量的增加而增加,呈现剂量效应。CLB急性、亚急性染毒实验提示CLB能快速促进成年大鼠的合成代谢从而增加体重。
2.CLB急性、亚急性染毒实验各实验组中的大鼠睾丸重量未见明显变化,同时其睾丸脏器系数也未见显著性变化。说明CLB急性(1 d)或亚急性染毒(14 d)后对成年大鼠睾丸重量的影响不明显。
3.CLB急性染毒(停药一天)实验表明20 mg kg-1 bw和40 mg kg-1 bw的CLB给药剂量能使大鼠睾丸组织StAR基因的mRNA表达水平迅速升高,但没有呈现相应的剂量效应。StAR基因的mRNA表达水平在停药七天后明显降低。提示CLB可能通过作用于p2肾上腺素受体迅速引起大鼠体内促激素的变化而影响StAR基因的mRNA表达变化。CLB急性染毒实验提示20 mg kg-1 bw的CLB给药剂量可能为成年大鼠睾丸β2肾上腺素受体的最大饱和剂量。同时提示StAR基因的mRNA表达变化亦与CLB在体内的代谢时间有关。
4 CLB亚急性染毒实验表明CLB均能使大鼠睾丸组织StAR mRNA表达水平降低且随CLB给药剂量的加大而表达降低,呈现剂量效应。CLB亚急性染毒停药七天能使StAR基因的mRNA表达水平有所升高但仍低于对照组水平。说明CLB (14 d)染毒能够降低大鼠睾丸基因StAR的mRNA表达。提示StAR基因的mRNA表达变化亦与CLB在体内的代谢时间有关。同时提示CLB亚急性染毒(14 d)可能通过影响大鼠睾丸组织内的β2肾上腺素受体的下调而影响体内促激素的变化从而最终影响大鼠睾丸StAR基因mRNA的表达水平下降。
Objective
The present study was carried out to investigate the effects of acute and subacute treatment with clenbuterol hydrochloride (CLB) on the expression of testicular steroidogenic acute regulatory protein (StAR) mRNA in adult male rats and to elucidate the possible mechanism (s) of genetic alterations caused byβ2-androgen agonists in testis. After that, it would be possible to provide the initial and referential data for further study about effects of growth promoting substances on male reproductive system and about growth promoters' potential hazards to environment.
Materials and Methods
1. Animals and Chemicals
Seventy adult male Sprague-Dawley rats (9-10 weeks old, weighing 200-220 g, specific pathogen-free grade) were housed in a clean facility. The animals were allowed access to commercial pellet food and tap water ad libitum and were maintained on a 12-h light/dark cycle throughout the experiment. CLB of each specific doses was dissolved in 0.9%NaCl solution up to 1 mL and was given to the animals on a specific time. All rats were pre-fed for 1 week to allow adaptation to the new environment.
2. Groups and the routes of administration
In the acute experiment (1 d), thirty rats were randomly divided into six groups (n=5) and were treated with CLB by gavage at dosages of:0 mg kg-1 bw (A and A'),20 mg kg-1 bw (B and B') and 40 mg kg-1 bw (C and C'), respectively. An equivalent volume of 0.9%NaCl solution was given to the vehicle groups (A, A') in the same way. Letter followed by the apostrophe means the group received a 7-d withdrawal period after treatment with CLB, respectively.
In the subacute experiment (14 d), forty rats were randomly assigned to eight groups (n=5) and were treated with CLB by gavage at dosages of:0 mg kg-1 bw day-1 (F and F'), 0.4 mg kg-1 bw day-1 (G and G'),2.0 mg kg-1 bw day-1 (H and H') and 18.5 mg kg-1 bw day-1 (I and I'), respectively. CLB was dissolved in 0.9%NaCl solution up to 1 mL and the vehicle groups (F, F') were received an equivalent volume of 0.9%NaCl solution by gavage. Letter followed by the apostrophe means the group received a 7-d withdrawal period after treatment with CLB, respectively.
Methods
The body weights and testes weights of each animal were recorded. The alterations in the mRNA expression of testicular steroidogenic acute regulatory (StAR) protein were determined by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). All data were expressed as mean±SD (x±s). Statistical analysis was performed with one-way analysis of variance (ANOVA), followed by t test between the treatment and control groups, which performed using SPSS 13.0 statistical software on computer. Statistical significance was accepted at the P＜0.05 level.
Results
1. Effects of CLB treatment on the body weights and testis wet weights.
In CLB acute (1 d) experiment, a significant increase (P＜0.05) was observed in the body weights of rats in groups B'and C' after a 7-d withdrawal period, no significant difference was seen in the groups B and C when compared to control group rats. Significant increases (P＜0.01) were observed in the body weights of rats treated with CLB in groups H, H', I and I'in CLB subacute experiment (14 d), statistical significance (P＜0.05) was also observed in groups G and G'with respect to the control group rats.
2. Effects of CLB treatment on the testis wet weights and the ratio of body weights to testis weights
No significant difference (P＞0.05) was observed in the testis wet weights between the control and CLB-treated groups in both acute and subacute experiment rats. The body weight/testis weight ratio was assessed and no statistical difference (P＞0.05) was found in either acute or subacute experiment animals as compared to rats in the control groups.
3. Effects of acute CLB treatment on the expression of testicular StAR mRNA in rats
In CLB acute experiment, the expression levels of StAR mRNA were markedly increased (P＜0.05) in the testis after administration of CLB at dosages of 20 mg kg-1 bw (B) and 40 mg kg-1 bw (C), but no dose-dependent effects of CLB treatment on the expression levels of StAR mRNA was observed, and StAR mRNA levels (B'and C') were returned to near normal levels after 7 d of CLB withdrawal compared to that of the control group rats.
4. Effects of subacute CLB treatment on the expression of testicular StAR mRNA in rats
The mRNA levels of StAR in rat testis showed a dose-dependent reduction in the sbuacute (14 d) CLB-treated groups and a trend in recovery of mRNA levels to normal level was noticed in the groups of 7-d CLB withdrawal at dosages of 0.4 mg kg-1 (G and G') and 2.0 mg kg-1 bw day-1 (H and H'). However, no recovery in the expression of StAR mRNA was observed in the groups that treated with CLB at the dosage of 18.5 mg kg-1 bw day-1 (I and I') even following a 7-d withdrawal period (P＜0.05) with respect to control group animals.
Conclusion
1. CLB induced a rapid increase in body weights of adult male rats after the CLB acute treatment (7 d of withdrawal) at both doses of 20 and 40 mg kg-1 bw, but no dose-dependent increase was observed. CLB induced a dose-dependent increase in body weights of adult male rats after the subacute CLB treatment. The increase in the body weights of rats may indicate that the single treatment with CLB to cause the maximum effects of increasing body weights is the does of 20 mg kg-1 bw or less. These results suggested that theβ2-adrenergic agonist CLB has potent repartitioning effects in animals through stimulating the anabolic hormones which will finally lead to the enhancement of body weights.
2. The weights of testes and the the ratio of body weights to testis weights were not significantly affected after CLB treatment. These results suggested that the CLB has little impact on the weights of testis of adult rats after an acute (1 d) or a subacute (14 d) treatment.
3. CLB induced a transient and promotive effect on the expression levels of testicular StAR mRNA after the CLB acute treatment at both dosages of 20 and 40 mg kg-1 bw, but not dose-dependent effects. These results may indicate that CLB induce the increase in mRNA level of StAR through acting on theβ2-adrenergic receptors in testis and then stimulating the trophic hormones secretion. These alterations in the expression levels of StAR mRNA elicited by CLB may also be attributed to the half-life of CLB in the rats. The results also may indicate that the dose of 20 mg kg-1 bw CLB is the maximum dosage forβ2-adrenergic receptors saturation dosage to rat testis or less.
4. In subacute exposure (14 d), CLB decreased the testicular expression of StAR mRNA and showed a dose-dependent and persistent and aggravating impact on the StAR mRNA levels. These alterations caused by CLB probably through a mechanism of action correlated to trophic hormones secretion affected by theβ2-adrenergic receptors down-regulation after a 14-d consecutive stimulation by CLB. These alterations in the expression levels of StAR mRNA elicited by CLB may also be attributed to the half-life of CLB in the rats.
研究β2肾上腺素受体激动剂盐酸克伦特罗(Clenbuterol hydrochloride, CLB)急性和亚急性染毒对成年大鼠睾丸类固醇激素合成急性调节蛋白(StAR)基因mRNA表达的影响,在分子水平探讨盐酸克伦特罗对睾丸遗传物质影响的相关机制,以期为进一步研究β2肾上腺素受体激动剂类药物对雄(男)性的生殖系统的影响及其环境危险度评价提供初步实验依据。
材料与方法
1.实验动物及试剂
CLB急性和亚急性染毒实验的动物均选取9-10周龄的SPF级的成年雄性Sprague-Dawley (SD)大鼠,体重200-220 g。所有大鼠均分笼饲养于标准动物房一周后用于实验,每日光照12 h,全价颗粒饲料喂养,自由摄食。每天固定时间染毒,染毒前禁食,染毒后喂食。每个剂量组的CLB均溶于0.9%的生理盐水至1 mL。
2.动物分组及染毒方式
70只大鼠随机均分为14组,每组5只动物。用于CLB急性染毒实验共6组。按照CLB给药浓度分别设为：对照组A和A’(0 mg k-1bw)、低剂量组B和B’(20 mg kg-1bw)和高剂量组C和C’(40 mg kg-1bw)。CLB均通过灌胃方式对大鼠给药一次,对照组A,A’以等量生理盐水(1 mL)代替CLB进行灌胃。字母A、B和C表示CLB急性染毒后停药一天组,字母加上标“’”表示CLB急性染毒后停药七天组。
用于CLB亚急性染毒实验的大鼠共8组,每组5只动物。按照CLB浓度分别设为：对照组F和F’(0 mg kg-1bw day-1)、低剂量组G和G’(0.4 mg kg-1bw day-1)、中剂量组H和H’(2.0 mg kg-1 bw day-1)、高剂量组I和I’(18.5 mg kg-1bw day-1)。CLB均通过灌胃方式对大鼠连续给药十四天,而对照组F和F’以等量生理盐水(1 mL)代替CLB进行灌胃。字母F、G、H和I表示CLB亚急性染毒后停药一天组,字母加上标“’”表示CLB亚急性染毒后停药七天组。
检测方法
分别记录各组大鼠在CLB给药前和处死前的体重；记录各组大鼠睾丸重量并计算其睾丸脏器系数；应用半定量反转录聚合酶链式反应(RT-PCR)检测睾丸组织中的类固醇激素合成急性调节蛋白(StAR)基因mRNA的表达变化。所有实验数据均采用分析统计软件SPSS 13.0进行单因素方差分析,组间比较采用t检验,P<0.05具有统计学意义。所有实验数据以均数±标准差((?)±s)表示。
结果
1.CLB急性、亚急性染毒大鼠体重的变化
与对照组相比,CLB急性染毒实验B、C组(停药一天)大鼠体重变化无明显差异(P>0.05),无统计学意义,但B’、C’组(停药七天)大鼠体重增加显著(P<0.05),有统计学意义；CLB亚急性染毒实验各实验组大鼠体重与对照组相比均明显增加,其中G,G’增加明显(P<0.05)有统计学意义,而H,H’,I和I’组体重增加极为显著(P<0.01),有统计学意义。
2.CLB急性、亚急性染毒实验大鼠睾丸重量及睾丸脏器系数的变化
CLB急性、亚急性染毒实验各组大鼠睾丸重量与对照组相比差异不显著(P>0.05),无统计学意义；CLB急性、亚急性染毒实验各组大鼠睾丸重量与体重的比值(睾丸脏器系数)与对照组相比有所降低,但差异不显著具(P>0.05),无统计学意义。3.CLB急性染毒实验大鼠睾丸StAR基因mRNA表达的变化
CLB急性染毒停药一天组B、C的StAR mRNA表达与对照组相比均显著升高(P＜0.05),有统计学意义；CLB急性染毒停药七天组B’,C’的StAR mRNA表达与对照组相比均略有升高,但差异不显著(P>0.05),不具统计学意义。
4.CLB亚急性染毒实验大鼠睾丸StAR基因mRNA表达的变化
CLB亚急性染毒实验停药一天的G、H、I三组的StAR mRNA表达水平与对照组相比均降低,降低程度随CLB的给药剂量增加而加大,其中I组降低显著(P<0.05),具有统计学意义；CLB亚急性染毒实验停药七天组G’和H’的StAR mRNA表达水平与对照组相比均降低,但差异不显著(P>0.05),不具统计学意义。但与相对应的G、H组相比其StARmRNA的表达水平随CLB给药剂量的降低而升高。I和I’组StAR mRNA表达水平与对照组相比呈现显著性降低(P<0.05),具有统计学意义。
结论
1.CLB急性染毒(停药七天)20 mg kg-1 bw和40 mg kg-1 bw均能使大鼠体重快速增加,但其体重增长速度没有呈现剂量效应。说明CLB能对大鼠体重产生影响的单次给药的最高剂量可能为20 mg kg-1 bw或者此剂量以下。CLB亚急性染毒实验(14 d)各实验组大鼠体重均显著增加,且随CLB给药剂量的增加而增加,呈现剂量效应。CLB急性、亚急性染毒实验提示CLB能快速促进成年大鼠的合成代谢从而增加体重。
2.CLB急性、亚急性染毒实验各实验组中的大鼠睾丸重量未见明显变化,同时其睾丸脏器系数也未见显著性变化。说明CLB急性(1 d)或亚急性染毒(14 d)后对成年大鼠睾丸重量的影响不明显。
3.CLB急性染毒(停药一天)实验表明20 mg kg-1 bw和40 mg kg-1 bw的CLB给药剂量能使大鼠睾丸组织StAR基因的mRNA表达水平迅速升高,但没有呈现相应的剂量效应。StAR基因的mRNA表达水平在停药七天后明显降低。提示CLB可能通过作用于p2肾上腺素受体迅速引起大鼠体内促激素的变化而影响StAR基因的mRNA表达变化。CLB急性染毒实验提示20 mg kg-1 bw的CLB给药剂量可能为成年大鼠睾丸β2肾上腺素受体的最大饱和剂量。同时提示StAR基因的mRNA表达变化亦与CLB在体内的代谢时间有关。
4 CLB亚急性染毒实验表明CLB均能使大鼠睾丸组织StAR mRNA表达水平降低且随CLB给药剂量的加大而表达降低,呈现剂量效应。CLB亚急性染毒停药七天能使StAR基因的mRNA表达水平有所升高但仍低于对照组水平。说明CLB (14 d)染毒能够降低大鼠睾丸基因StAR的mRNA表达。提示StAR基因的mRNA表达变化亦与CLB在体内的代谢时间有关。同时提示CLB亚急性染毒(14 d)可能通过影响大鼠睾丸组织内的β2肾上腺素受体的下调而影响体内促激素的变化从而最终影响大鼠睾丸StAR基因mRNA的表达水平下降。
Objective
The present study was carried out to investigate the effects of acute and subacute treatment with clenbuterol hydrochloride (CLB) on the expression of testicular steroidogenic acute regulatory protein (StAR) mRNA in adult male rats and to elucidate the possible mechanism (s) of genetic alterations caused byβ2-androgen agonists in testis. After that, it would be possible to provide the initial and referential data for further study about effects of growth promoting substances on male reproductive system and about growth promoters' potential hazards to environment.
Materials and Methods
1. Animals and Chemicals
Seventy adult male Sprague-Dawley rats (9-10 weeks old, weighing 200-220 g, specific pathogen-free grade) were housed in a clean facility. The animals were allowed access to commercial pellet food and tap water ad libitum and were maintained on a 12-h light/dark cycle throughout the experiment. CLB of each specific doses was dissolved in 0.9%NaCl solution up to 1 mL and was given to the animals on a specific time. All rats were pre-fed for 1 week to allow adaptation to the new environment.
2. Groups and the routes of administration
In the acute experiment (1 d), thirty rats were randomly divided into six groups (n=5) and were treated with CLB by gavage at dosages of:0 mg kg-1 bw (A and A'),20 mg kg-1 bw (B and B') and 40 mg kg-1 bw (C and C'), respectively. An equivalent volume of 0.9%NaCl solution was given to the vehicle groups (A, A') in the same way. Letter followed by the apostrophe means the group received a 7-d withdrawal period after treatment with CLB, respectively.
In the subacute experiment (14 d), forty rats were randomly assigned to eight groups (n=5) and were treated with CLB by gavage at dosages of:0 mg kg-1 bw day-1 (F and F'), 0.4 mg kg-1 bw day-1 (G and G'),2.0 mg kg-1 bw day-1 (H and H') and 18.5 mg kg-1 bw day-1 (I and I'), respectively. CLB was dissolved in 0.9%NaCl solution up to 1 mL and the vehicle groups (F, F') were received an equivalent volume of 0.9%NaCl solution by gavage. Letter followed by the apostrophe means the group received a 7-d withdrawal period after treatment with CLB, respectively.
Methods
The body weights and testes weights of each animal were recorded. The alterations in the mRNA expression of testicular steroidogenic acute regulatory (StAR) protein were determined by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). All data were expressed as mean±SD (x±s). Statistical analysis was performed with one-way analysis of variance (ANOVA), followed by t test between the treatment and control groups, which performed using SPSS 13.0 statistical software on computer. Statistical significance was accepted at the P＜0.05 level.
Results
1. Effects of CLB treatment on the body weights and testis wet weights.
In CLB acute (1 d) experiment, a significant increase (P＜0.05) was observed in the body weights of rats in groups B'and C' after a 7-d withdrawal period, no significant difference was seen in the groups B and C when compared to control group rats. Significant increases (P＜0.01) were observed in the body weights of rats treated with CLB in groups H, H', I and I'in CLB subacute experiment (14 d), statistical significance (P＜0.05) was also observed in groups G and G'with respect to the control group rats.
2. Effects of CLB treatment on the testis wet weights and the ratio of body weights to testis weights
No significant difference (P＞0.05) was observed in the testis wet weights between the control and CLB-treated groups in both acute and subacute experiment rats. The body weight/testis weight ratio was assessed and no statistical difference (P＞0.05) was found in either acute or subacute experiment animals as compared to rats in the control groups.
3. Effects of acute CLB treatment on the expression of testicular StAR mRNA in rats
In CLB acute experiment, the expression levels of StAR mRNA were markedly increased (P＜0.05) in the testis after administration of CLB at dosages of 20 mg kg-1 bw (B) and 40 mg kg-1 bw (C), but no dose-dependent effects of CLB treatment on the expression levels of StAR mRNA was observed, and StAR mRNA levels (B'and C') were returned to near normal levels after 7 d of CLB withdrawal compared to that of the control group rats.
4. Effects of subacute CLB treatment on the expression of testicular StAR mRNA in rats
The mRNA levels of StAR in rat testis showed a dose-dependent reduction in the sbuacute (14 d) CLB-treated groups and a trend in recovery of mRNA levels to normal level was noticed in the groups of 7-d CLB withdrawal at dosages of 0.4 mg kg-1 (G and G') and 2.0 mg kg-1 bw day-1 (H and H'). However, no recovery in the expression of StAR mRNA was observed in the groups that treated with CLB at the dosage of 18.5 mg kg-1 bw day-1 (I and I') even following a 7-d withdrawal period (P＜0.05) with respect to control group animals.
Conclusion
1. CLB induced a rapid increase in body weights of adult male rats after the CLB acute treatment (7 d of withdrawal) at both doses of 20 and 40 mg kg-1 bw, but no dose-dependent increase was observed. CLB induced a dose-dependent increase in body weights of adult male rats after the subacute CLB treatment. The increase in the body weights of rats may indicate that the single treatment with CLB to cause the maximum effects of increasing body weights is the does of 20 mg kg-1 bw or less. These results suggested that theβ2-adrenergic agonist CLB has potent repartitioning effects in animals through stimulating the anabolic hormones which will finally lead to the enhancement of body weights.
2. The weights of testes and the the ratio of body weights to testis weights were not significantly affected after CLB treatment. These results suggested that the CLB has little impact on the weights of testis of adult rats after an acute (1 d) or a subacute (14 d) treatment.
3. CLB induced a transient and promotive effect on the expression levels of testicular StAR mRNA after the CLB acute treatment at both dosages of 20 and 40 mg kg-1 bw, but not dose-dependent effects. These results may indicate that CLB induce the increase in mRNA level of StAR through acting on theβ2-adrenergic receptors in testis and then stimulating the trophic hormones secretion. These alterations in the expression levels of StAR mRNA elicited by CLB may also be attributed to the half-life of CLB in the rats. The results also may indicate that the dose of 20 mg kg-1 bw CLB is the maximum dosage forβ2-adrenergic receptors saturation dosage to rat testis or less.
4. In subacute exposure (14 d), CLB decreased the testicular expression of StAR mRNA and showed a dose-dependent and persistent and aggravating impact on the StAR mRNA levels. These alterations caused by CLB probably through a mechanism of action correlated to trophic hormones secretion affected by theβ2-adrenergic receptors down-regulation after a 14-d consecutive stimulation by CLB. These alterations in the expression levels of StAR mRNA elicited by CLB may also be attributed to the half-life of CLB in the rats.
引文
[1]Rothwell NJ, Stock MJ. Modification of body composition by clenbuterol in normal and dystrophic (mdx) mice. Biosci Rep,1985,5(9):755-60.
[2]Mitchell GA, Dunnavan G. Illegal use of beta-adrenergic agonists in the United States. J Anim Sci,1998,76(1):208-11.
[3]Kuiper HA, Noordam MY, van Dooren-Flipsen MM, Schilt R, and Roos AH. Illegal use of beta-adrenergic agonists:European Community. J Anim Sci,1998,76(1):195-207.
[4]Barbosa J, Cruz C, Martins J, Silva JM, Neves C, Alves C, and Da Silveira MI. Food poisoning by clenbuterol in Portugal. Food Addit Contam,2005,22(6):563-6.
[5]Blanco A, Flores-Acuna F, Roldan-Villalobos R, and Monterde JG. Testicular damage from anabolic treatments with the beta(2)-adrenergic agonist clenbuterol in pigs:a light and electron microscope study. Vet J,2002,163(3):292-8.
[6]Kruger G, Keck J, Noll K, and Pieper H. Synthesis of further amino-halogen-substituted phenyl-aminoethanols. Arzneimittelforschung,1984,34(11 A):1612-24.
[7]Rose MD, Shearer G, and Farrington WH. The effect of cooking on veterinary drug residues in food:1. Clenbuterol. Food Addit Contam,1995,12(1):67-76.
[8]Biolatti B, Bollo E, Re G, Appino S, Tartari E, Benatti G, Elliott CT, and McCaughey WJ. Pathology and residues in veal calves treated experimentally with clenbuterol. Res Vet Sci, 1994,57(3):365-71.
[9]Meyer HH, Rinke LM. The pharmacokinetics and residues of clenbuterol in veal calves. J Anim Sci,1991,69(11):4538-44.
[10]Yamamoto I, Iwata K, and Nakashima M. Pharmacokinetics of plasma and urine clenbuterol in man, rat, and rabbit. J Pharmacobiodyn,1985,8(5):385-91.
[11]van der Merwe PJ, Toerien S, and Burger WP. Pharmacokinetics of clenbuterol in the ostrich. Analyst,1998,123(12):2715-7.
[12]Dursch I, Meyer HH, and Karg H. Accumulation of the beta-agonist clenbuterol by pigmented tissues in rat eye and hair of veal calves. J Anim Sci,1995,73(7):2050-3.
[13]Smith DJ, Paulson GD. Distribution, elimination, and residues of [14C] clenbuterol HCl in Holstein calves. J Anim Sci,1997,75(2):454-61.
[14]Smith DJ. Total radioactive residues and clenbuterol residues in swine after dietary administration of [14C] clenbuterol for seven days and preslaughter withdrawal periods of zero, three, or seven days. J Anim Sci,2000,78(11):2903-12.
[15]Elliott CT, Crooks SR, McEvoy JG, McCaughey WJ, Hewitt SA, Patterson D, and Kilpatrick D. Observations on the effects of long-term withdrawal on carcass composition and residue concentrations in clenbuterol-medicated cattle. Vet Res Commun,1993,17(6): 459-68.
[16]Zalko D, Bories G, and Tulliez J. Metabolic fate of clenbuterol in calves. J Agric Food Chem,1998,46(5):1935-1943.
[17]Smith DJ. Total radioactive residues and clenbuterol residues in edible tissues, and the stereochemical composition of clenbuterol in livers of broilers after exposure to three levels of dietary [14C] clenbuterol HCl and three preslaughter withdrawal periods. J Anim Sci,1998,76(12):3043-53.
[18]Hassett LJ, Sloss V. The use of Clenbuterol to produce relaxation of the myometrium during caesarean operation in cattle. Aust Vet J,1984,61(12):401-3.
[19]Greene HJ. Clinical study of the use of clenbuterol for postponing parturition in cows. Vet Rec,1981,109(13):283-5.
[20]Denooij PP. The Use of Clenbuterol for Obstetrical Procedures in Forty Cows and One Horse. Can Vet J,1984,25(9):357-359.
[21]Sasse IIL, Hajer R. NAB 365, a beta 2-receptor sympathomimetic agent:clinical experience in horses with lung disease. Journal of Veterinary Pharmacology and Therapeutics,1978,1(3):241-244.
[22]Maltin CA, Delday MI, Watson JS, Heys SD, Nevison IM, Ritchie IK, and Gibson PH. Clenbuterol, a beta-adrenoceptor agonist, increases relative muscle strength in orthopaedic patients. Clin Sci (Lond),1993,84(6):651-4.
[23]Noguchi M, Eguchi Y, Ichiki J, Yahara J, and Noda S. Therapeutic efficacy of clenbuterol for urinary incontinence after radical prostatectomy. Int J Urol,1997,4(5):480-3.
[24]Culmsee C, Stumm RK, Schafer MK, Weihe E, and Krieglstein J. Clenbuterol induces growth factor mRNA, activates astrocytes, and protects rat brain tissue against ischemic damage. Eur J Pharmacol,1999,379(1):33-45.
[25]Semkova I, Schilling M, Henrich-Noack P, Rami A, and Krieglstein J. Clenbuterol protects mouse cerebral cortex and rat hippocampus from ischemic damage and attenuates glutamate neurotoxicity in cultured hippocampal neurons by induction of NGF. Brain Res, 1996,717(1-2):44-54.
[26]Andre C, Couton D, Gaston J, Erraji L, Renia L, Varlet P, and Guillet JG. beta2-adrenergic receptor-selective agonist clenbuterol prevents Fas-induced liver apoptosis and death in mice. Am J Physiol,1999,276(3 Pt 1):G647-54.
[27]徐向阳,顾玉东.氨哮素对神经切断再缝合后肌肉功能恢复的影响.中华手外科杂志,1996,12(A00)：46-48.
[28]常秉文,刘永志.盐酸克伦特罗及其危害.内蒙古畜牧科学,2002,23(002)：38-39.
[29]李俊杰.浅谈饲有瘦肉精猪肉对人体危害及其检疫.福建畜牧兽医,2001,23(005)：53-53.
[30]Carter WJ, Lynch ME. Effect of clenbuterol on recovery of muscle mass and carcass protein content following dietary protein depletion in young and old rats. J Gerontol,1994, 49(4):B162-8.
[31]Cardoso LA, Stock MJ. Effect of clenbuterol on growth and body composition during food restriction in rats. J Anim Sci,1996,74(9):2245-52.
[32]Hamby PL, Stouffer JR, and Smith SB. Muscle metabolism and real-time ultrasound measurement of muscle and subcutaneous adipose tissue growth in lambs fed diets containing a beta-agonist. J Anim Sci,1986,63(5):1410-7.
[33]Nials AT, Coleman RA, Johnson M, Magnussen H, Rabe KF, and Vardey CJ. Effects of beta-adrenoceptor agonists in human bronchial smooth muscle. British journal of pharmacology,1993,110(3):1112.
[34]Brockway JM, MacRae JC, and Williams PE. Side effects of clenbuterol as a repartitioning agent. The Veterinary Record,1987,120(16):381-383.
[35]Burniston JG, Ng Y, Clark WA, Colyer J, Tan LB, and Goldspink DF. Myotoxic effects of clenbuterol in the rat heart and soleus muscle. J Appl Physiol,2002,93(5):1824-32.
[36]Eisemann JH, Huntington GB. Effects of dietary clenbuterol on net flux across the portal-drained viscera, liver and hindquarters of steers (Bos taurus). Comp Biochem Physiol C,1993,104(3):401-6.
[37]Hoffman RJ, Hoffman RS, Freyberg CL, Poppenga RH, and Nelson LS. Clenbuterol ingestion causing prolonged tachycardia, hypokalemia, and hypophosphatemia with confirmation by quantitative levels. J Toxicol Clin Toxicol,2001,39(4):339-44.
[38]Gross WB, Siegel PB. Environment-genetic influences on immunocompetence. J Anim Sci,1988,66(8):2091-4.
[39]Kelley KW. Stress and immune function:a bibliographic review. Ann Rech Vet,1980, 11(4):445.78.
[40]Sanders VM, Munson AE. Role of alpha adrenoceptor activation in modulating the murine primary antibody response in vitro. J Pharmacol Exp Ther,1985,232(2):395-400.
[41]Cubria JC, Reguera R, Balana-Fouce R, Ordonez C, and Ordonez D. Polyamine-mediated heart hypertrophy induced by clenbuterol in the mouse. J Pharm Pharmacol,1998,50(1): 91-6.
[42]Gojmerac T, Pleadin J, Zuric M, Mirko L, and Stipica C. Effects of repeated growth-promoting doses of clenbuterol on the hepatic function of female pigs. Vet Hum Toxicol,2002,44(5):269-71.
[43]Illera JC, Silvan G, Blass A, Martinez MM, and Illera M. The effect of clenbuterol on adrenal function in rats. Analyst,1998,123(12):2521-4.
[44]Biolatti B, Castagnaro M, Bollo E, Appino S, and Re G. Genital lesions following long-term administration of clenbuterol in female pigs. Vet Pathol,1994,31(1):82-92.
[45]Re G, Badino P, Novelli A, and Girardi C. Down-regulation of beta-adrenergic receptors and up-regulation of estrogen and progesterone receptors induced in the reproductive system of female veal calves by dietary clenbuterol Am J Vet Res,1995,56(11):1493-7.
[46]Cagampang FR, Ohkura S, Tsukamura H, Coen CW, Ota K, and Maeda K. Alpha 2-adrenergic receptors are involved in the suppression of luteinizing hormone release during acute fasting in the ovariectomized estradiol-primed rats. Neuroendocrinology, 1992,56(5):724-8.
[47]Jarzab B, Kaminski M, Gubala E, Achtelik W, Wagiel J, and Dohler KD. Postnatal treatment of rats with the beta 2-adrenergic agonist salbutamol influences the volume of the sexually dimorphic nucleus in the preoptic area Brain Res,1990,516(2):257-62.
[48]McElligott MA, Barreto A, Jr., and Chaung LY. Effect of continuous and intermittent clenbuterol feeding on rat growth rate and muscle. Comp Biochem Physiol C,1989,92(1): 135-8.
[49]Clermont Y, Harvey SC. Duration of the Cycle of the Seminiferous Epithelium of Normal, Hypophysectomized and Hypophysectomized-Hormone Treated Albino Rats. Endocrinology,1965,76:80-9.
[50]Hess RA, Schaeffer DJ, Eroschenko VP, and Keen JE. Frequency of the stages in the cycle of the seminiferous epithelium in the rat. Biol Reprod,1990,43(3):517-24.
[51]Zalko D, Debrauwer L, Bories G, and Tulliez J. Metabolism of clenbuterol in rats. Drug Metab Dispos,1998,26(9):891-9.
[52]贾强,张振玲,王筱芬,史岩,谢林.盐酸克伦特罗的急性毒性和致突变试验.毒理学杂志,2005,19(001)：75-76.
[53]Christenson LK, Strauss JF. Steroidogenic acute regulatory protein:an update on its regulation and mechanism of action. Arch Med Res,2001,32(6):576-86.
[54]Irusta G, Parborell F, Peluffo M, Manna PR, Gonzalez-Calvar SI, Calandra R, Stocco DM, and Tesone M. Steroidogenic acute regulatory protein in ovarian follicles of gonadotropin-stimulated rats is regulated by a gonadotropin-releasing hormone agonist. Biol Reprod,2003,68(5):1577-83.
[55]Lin D, Sugawara T, Strauss JF,3rd, Clark BJ, Stocco DM, Saenger P, Rogol A, and Miller WL. Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis. Science,1995,267(5205):1828-31.
[56]Stocco DM. The role of the StAR protein in steroidogenesis:challenges for the future. J Endocrinol,2000,164(3):247-53.
[57]Hales DB, Sha LL, and Payne AH. Testosterone inhibits cAMP-induced de Novo synthesis of Leydig cell cytochrome P-450 (17 alpha) by an androgen receptor-mediated mechanism J Biol Chem,1987,262(23):11200-6.
[58]Manna PR, Dyson MT, Eubank DW, Clark BJ, Lalli E, Sassone-Corsi P, Zeleznik AJ, and Stocco DM. Regulation of steroidogenesis and the steroidogenic acute regulatory protein by a member of the cAMP response-element binding protein family. Mol Endocrinol,2002,16(1):184-99.
[59]Nishimura R, Shibaya M, Skarzynski DJ, and Okuda K. Progesterone stimulation by LH involves the phospholipase-C pathway in bovine luteal cells. J Reprod Dev,2004,50(2): 257-61.
[60]Shen WJ, Patel S, Natu V, Hong R, Wang J, Azhar S, and Kraemer FB. Interaction of hormone-sensitive lipase with steroidogenic acute regulatory protein:facilitation of cholesterol transfer in adrenal. J Biol Chem,2003,278(44):43870-6.
[61]Liu MY, Leu SF, Yang HY, and Huang BM. Inhibitory mechanisms of lead on steroidogenesis in MA-10 mouse Leydig tumor cells. Arch Androl,2003,49(1):29-38.
[62]Manna PR, Huhtaniemi IT, Wang XJ, Eubank DW, and Stocco DM. Mechanisms of epidermal growth factor signaling:regulation of steroid biosynthesis and the steroidogenic acute regulatory protein in mouse Leydig tumor cells. Biol Reprod,2002,67(5):1393-404.
[63]Manna PR, Roy P, Clark BJ, Stocco DM, and Huhtaniemi IT. Interaction of thyroid hormone and steroidogenic acute regulatory (StAR) protein in the regulation of murine Leydig cell steroidogenesis. J Steroid BiochemMol Biol,2001,76(1-5):167-77.
[64]Stocco DM, Clark BJ. Regulation of the acute production of steroids in steroidogenic cells. Endocr Rev,1996,17(3):221-44.
[65]Clark BJ, Wells J, King SR, and Stocco DM. The purification, cloning, and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR). J Biol Chem, 1994,269(45):28314-22.
[66]Pollack SE, Furth EE, Kallen CB, Arakane F, Kiriakidou M, Kozarsky KF, and Strauss JF. Localization of the steroidogenic acute regulatory protein in human tissues. J Clin Endocrinol Metab,1997,82(12):4243-51.
[67]Pilon N, Daneau I, Brisson C, Ethier JF, Lussier JG, and Silversides DW. Porcine and bovine steroidogenic acute regulatory protein (StAR) gene expression during gestation. Endocrinology,1997,138(3):1085-91.
[68]Kiriakidou M, McAllister JM, Sugawara T, and Strauss JF. Expression of steroidogenic acute regulatory protein (StAR) in the human ovary. J Clin Endocrinol Metab,1996, 81(11):4122-8.
[69]Bosmann HB, Hales KH, Li X, Liu Z, Stocco DM, and Hales DB. Acute in vivo inhibition of testosterone by endotoxin parallels loss of steroidogenic acute regulatory (StAR) protein in Leydig cells. Endocrinology,1996,137(10):4522-5.
[70]Leers-Sucheta S, Stocco DM, and Azhar S. Down-regulation of steroidogenic acute regulatory (StAR) protein in rat Leydig cells:implications for regulation of testosterone production during aging. Mech Ageing Dev,1999,107(2):197-203.
[71]Gregory CW, DePhilip RM. Detection of steroidogenic acute regulatory protein (stAR) in mitochondria of cultured rat Sertoli cells incubated with follicle-stimulating hormone. Biol Reprod,1998,58(2):470-4.
[72]Ford SL, Reinhart AJ, Lukyanenko Y, Hutson JC, and Stocco DM. Pregnenolone synthesis in immature rat Sertoli cells. Molecular and cellular endocrinology,1999,157(1-2):87-94.
[73]Splan RK, Pilcik BR, Tomsen UJ, Arango JA, Ruiz-Flores A, Hallock JW, Allan MF, Karnati SK, Vega V, Lindsey BR, Moody DE, and Pomp D. Rapid communication: mapping of the Steroidogenic Acute Regulatory Protein (StAR) gene to porcine chromosome 15 by linkage analysis using a novel PCR-RFLP. J Anim Sci,1998,76(2): 658-9.
[74]Clark BJ. Hormonal and developmental regulation of the steroidogenic acute regulatory protein. Molecular Endocrinology,1995,9(10):1346-1355.
[75]Stocco DM. StAR protein and the regulation of steroid hormone biosynthesis. Annu Rev Physiol,2001,63:193-213.
[76]Hasegawa T, Zhao L, Caron KM, Majdic G, Suzuki T, Shizawa S, Sasano H, and Parker KL. Developmental roles of the steroidogenic acute regulatory protein (StAR) as revealed by StAR knockout mice. Mol Endocrinol,2000,14(9):1462-71.
[77]赵荣坡,熊承良.弱精子症,少弱精子症患者血清,精浆和精子锌含量分析.中华男科学杂志,2005,11(009)：680-682.
[78]Walsh LP, McCormick C, Martin C, and Stocco DM. Roundup inhibits steroidogenesis by disrupting steroidogenic acute regulatory (StAR) protein expression Environ Health Perspect,2000,108(8):769-76.
[79]Cherradi N, Rossier MF, Vallotton MB, Timberg R, Friedberg I, Orly J, Wang XJ, Stocco DM, and Capponi AM. Submitochondrial distribution of three key steroidogenic proteins (steroidogenic acute regulatory protein and cytochrome p450scc and 3beta-hydroxysteroid dehydrogenase isomerase enzymes) upon stimulation by intracellular calcium in adrenal glomerulosa cells. J Biol Chem,1997,272(12):7899-907.
[80]Nishikawa T, Sasano H, Omura M, and Suematsu S. Regulation of expression of the steroidogenic acute regulatory (StAR) protein by ACTH in bovine adrenal fasciculata cells. Biochem Biophys Res Commun,1996,223(1):12-8.
[81]Pescador N, Houde A, Stocco DM, and Murphy BD. Follicle-stimulating hormone and intracellular second messengers regulate steroidogenic acute regulatory protein messenger ribonucleic acid in luteinized porcine granulosa cells. Biol Reprod,1997,57(3):660-8.
[82]Balasubramanian K, Lavoie HA, Garmey JC, Stocco DM, and Veldhuis JD. Regulation of porcine granulosa cell steroidogenic acute regulatory protein (StAR) by insulin-like growth factor I:synergism with follicle-stimulating hormone or protein kinase A agonist. Endocrinology,1997,138(1):433-9.
[83]Mauduit C, Gasnier F, Rey C, Chauvin MA, Stocco DM, Louisot P, and Benahmed M. Tumor necrosis factor-alpha inhibits leydig cell steroidogenesis through a decrease in steroidogenic acute regulatory protein expression. Endocrinology,1998,139(6):2863-8.
[84]Luo L, Chen H, Stocco DM, and Zirkin BR. Leydig cell protein synthesis and steroidogenesis in response to acute stimulation by luteinizing hormone in rats. Biol Reprod,1998,59(2):263-70.
[85]Brand C, Cherradi N, Defaye G, Chinn A, Chambaz EM, Feige JJ, and Bailly S. Transforming growth factor betal decreases cholesterol supply to mitochondria via repression of steroidogenic acute regulatory protein expression. J Biol Chem,1998, 273(11):6410-6.
[86]Chung PH, Sandhoff TW, and McLean MP. Hormone and prostaglandin F2 alpha regulation of messenger ribonucleic acid encoding steroidogenic acute regulatory protein in human corpora lutea Endocrine,1998,8(2):153-60.
[87]Lin T, Hu J, Wang D, and Stocco DM. Interferon-gamma inhibits the steroidogenic acute regulatory protein messenger ribonucleic acid expression and protein levels in primary cultures of rat Leydig cells. Endocrinology,1998,139(5):2217-22.
[88]Walsh LP, Stocco DM. The role of the steroidogenic acute regulatory (StAR) protein in environmental endocrine disruptor inhibited steroidogenesis. Proc.52nd Yamada Conf. Molecular Steroidogenesis,2000.
[89]Walsh LP, Stocco DM. Effects of lindane on steroidogenesis and steroidogenic acute regulatory protein expression. Biol Reprod,2000,63(4):1024-33.
[90]Walsh LP, Webster DR, and Stocco DM. Dimethoate inhibits steroidogenesis by disrupting transcription of the steroidogenic acute regulatory (StAR) gene. Journal of Endocrinology, 2000,167(2):253-263.
[91]Aleem M, Padwal V, Choudhari J, Balasinor N, Parte P, and Gill-Sharma M. Cyproterone acetate affects protamine gene expression in the testis of adult male rat. Contraception, 2005,71(5):379-91.
[92]Peters B, Teubner P, Clausmeyer S, Puschner T, Maser-Gluth C, Wrede HJ, Kranzlin B, and Peters J. StAR expression and the long-term aldosterone response to high-potassium diet in Wistar-Kyoto and spontaneously hypertensive rats. Am J Physiol Endocrinol Metab,2007,292(1):E16-23.
[93]Yang YT, McElligott MA. Multiple actions of beta-adrenergic agonists on skeletal muscle and adipose tissue. Biochem J,1989,261(1):1-10.
[94]MacRae JC, Skene PA, Connell A, Buchan V, and Lobley GE. The action of the beta-agonist clenbuterol on protein and energy metabolism in fattening wether lambs. Br J Nutr,1988,59(3):457-65.
[95]Eisemann JH, Huntington GB, and Ferrell CL. Effects of dietary clenbuterol on metabolism of the hindquarters in steers. J Anim Sci,1988,66(2):342-53.
[96]周光宏,孙晨华.克伦特罗对鸭的产肉性能和有关代谢指标的影响.南京农业大学学报,1991,14(004)：81-86.
[97]王若军,郭年藩.β-肾上腺素能兴奋剂的作用机理及应用效果(上).国外畜牧科技,1993,20(006)：20-21.
[98]王若军,郭年藩.β-肾上腺素能兴奋剂的作用机理及应用效果的研究(下).国外畜牧科技,1994,21(002)：32-34.
[99]王若军,郭年藩.β-肾上腺素能兴奋剂的作用机理及应用效果(中).国外畜牧科技,1994,21(001)：19-21.
[100]Anakwe OO, Murphy PR, and Moger WH. Characterization of beta-adrenergic binding sites on rodent Leydig cells. Biol Reprod,1985,33(4):815-26.
[101]Poyet P, Labrie F. Characterization of beta-adrenergic receptors in dispersed rat testicular interstitial cells. J Androl,1987,8(1):7-13.
[102]Renier G, Gaulin J, Gibb W, Collu R, and Ducharme JR. Effect of catecholamines on porcine Sertoli and Leydig cells in primary culture. Can J Physiol Pharmacol,1987, 65(10):2053-8.
[103]Badino P, Odore R, and Re G. Are so many adrenergic receptor subtypes really present in domestic animal tissues? A pharmacological perspective. Vet J,2005,170(2):163-74.
[104]Chikhou F, Moloney AP, Austin FH, Roche JF, and Enright WJ. Effects of cimaterol administration on plasma concentrations of various hormones and metabolites in Friesian steers. Domest Anim Endocrinol,1991,8(4):471-80.
[105]Kenakin T. Efficacy in drug receptor theory:outdated concept or under-valued tool? Trends Pharmacol Sci,1999,20(10):400-5.
[106]Burns CJ, Gyles SL, Persaud SJ, Sugden D, Whitehouse BJ, and Jones PM. Phosphoprotein phosphatases regulate steroidogenesis by influencing StAR gene transcription. Biochem Biophys Res Commun,2000,273(1):35-9.
[107]Liggett SB. Update on current concepts of the molecular basis of beta2-adrenergic receptor signaling. J Allergy Clin Immunol,2002,110(6 Suppl):S223-7.
[108]Badino P, Odore R, Pagliasso S, Schiavone A, Girardi C, and Re G Steroid and beta-adrenergic receptor modifications in target organs of broiler chickens fed with a diet containing beta2-adrenergic agents. Food ChemToxicol,2008,46(6):2239-43.
[2]Mitchell GA, Dunnavan G. Illegal use of beta-adrenergic agonists in the United States. J Anim Sci,1998,76(1):208-11.
[3]Kuiper HA, Noordam MY, van Dooren-Flipsen MM, Schilt R, and Roos AH. Illegal use of beta-adrenergic agonists:European Community. J Anim Sci,1998,76(1):195-207.
[4]Barbosa J, Cruz C, Martins J, Silva JM, Neves C, Alves C, and Da Silveira MI. Food poisoning by clenbuterol in Portugal. Food Addit Contam,2005,22(6):563-6.
[5]Blanco A, Flores-Acuna F, Roldan-Villalobos R, and Monterde JG. Testicular damage from anabolic treatments with the beta(2)-adrenergic agonist clenbuterol in pigs:a light and electron microscope study. Vet J,2002,163(3):292-8.
[6]Kruger G, Keck J, Noll K, and Pieper H. Synthesis of further amino-halogen-substituted phenyl-aminoethanols. Arzneimittelforschung,1984,34(11 A):1612-24.
[7]Rose MD, Shearer G, and Farrington WH. The effect of cooking on veterinary drug residues in food:1. Clenbuterol. Food Addit Contam,1995,12(1):67-76.
[8]Biolatti B, Bollo E, Re G, Appino S, Tartari E, Benatti G, Elliott CT, and McCaughey WJ. Pathology and residues in veal calves treated experimentally with clenbuterol. Res Vet Sci, 1994,57(3):365-71.
[9]Meyer HH, Rinke LM. The pharmacokinetics and residues of clenbuterol in veal calves. J Anim Sci,1991,69(11):4538-44.
[10]Yamamoto I, Iwata K, and Nakashima M. Pharmacokinetics of plasma and urine clenbuterol in man, rat, and rabbit. J Pharmacobiodyn,1985,8(5):385-91.
[11]van der Merwe PJ, Toerien S, and Burger WP. Pharmacokinetics of clenbuterol in the ostrich. Analyst,1998,123(12):2715-7.
[12]Dursch I, Meyer HH, and Karg H. Accumulation of the beta-agonist clenbuterol by pigmented tissues in rat eye and hair of veal calves. J Anim Sci,1995,73(7):2050-3.
[13]Smith DJ, Paulson GD. Distribution, elimination, and residues of [14C] clenbuterol HCl in Holstein calves. J Anim Sci,1997,75(2):454-61.
[14]Smith DJ. Total radioactive residues and clenbuterol residues in swine after dietary administration of [14C] clenbuterol for seven days and preslaughter withdrawal periods of zero, three, or seven days. J Anim Sci,2000,78(11):2903-12.
[15]Elliott CT, Crooks SR, McEvoy JG, McCaughey WJ, Hewitt SA, Patterson D, and Kilpatrick D. Observations on the effects of long-term withdrawal on carcass composition and residue concentrations in clenbuterol-medicated cattle. Vet Res Commun,1993,17(6): 459-68.
[16]Zalko D, Bories G, and Tulliez J. Metabolic fate of clenbuterol in calves. J Agric Food Chem,1998,46(5):1935-1943.
[17]Smith DJ. Total radioactive residues and clenbuterol residues in edible tissues, and the stereochemical composition of clenbuterol in livers of broilers after exposure to three levels of dietary [14C] clenbuterol HCl and three preslaughter withdrawal periods. J Anim Sci,1998,76(12):3043-53.
[18]Hassett LJ, Sloss V. The use of Clenbuterol to produce relaxation of the myometrium during caesarean operation in cattle. Aust Vet J,1984,61(12):401-3.
[19]Greene HJ. Clinical study of the use of clenbuterol for postponing parturition in cows. Vet Rec,1981,109(13):283-5.
[20]Denooij PP. The Use of Clenbuterol for Obstetrical Procedures in Forty Cows and One Horse. Can Vet J,1984,25(9):357-359.
[21]Sasse IIL, Hajer R. NAB 365, a beta 2-receptor sympathomimetic agent:clinical experience in horses with lung disease. Journal of Veterinary Pharmacology and Therapeutics,1978,1(3):241-244.
[22]Maltin CA, Delday MI, Watson JS, Heys SD, Nevison IM, Ritchie IK, and Gibson PH. Clenbuterol, a beta-adrenoceptor agonist, increases relative muscle strength in orthopaedic patients. Clin Sci (Lond),1993,84(6):651-4.
[23]Noguchi M, Eguchi Y, Ichiki J, Yahara J, and Noda S. Therapeutic efficacy of clenbuterol for urinary incontinence after radical prostatectomy. Int J Urol,1997,4(5):480-3.
[24]Culmsee C, Stumm RK, Schafer MK, Weihe E, and Krieglstein J. Clenbuterol induces growth factor mRNA, activates astrocytes, and protects rat brain tissue against ischemic damage. Eur J Pharmacol,1999,379(1):33-45.
[25]Semkova I, Schilling M, Henrich-Noack P, Rami A, and Krieglstein J. Clenbuterol protects mouse cerebral cortex and rat hippocampus from ischemic damage and attenuates glutamate neurotoxicity in cultured hippocampal neurons by induction of NGF. Brain Res, 1996,717(1-2):44-54.
[26]Andre C, Couton D, Gaston J, Erraji L, Renia L, Varlet P, and Guillet JG. beta2-adrenergic receptor-selective agonist clenbuterol prevents Fas-induced liver apoptosis and death in mice. Am J Physiol,1999,276(3 Pt 1):G647-54.
[27]徐向阳,顾玉东.氨哮素对神经切断再缝合后肌肉功能恢复的影响.中华手外科杂志,1996,12(A00)：46-48.
[28]常秉文,刘永志.盐酸克伦特罗及其危害.内蒙古畜牧科学,2002,23(002)：38-39.
[29]李俊杰.浅谈饲有瘦肉精猪肉对人体危害及其检疫.福建畜牧兽医,2001,23(005)：53-53.
[30]Carter WJ, Lynch ME. Effect of clenbuterol on recovery of muscle mass and carcass protein content following dietary protein depletion in young and old rats. J Gerontol,1994, 49(4):B162-8.
[31]Cardoso LA, Stock MJ. Effect of clenbuterol on growth and body composition during food restriction in rats. J Anim Sci,1996,74(9):2245-52.
[32]Hamby PL, Stouffer JR, and Smith SB. Muscle metabolism and real-time ultrasound measurement of muscle and subcutaneous adipose tissue growth in lambs fed diets containing a beta-agonist. J Anim Sci,1986,63(5):1410-7.
[33]Nials AT, Coleman RA, Johnson M, Magnussen H, Rabe KF, and Vardey CJ. Effects of beta-adrenoceptor agonists in human bronchial smooth muscle. British journal of pharmacology,1993,110(3):1112.
[34]Brockway JM, MacRae JC, and Williams PE. Side effects of clenbuterol as a repartitioning agent. The Veterinary Record,1987,120(16):381-383.
[35]Burniston JG, Ng Y, Clark WA, Colyer J, Tan LB, and Goldspink DF. Myotoxic effects of clenbuterol in the rat heart and soleus muscle. J Appl Physiol,2002,93(5):1824-32.
[36]Eisemann JH, Huntington GB. Effects of dietary clenbuterol on net flux across the portal-drained viscera, liver and hindquarters of steers (Bos taurus). Comp Biochem Physiol C,1993,104(3):401-6.
[37]Hoffman RJ, Hoffman RS, Freyberg CL, Poppenga RH, and Nelson LS. Clenbuterol ingestion causing prolonged tachycardia, hypokalemia, and hypophosphatemia with confirmation by quantitative levels. J Toxicol Clin Toxicol,2001,39(4):339-44.
[38]Gross WB, Siegel PB. Environment-genetic influences on immunocompetence. J Anim Sci,1988,66(8):2091-4.
[39]Kelley KW. Stress and immune function:a bibliographic review. Ann Rech Vet,1980, 11(4):445.78.
[40]Sanders VM, Munson AE. Role of alpha adrenoceptor activation in modulating the murine primary antibody response in vitro. J Pharmacol Exp Ther,1985,232(2):395-400.
[41]Cubria JC, Reguera R, Balana-Fouce R, Ordonez C, and Ordonez D. Polyamine-mediated heart hypertrophy induced by clenbuterol in the mouse. J Pharm Pharmacol,1998,50(1): 91-6.
[42]Gojmerac T, Pleadin J, Zuric M, Mirko L, and Stipica C. Effects of repeated growth-promoting doses of clenbuterol on the hepatic function of female pigs. Vet Hum Toxicol,2002,44(5):269-71.
[43]Illera JC, Silvan G, Blass A, Martinez MM, and Illera M. The effect of clenbuterol on adrenal function in rats. Analyst,1998,123(12):2521-4.
[44]Biolatti B, Castagnaro M, Bollo E, Appino S, and Re G. Genital lesions following long-term administration of clenbuterol in female pigs. Vet Pathol,1994,31(1):82-92.
[45]Re G, Badino P, Novelli A, and Girardi C. Down-regulation of beta-adrenergic receptors and up-regulation of estrogen and progesterone receptors induced in the reproductive system of female veal calves by dietary clenbuterol Am J Vet Res,1995,56(11):1493-7.
[46]Cagampang FR, Ohkura S, Tsukamura H, Coen CW, Ota K, and Maeda K. Alpha 2-adrenergic receptors are involved in the suppression of luteinizing hormone release during acute fasting in the ovariectomized estradiol-primed rats. Neuroendocrinology, 1992,56(5):724-8.
[47]Jarzab B, Kaminski M, Gubala E, Achtelik W, Wagiel J, and Dohler KD. Postnatal treatment of rats with the beta 2-adrenergic agonist salbutamol influences the volume of the sexually dimorphic nucleus in the preoptic area Brain Res,1990,516(2):257-62.
[48]McElligott MA, Barreto A, Jr., and Chaung LY. Effect of continuous and intermittent clenbuterol feeding on rat growth rate and muscle. Comp Biochem Physiol C,1989,92(1): 135-8.
[49]Clermont Y, Harvey SC. Duration of the Cycle of the Seminiferous Epithelium of Normal, Hypophysectomized and Hypophysectomized-Hormone Treated Albino Rats. Endocrinology,1965,76:80-9.
[50]Hess RA, Schaeffer DJ, Eroschenko VP, and Keen JE. Frequency of the stages in the cycle of the seminiferous epithelium in the rat. Biol Reprod,1990,43(3):517-24.
[51]Zalko D, Debrauwer L, Bories G, and Tulliez J. Metabolism of clenbuterol in rats. Drug Metab Dispos,1998,26(9):891-9.
[52]贾强,张振玲,王筱芬,史岩,谢林.盐酸克伦特罗的急性毒性和致突变试验.毒理学杂志,2005,19(001)：75-76.
[53]Christenson LK, Strauss JF. Steroidogenic acute regulatory protein:an update on its regulation and mechanism of action. Arch Med Res,2001,32(6):576-86.
[54]Irusta G, Parborell F, Peluffo M, Manna PR, Gonzalez-Calvar SI, Calandra R, Stocco DM, and Tesone M. Steroidogenic acute regulatory protein in ovarian follicles of gonadotropin-stimulated rats is regulated by a gonadotropin-releasing hormone agonist. Biol Reprod,2003,68(5):1577-83.
[55]Lin D, Sugawara T, Strauss JF,3rd, Clark BJ, Stocco DM, Saenger P, Rogol A, and Miller WL. Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis. Science,1995,267(5205):1828-31.
[56]Stocco DM. The role of the StAR protein in steroidogenesis:challenges for the future. J Endocrinol,2000,164(3):247-53.
[57]Hales DB, Sha LL, and Payne AH. Testosterone inhibits cAMP-induced de Novo synthesis of Leydig cell cytochrome P-450 (17 alpha) by an androgen receptor-mediated mechanism J Biol Chem,1987,262(23):11200-6.
[58]Manna PR, Dyson MT, Eubank DW, Clark BJ, Lalli E, Sassone-Corsi P, Zeleznik AJ, and Stocco DM. Regulation of steroidogenesis and the steroidogenic acute regulatory protein by a member of the cAMP response-element binding protein family. Mol Endocrinol,2002,16(1):184-99.
[59]Nishimura R, Shibaya M, Skarzynski DJ, and Okuda K. Progesterone stimulation by LH involves the phospholipase-C pathway in bovine luteal cells. J Reprod Dev,2004,50(2): 257-61.
[60]Shen WJ, Patel S, Natu V, Hong R, Wang J, Azhar S, and Kraemer FB. Interaction of hormone-sensitive lipase with steroidogenic acute regulatory protein:facilitation of cholesterol transfer in adrenal. J Biol Chem,2003,278(44):43870-6.
[61]Liu MY, Leu SF, Yang HY, and Huang BM. Inhibitory mechanisms of lead on steroidogenesis in MA-10 mouse Leydig tumor cells. Arch Androl,2003,49(1):29-38.
[62]Manna PR, Huhtaniemi IT, Wang XJ, Eubank DW, and Stocco DM. Mechanisms of epidermal growth factor signaling:regulation of steroid biosynthesis and the steroidogenic acute regulatory protein in mouse Leydig tumor cells. Biol Reprod,2002,67(5):1393-404.
[63]Manna PR, Roy P, Clark BJ, Stocco DM, and Huhtaniemi IT. Interaction of thyroid hormone and steroidogenic acute regulatory (StAR) protein in the regulation of murine Leydig cell steroidogenesis. J Steroid BiochemMol Biol,2001,76(1-5):167-77.
[64]Stocco DM, Clark BJ. Regulation of the acute production of steroids in steroidogenic cells. Endocr Rev,1996,17(3):221-44.
[65]Clark BJ, Wells J, King SR, and Stocco DM. The purification, cloning, and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR). J Biol Chem, 1994,269(45):28314-22.
[66]Pollack SE, Furth EE, Kallen CB, Arakane F, Kiriakidou M, Kozarsky KF, and Strauss JF. Localization of the steroidogenic acute regulatory protein in human tissues. J Clin Endocrinol Metab,1997,82(12):4243-51.
[67]Pilon N, Daneau I, Brisson C, Ethier JF, Lussier JG, and Silversides DW. Porcine and bovine steroidogenic acute regulatory protein (StAR) gene expression during gestation. Endocrinology,1997,138(3):1085-91.
[68]Kiriakidou M, McAllister JM, Sugawara T, and Strauss JF. Expression of steroidogenic acute regulatory protein (StAR) in the human ovary. J Clin Endocrinol Metab,1996, 81(11):4122-8.
[69]Bosmann HB, Hales KH, Li X, Liu Z, Stocco DM, and Hales DB. Acute in vivo inhibition of testosterone by endotoxin parallels loss of steroidogenic acute regulatory (StAR) protein in Leydig cells. Endocrinology,1996,137(10):4522-5.
[70]Leers-Sucheta S, Stocco DM, and Azhar S. Down-regulation of steroidogenic acute regulatory (StAR) protein in rat Leydig cells:implications for regulation of testosterone production during aging. Mech Ageing Dev,1999,107(2):197-203.
[71]Gregory CW, DePhilip RM. Detection of steroidogenic acute regulatory protein (stAR) in mitochondria of cultured rat Sertoli cells incubated with follicle-stimulating hormone. Biol Reprod,1998,58(2):470-4.
[72]Ford SL, Reinhart AJ, Lukyanenko Y, Hutson JC, and Stocco DM. Pregnenolone synthesis in immature rat Sertoli cells. Molecular and cellular endocrinology,1999,157(1-2):87-94.
[73]Splan RK, Pilcik BR, Tomsen UJ, Arango JA, Ruiz-Flores A, Hallock JW, Allan MF, Karnati SK, Vega V, Lindsey BR, Moody DE, and Pomp D. Rapid communication: mapping of the Steroidogenic Acute Regulatory Protein (StAR) gene to porcine chromosome 15 by linkage analysis using a novel PCR-RFLP. J Anim Sci,1998,76(2): 658-9.
[74]Clark BJ. Hormonal and developmental regulation of the steroidogenic acute regulatory protein. Molecular Endocrinology,1995,9(10):1346-1355.
[75]Stocco DM. StAR protein and the regulation of steroid hormone biosynthesis. Annu Rev Physiol,2001,63:193-213.
[76]Hasegawa T, Zhao L, Caron KM, Majdic G, Suzuki T, Shizawa S, Sasano H, and Parker KL. Developmental roles of the steroidogenic acute regulatory protein (StAR) as revealed by StAR knockout mice. Mol Endocrinol,2000,14(9):1462-71.
[77]赵荣坡,熊承良.弱精子症,少弱精子症患者血清,精浆和精子锌含量分析.中华男科学杂志,2005,11(009)：680-682.
[78]Walsh LP, McCormick C, Martin C, and Stocco DM. Roundup inhibits steroidogenesis by disrupting steroidogenic acute regulatory (StAR) protein expression Environ Health Perspect,2000,108(8):769-76.
[79]Cherradi N, Rossier MF, Vallotton MB, Timberg R, Friedberg I, Orly J, Wang XJ, Stocco DM, and Capponi AM. Submitochondrial distribution of three key steroidogenic proteins (steroidogenic acute regulatory protein and cytochrome p450scc and 3beta-hydroxysteroid dehydrogenase isomerase enzymes) upon stimulation by intracellular calcium in adrenal glomerulosa cells. J Biol Chem,1997,272(12):7899-907.
[80]Nishikawa T, Sasano H, Omura M, and Suematsu S. Regulation of expression of the steroidogenic acute regulatory (StAR) protein by ACTH in bovine adrenal fasciculata cells. Biochem Biophys Res Commun,1996,223(1):12-8.
[81]Pescador N, Houde A, Stocco DM, and Murphy BD. Follicle-stimulating hormone and intracellular second messengers regulate steroidogenic acute regulatory protein messenger ribonucleic acid in luteinized porcine granulosa cells. Biol Reprod,1997,57(3):660-8.
[82]Balasubramanian K, Lavoie HA, Garmey JC, Stocco DM, and Veldhuis JD. Regulation of porcine granulosa cell steroidogenic acute regulatory protein (StAR) by insulin-like growth factor I:synergism with follicle-stimulating hormone or protein kinase A agonist. Endocrinology,1997,138(1):433-9.
[83]Mauduit C, Gasnier F, Rey C, Chauvin MA, Stocco DM, Louisot P, and Benahmed M. Tumor necrosis factor-alpha inhibits leydig cell steroidogenesis through a decrease in steroidogenic acute regulatory protein expression. Endocrinology,1998,139(6):2863-8.
[84]Luo L, Chen H, Stocco DM, and Zirkin BR. Leydig cell protein synthesis and steroidogenesis in response to acute stimulation by luteinizing hormone in rats. Biol Reprod,1998,59(2):263-70.
[85]Brand C, Cherradi N, Defaye G, Chinn A, Chambaz EM, Feige JJ, and Bailly S. Transforming growth factor betal decreases cholesterol supply to mitochondria via repression of steroidogenic acute regulatory protein expression. J Biol Chem,1998, 273(11):6410-6.
[86]Chung PH, Sandhoff TW, and McLean MP. Hormone and prostaglandin F2 alpha regulation of messenger ribonucleic acid encoding steroidogenic acute regulatory protein in human corpora lutea Endocrine,1998,8(2):153-60.
[87]Lin T, Hu J, Wang D, and Stocco DM. Interferon-gamma inhibits the steroidogenic acute regulatory protein messenger ribonucleic acid expression and protein levels in primary cultures of rat Leydig cells. Endocrinology,1998,139(5):2217-22.
[88]Walsh LP, Stocco DM. The role of the steroidogenic acute regulatory (StAR) protein in environmental endocrine disruptor inhibited steroidogenesis. Proc.52nd Yamada Conf. Molecular Steroidogenesis,2000.
[89]Walsh LP, Stocco DM. Effects of lindane on steroidogenesis and steroidogenic acute regulatory protein expression. Biol Reprod,2000,63(4):1024-33.
[90]Walsh LP, Webster DR, and Stocco DM. Dimethoate inhibits steroidogenesis by disrupting transcription of the steroidogenic acute regulatory (StAR) gene. Journal of Endocrinology, 2000,167(2):253-263.
[91]Aleem M, Padwal V, Choudhari J, Balasinor N, Parte P, and Gill-Sharma M. Cyproterone acetate affects protamine gene expression in the testis of adult male rat. Contraception, 2005,71(5):379-91.
[92]Peters B, Teubner P, Clausmeyer S, Puschner T, Maser-Gluth C, Wrede HJ, Kranzlin B, and Peters J. StAR expression and the long-term aldosterone response to high-potassium diet in Wistar-Kyoto and spontaneously hypertensive rats. Am J Physiol Endocrinol Metab,2007,292(1):E16-23.
[93]Yang YT, McElligott MA. Multiple actions of beta-adrenergic agonists on skeletal muscle and adipose tissue. Biochem J,1989,261(1):1-10.
[94]MacRae JC, Skene PA, Connell A, Buchan V, and Lobley GE. The action of the beta-agonist clenbuterol on protein and energy metabolism in fattening wether lambs. Br J Nutr,1988,59(3):457-65.
[95]Eisemann JH, Huntington GB, and Ferrell CL. Effects of dietary clenbuterol on metabolism of the hindquarters in steers. J Anim Sci,1988,66(2):342-53.
[96]周光宏,孙晨华.克伦特罗对鸭的产肉性能和有关代谢指标的影响.南京农业大学学报,1991,14(004)：81-86.
[97]王若军,郭年藩.β-肾上腺素能兴奋剂的作用机理及应用效果(上).国外畜牧科技,1993,20(006)：20-21.
[98]王若军,郭年藩.β-肾上腺素能兴奋剂的作用机理及应用效果的研究(下).国外畜牧科技,1994,21(002)：32-34.
[99]王若军,郭年藩.β-肾上腺素能兴奋剂的作用机理及应用效果(中).国外畜牧科技,1994,21(001)：19-21.
[100]Anakwe OO, Murphy PR, and Moger WH. Characterization of beta-adrenergic binding sites on rodent Leydig cells. Biol Reprod,1985,33(4):815-26.
[101]Poyet P, Labrie F. Characterization of beta-adrenergic receptors in dispersed rat testicular interstitial cells. J Androl,1987,8(1):7-13.
[102]Renier G, Gaulin J, Gibb W, Collu R, and Ducharme JR. Effect of catecholamines on porcine Sertoli and Leydig cells in primary culture. Can J Physiol Pharmacol,1987, 65(10):2053-8.
[103]Badino P, Odore R, and Re G. Are so many adrenergic receptor subtypes really present in domestic animal tissues? A pharmacological perspective. Vet J,2005,170(2):163-74.
[104]Chikhou F, Moloney AP, Austin FH, Roche JF, and Enright WJ. Effects of cimaterol administration on plasma concentrations of various hormones and metabolites in Friesian steers. Domest Anim Endocrinol,1991,8(4):471-80.
[105]Kenakin T. Efficacy in drug receptor theory:outdated concept or under-valued tool? Trends Pharmacol Sci,1999,20(10):400-5.
[106]Burns CJ, Gyles SL, Persaud SJ, Sugden D, Whitehouse BJ, and Jones PM. Phosphoprotein phosphatases regulate steroidogenesis by influencing StAR gene transcription. Biochem Biophys Res Commun,2000,273(1):35-9.
[107]Liggett SB. Update on current concepts of the molecular basis of beta2-adrenergic receptor signaling. J Allergy Clin Immunol,2002,110(6 Suppl):S223-7.
[108]Badino P, Odore R, Pagliasso S, Schiavone A, Girardi C, and Re G Steroid and beta-adrenergic receptor modifications in target organs of broiler chickens fed with a diet containing beta2-adrenergic agents. Food ChemToxicol,2008,46(6):2239-43.