Polycyclic Aromatic Hydrocarbons Present in Cigarette Smoke Cause Bone Loss in an Ovariectomized Rat Model L. L. LEE, 1,2 J. S. C. LEE, 1,2 S. D. WALDMAN, 1,3 R. F. CASPER, 1,2,4 and M. D. GRYNPAS 1,2,3 1 Samuel Lunenfeld Research Institute of Mount Sinai Hospital, Toronto, ON, Canada 2 Departments of Laboratory Medicine and Pathobiology, 3 Institute of Biomaterials and Biomedical Engineering, and 4 Department of Obstetrics and Gynecology, University of Toronto, Toronto, ON, Canada A number of epidemiological studies have suggested that cigarette smoking is a risk factor for osteoporosis. Benzo- (a)pyrene (BaP) and 7,12-dimethylbenz(a)anthracene (DMBA) are polycyclic aromatic hydrocarbons (PAHs) found in the tar fraction of cigarette smoke, as well as in car exhaust and furnace gases. We hypothesized that BaP and DMBA are responsible, through interaction with the aryl hydrocarbon receptor (AhR), for the bone loss and fragility seen in smoking-related osteoporosis. In this study four groups of 9-month-old Sprague-Dawley rats were examined. An intact group served as controls. A second control was the ovariectomized (ovx) group. The third group (ovx  E 2 ) were ovariectomized and also given a continuous basal dose of estrogen by implanted estrogen pellet (0.085 mg of 17-estradiol per rat). The fourth group (ovx  E 2  BaP/DMBA) was ovariectomized with an estradiol pellet, and received subcutaneous injections of 250 g/kg of BaP/DMBA weekly for 15 weeks. The loss of ovarian function allowed the study of a direct effect of BaP/DMBA on bone while the concomitant estrogen reple- tion prevented ovx-related bone loss. Dual-energy X-ray absorptiometry (DEXA), histomorphometry, image analy- sis, and mechanical testing were used to determine the effect of the treatments on bone. The DEXA results showed a significant (p < 0.05) decrease in bone mineral density compared with intact controls with both ovx alone and with ovx  E 2  BaP/DMBA treatment. The ovx  E 2 rats were similar to the intact controls. The osteoid parameters showed a significant increase (p < 0.05) with BaP/DMBA addition vs. intact controls, mimicking the ovx rats. The ovx  E 2 rats had osteoid parameters comparable to those of intact rats. Bone connectivity was decreased in the ovx and ovx  E 2  BaP/DMBA animals. Connectivity of the ovx  E 2 rats was comparable to that of intact animals. A decrease in failure force was seen in three-point bending for the ovx  E 2  BaP/DMBA group and in vertebral compression in both the ovx and ovx  E 2  BaP/DMBA groups vs. intact controls. The mechanical properties of the ovx  E 2 rats were similar to those of intact rats. These results demonstrate that BaP/DMBA causes a loss of bone mass and bone strength, possibly through an increase in bone turnover. This is the first in vivo study linking environmental toxicants, found in the tar fraction of cig- arette smoke and in urban air pollution, to loss of bone mass and strength in estrogen-replete ovx rats. (Bone 30: 917–923; 2002) © 2002 by Elsevier Science Inc. All rights reserved. Key Words: Polycyclic aromatic hydrocarbons; Benzo(a)pyrene (BaP); 7,12-Dimethylbenz(a)anthracene (DMBA); Cigarette smoke; Ovariectomy (ovx); Estrogen; Osteoporosis. Introduction Epidemiological studies have focused on the deleterious effects of smoking on human health. Of particular interest to our present study was the number of epidemiological reports that implicated cigarette smoke as a serious risk factor for postmenopausal osteoporosis. It has been well documented that cigarette smoke decreases bone mineral density 20,34 and increases the risk of bone fracture. 10 Cigarette smoking in women has also been associated with an earlier age of natural menopause 13 and re- duced fertility. 39 Collectively, the literature suggests that the toxic effect of cigarette smoke may be mediated through a deficiency in estrogen levels. It is possible that one or more chemicals in cigarette smoke may directly disrupt estrogen ac- tivity, thereby affecting the bone. Despite the knowledge that cigarette smoking is a well- established risk factor, the mechanism responsible for the ad- verse effects of cigarette smoke on bone has yet to be deter- mined. One difficulty lies in the fact that cigarette smoke is composed of over 4000 different chemicals. 4 In the present study, we focused on the role of polycyclic aromatic hydrocar- bons (PAHs) that interact with the aryl hydrocarbon receptor (AhR) as the potential mediators of the toxic effects of cigarette smoking. Benzo(a)pyrene (BaP) and 7,12-dimethylbenz(a)an- thracene (DMBA) are two PAHs found in high concentrations (40 –100 ng per cigarette) in the tar fraction of cigarette smoke. 4,37 BaP is also an environmental toxicant found in diesel engine emissions, automobile exhaust, furnace gases, and smoked foods. 29 The biological effects of PAH are mediated by the aryl hydrocarbon receptor (AhR). AhR is a 110 kDa protein of the basic helix-loop-helix/PAS family of transcription factors. AhR is present in the cytosol of mammalian cells of almost all organs and tissues bound to heat shock protein-90 (HSP-90). 31 With ligand binding, AhR dissociates from HSP-90 and the ligand- Address for correspondence and reprints: Dr. Marc D. Grynpas, Samuel Lunenfeld Research Institute, 600 University Avenue, Room 840, To- ronto M5G 1X5, Canada. E-mail: grynpas@mshri.on.ca Bone Vol. 30, No. 6 June 2002:917–923 917 © 2002 by Elsevier Science Inc. 8756-3282/02/$22.00 All rights reserved. PII S8756-3282(02)00726-3