Li QN et al / Acta Pharmacol Sin 2003 Jun; 24 (6): 599-604
LI Qing-Nan2,4, HU Bin, HUANG Lian-Fang, CHEN Yan, WENG Ling-Ling3, ZHENG Hu3, CHEN Huai-Qing4
2 Bone Biology Laboratory, Guangdong Medical College, Zhanjiang 524023; 3School of Pharmacology; 4 Bio-Medical-Engineering Laboratory, Western China University of Medical Sciences, Chengdu 610041, China
1 Project supported by the National Natural Science Foundation of China, No 39430120.
2 Correspendence to Dr LI Qing-Nan. Phn 86-759-238-8578. Fax 86-759-228-4104. E-mail qingnan@gdmc.edu.cn
Received 2002-10-21 Accepted 2002-12-13
KEY WORDS tetracycline; ovariectomy; metabolic bone diseases; tibia; uterus
ABSTRACT
AIM: To study the effects of low doses of hydrochloride tetracycline (Tc) on bone metabolism and uterus in the ovariectomized (Ova) rats. METHODS: Forty 3-month-old rats were randomly divided into 5 groups: sham group, Ova group, Tc1 group (1.2 mg×kg-1× d-1), Tc2 group (4.8 mg×kg-1× d-1), and estrone group (1.48 mg×kg-1× d-1), oral fed for 3 months. The proximal tibia metaphyses were processed undecalcified for quantitative bone histomorphometry and the soft tissues were processed in paraffin for pathological observation. RESULTS: Placebo-treated (lactose) Ova rats were characterized by trabecular area (TA) decreasing and their architecture worsening compared with sham controls, and bone resorption was over formation with high bone turnover. The uteri were atrophy. (2) In estrone-treated group, TA and trabecular numbers were significantly increased and the trabecular separation decreased vs Ova group. Estrone slowed down Ova-inducing bone high turnover. But the size, weight, and the endometrium of the uteri in this group were increased vs Ova group. (3) TA was increased in both Tc1 and Tc2 groups compared with Ova rats. Tc maintained bone formation indices almost at Ova level, and only decreased mineral apposition rate (MAR) in Tc1 group, and declined bone resorption perimeter. The uteri and the cell of liver and kidney almost maintained at Ova level; Tc2 decreased labeling perimeter and increased MAR in comparison with Tc1 group. The uteri were atrophy, whose size maintained at Ova level; yellow labeling was not found in bone with these doses of Tc, while yellow labeling could be seen with the doses of 30 mg×kg-1× d-1 of Tc for bone marker.CONCLUSION: The two doses of Tc have similar effects on preventing bone loss in Ova rats while the bone formation and uterus are not affected. However, Tc2 does not have more effects on increasing bone mass, Tc2 causes less mild damages to the liver and kidneys.
INTRODUCTION
Osteoporosis is a common disease characterized by an absolute decrease in bone mass leading to skel etal fractures with minimal trauma, which has serious health consequences for older individuals of both genders because of often-prolonged hospitalization, loss of independence, and increased risk of death. Thus, drugs that are able to inhibit bone resorption are commonly used in the treatment of postmenopausal bone loss, such as estrogen and bisphosphonates. However, the severity of the side effects and problems associated with long-term estrogen limited its use. Bisphosphonates are strong antiresorptive agents which inhibit bone turn over and therefore, may induce microdamage[1]. There are no ideal medicines for osteoporosis in the world right now. Hydrochloride tetracycline (Tc), as one kind of the antibiotic and fluorochrome dye, can be chelated by bone, which can determine where and how fast bone is forming[1-3]. Yellow labeling can be seen with Tc in high dose (30 mg×kg-1× d-1) for bone marker under epifluorescent microscope. But Tc may have an effect on bone formation for chelation characteristic of tetracycline. The hypothesis of this study is to test that Tc may be superior to antiresorptive agents for augmentation of cancellous bone mass, and that the mechanism of Tc is to decrease bone resorption, but not to decrease bone formation in the ovariectomized (Ova) rats. Early reports showed that Tc had side effects in liver, kidney, bone, and teeth when used in high dose and long time[4,5]. However, Tc has a bone activity, it is essential and possible to find the lowest dose which is not only beneficial to bone quality, but also still safe for long-term use. The purpose of this study is to design whether Tc in very low doses (1.2 and 4.8 mg×kg-1× d-1) prevents osteopenia, not associated with decreasing bone turnover, but decreasing bone resorption and maintaining bone formation indices in Ova rats without side effects on uterus, liver, kidney, teeth, etc.
MATERIALS AND METHODS
Rats Sprague-Dawley rats,
, n=50, weighing190 g±18 g, 3-month-old (Guangdong Experimental
Animal Center, China, Clean animals GD99A059) were
acclimated to local vivarium conditions for 2 weeks.
Each rat was housed in 69 cm×30 cm×20 cm cage and
allowed free access to water and pelleted diets. Forty
rats were ovariectomized and ten rats were sham
operated from a dorsal approach[6]. Rats were divided into 5
groups: Sham group, Ova group, Tc1 group,Tc2 group,
and estrone group.
Sham and Ova controls received ig the vehicle(lactose), the other groups were fed the following drugs: Tc (1.2 and 4.8 mg×kg-1× d-1) (Sichuan Pharmaceutical Company), estrone (1.48 mg×kg-1× d-1) (School of Pharmacy, Western China University of Medical Sciences), experimental period was 90 d.
Labeling administration All rats received sc injections of tetracycline (Sichuan Pharmaceutical Company) 30 mg/kg at d 14 and d 13 and calcein (Sigma Chemical Co, St Louis, MO, USA) 5 mg/kg at d 4 and d 3 before sacrifice[6].
Autopsy and sample preparation Rats were sacrificed by heart puncture under sodium pentobarbitital anesthesia (40 mg/kg). The left tibia was removed, dissected free of soft tissue, and cut into three equal parts which were then undecalcified embedding and sectioned in hard tissue Microtone (Leica 2155, Germany).
Bone histomorphometric analyses Measurements were performed with a digitizing system consisting of a light and epifluorescent microscope. The system was coupled to an Apple Macintosh computer with a morphometry program "Stereology" (KSS Computer Engineers, Magna, UT)[7]. Static parameters including total tissue area, trabecular bone area, and perimeter were measured and used to calculate the percent trabecular bone area (TA) and trabecular thickness (TT), number (TN), and separation (TS). Dynamic parameters included single- and double-labeled perimeters, interlabeled width and osteoclast number. The above parameters were used to calculate the percent of labeled perimeters (LP), mineral apposition rate (MAR), trabecular bone area-based bone formation rate (BFR/BV), tissue area-based bone formation rate (BFR/TV), osteoclast number per mm on trabecular bone surface (ON)[8].
Soft tissue observation Liver, kidney, and uterus
were corrected and regularly embedded in paraffin and
sectioned 5 
m in microtone, H-E stain for
pathological observation.
Statistic analysis Data were expressed as mean± SD and analyzed by Dunnett t-test. The % was calculated from X2/X1 ×100-100.
RESULTS
Bone histomorphometric changes
Ova group A decrease in TA and TN with an increase in TS were observed at d 90. Both the bone formation parameter and osteoclast number were increased in Ova compared with the sham-operated controls (Tab 1).
Estrone group TA and TN were significantly increased in estrone-treated group compared with Ova-operated group and its trabecular separation decreased but did not reach up to sham level. Estrone decreased bone formation indices (LP, MAR, and BFR/BV) and osteoclast number in Ova rats (Tab 1). These data showed that estrone slowed down Ova-inducing bone high turnover.
Tc1 group Treatment with Tc1 for 90 d increased TA (+73 %), TN (+132 %) and decreased TS (-63 %) in proximal tibia metaphyses (PTM) of Ova rats, which did not reach up to sham level. For dynamic data, Tc used for 90 d did not decrease bone formation indices (LP %, BFR/BV), which were much higher than that in estrone group and almost maintained at Ova level, but only MAR decreased, which was still lower than that in estrone group. Osteoclast number was insignificantly decreased in Tc1 rats (Tab 1). No yellow labeling was investigated in this dose (1.2 mg/kg) except high dose for bone marker under epifluorescent microscope.
Tc2 group Treatment with Tc2 for 90 d had the same effect on TA as Tc1, but for dynamic data,Tc2 decreased LP and BFR and increased MAR, compared with Tc1 group. The osteoclast number was significantly decreased (-61 %) in Tc2 rats. No yellow labeling was investigated in this dose ( 4.8 mg/kg) except high dose for bone marker under epifluorescent microscope (Fig 1).
Tab 1. Effects of tetracycline (Tc) on ovariectomized rats. Mean±SD. bP<0.05 vs sham. eP<0.05 vs Ova. hP<0.05 vs Tc1.
Tc1: 1.2 mg/kg; Tc2: 4.8 mg/kg. % Sham: change in percent vs sham; % Ova: change in percent vs Ova; % Tc1: change in percent vs Tc1. TA: trabecular bone area; TT: trabecular thickness; TN: trabecular number; TS: trabecular separation; LP: labeled perimeters; MAR:mineral apposition rate; BFR/BV: trabecular bone area-based bone formation rate; BFR/TV: tissue area-based bone formation rate; ON: trabecular bone surface.
Fig 1. Trabecular bone of Tc2 group under fluorescent microscope ( H-E stain, ×200). No yellow labeling was investigated in trabecular bone (a) except Tc high dose mg/kg for bone marker (b).
Pathological changes (Fig 2)
Ova group The mild hydropic degeneration was encountered in the liver cells and the epithelial cells of the proximal convoluted tubules of the kidney. The uteri were atrophy. It was noted that the size of the uteri was significantly small and the endometrium was relatively thiner in which there were fewer endometrial glands and stroma.
Estrone group The uteri in estrone group were increased in size and weight vs Ova group, the hydropic degeneration was observed in the liver cells and the epithelial cells of the tubules of the kidney. The normal endometrium of uterine had been totally replaced by stratified squamous cells in 4 cases out of 9 (squamous metaplasia).
Tc2 group The hydropic degeneration was seen in the liver cells and the epithelial cells of the tubules of the kidney. Very mild fatty degeneration in the cells of liver was seen in 4 cases out of 9. The changes of uteri in Tc2 group were as the same as those in Ova group.
Fig 2. Cross-section of uterus and endometrium. Endometrium in sham (A) and estrone (C) was much thicker than that in Ova (B), Tc1 (D), and Tc2 (E).
DISCUSSION
Tc in this study was found to be effective like estrogen on increasing trabecular bone mass in Ova rats. But the mechanisms between Tc and estrogen to prevent bone loss were different. Tc is a substance that binds to calcium in bone surface and the mechanism of Tc preventing bone loss is to decrease bone resorption, and in the meantime maintains bone formation indices in Ova rats. The effects of Tc are similar to anabolic medicine, such as parathyroid hormone[9]. However, estrogen is a typical antiresorptive drug to decrease bone turnover, not only decreasing bone resorption but also decreasing bone formation.
The dynamic data indicated that Tc1 maintained bone formation indices almost at Ova level, such as LP, which represents osteoblast recruitment. This result is the same as that[2]. But MAR, which represents osteoblast activity has significantly been decreased in Tc1 compared with Ova group, and it is significantly lower than that in the estrogen group. Final result of Tc1 is to increase bone formation rate and reduce bone loss in Ova rats. Tc2 has the same effects on static data (TA, TN, and TS) compared with Tc1, but dynamic data are different from Tc1. Tc2 decreases LP and BFR/BV, but increases MAR. This occurrence shows that Tc2 increases the osteoblast activity, while Tc1 increases osteoblast recruitment. Why Tc1 and Tc2 have the different bone activity on osteoblast is an interesting topic, which needs further study. For the bone mass Tc2 almost has the same effects as Tc1. This result is probably due to the dose differences of Tc being too small. The further study is neccessary to test more dose effects of Tc on bone. However, osteoclast numbers of both groups are significantly decreased in Ova rats. Tc2 had more suppressive effects on osteoclast than that in Tc1 group. The mechanism of Tc inhibiting osteoclast might be associated with its induction of apoptosis in osteoclast[10].
Tc 30 mg×kg-1× d-1 is used mostly in a method to be labeled as a bone marker. If Tc is used in a dose lower than 30 mg×kg-1× d-1 it will not have a yellow marker or the marker will not be clearly seen on bone under epifluorescent microscope in early studies[4,5]. The lower doses of Tc in this study are 5 %-16 % dose of Tc as a bone marker and 0.24 %-0.72 % dose of Tc as an antibiotic in clinic, which means this lower dose of Tc is impossible to make bone and teeth yellow in adult rats, but for young rats is not clear. The data of lower doses of Tc did not affect the labeling in higher dose as a bone marker (Fig 1). Because the doses of Tc in this study are very low, our hypothesis is that Tc is impossible to cause serious side effects on liver and kidney. The results of pathological section of the liver and kidney have been verified. The most advantage of these doses of Tc is that it does not cause the proliferation of the uteri (Fig 2). It is noted that the sizes of the uteri are significantly small and the endometrium is relatively thiner in which there are fewer endometrial glands and stroma, while estrogen does enlarge uteri sizes due to their proliferation which may induce cancer of the uteri.
The results provide basic information about the low doses and long-term effects of Tc on bone mass and bone structure, and can be used in designing the future experiments. Tc structure can be modified to benefit the bone without severe side-effects on bone, teeth, liver, and kidney, etc[11].
In summary, this study indicated that two doses of hydrochloride tetracycline had the effects on prevention of bone loss as well as showed no harms to liver, kidneys, and uteri in ovariectomized rats. The study showed an obvious advantage of Tc than estrogen, which induces uterus weight even it does prevent the bone loss. This is the result of fundamental differences of the mechanism in prevention of bone loss between Tc and estrogen. As Tc does not slow down the bone formation, it indicates that low dosage of hydrochloride tetracycline may be a good medicine in the prevention of bone loss in osteopenia induced by ovariectomy.
ACKNOWLEDGMENTS The authors are grateful to Prof FU Zhi-Gang (Department of Foreign Language Teaching, Guangdong Medical College) for his excellent English editorial assistance, and Prof CHEN Xiao-Yi (Department of Pathology, Guangdong Medical College) for her investigation in soft tissue.
REFERENCES