Association between P2X7 Receptor Polymorphisms and Bone Status in Mice.

Macrophages from mouse strains with the naturally occurring mutation P451L in the purinergic receptor P2X7 have impaired responses to agonists (1). Because P2X7 receptors are expressed in bone cells and are implicated in bone physiology, we asked whether strains with the P451L mutation have a different bone phenotype. By sequencing the most common strains of inbred mice, we found that only a few strains (BALB, NOD, NZW, and 129) were harboring the wild allelic version of the mutation (P451) in the gene for the purinergic receptor P2X7. The strains were compared by means of dual energy X-ray absorptiometry (DXA), bone markers, and three-point bending. Cultured osteoclasts were used in the ATP-induced pore formation assay. We found that strains with the P451 allele (BALB/cJ and 129X1/SvJ) had stronger femurs and higher levels of the bone resorption marker C-telopeptide collagen (CTX) compared to C57Bl/6 (B6) and DBA/2J mice. In strains with the 451L allele, pore-formation activity in osteoclasts in vitro was lower after application of ATP. In conclusion, two strains with the 451L allele of the naturally occurring mutation P451L, have weaker bones and lower levels of CTX, suggesting lower resorption levels in these animals, which could be related to the decreased ATP-induced pore formation observed in vitro. The importance of these findings for the interpretation of the earlier reported effects of P2X7 in mice is discussed, along with strategies in developing a murine model for testing the therapeutic effects of P2X7 agonists and antagonists upon postmenopausal osteoporosis.

Genetic Background Strongly Influences the Bone Phenotype of P2X7 Receptor Knockout Mice.

The purinergic P2X7 receptor is expressed by bone cells and has been shown to be important in both bone formation and bone resorption. In this study we investigated the importance of the genetic background of the mouse strains on which the P2X7 knock-out models were based by comparing bone status of a new BALB/cJ P2X7(-/-) strain with a previous one based on the C57BL/6 strain. Female four-month-old mice from both strains were DXA scanned on a PIXImus densitometer; femurs were collected for bone strength measurements and serum for bone marker analysis. Bone-related parameters that were altered only slightly in the B6 P2X7(-/-) became significantly altered in the BALB/cJ P2X7(-/-) when compared to their wild type littermates. The BALB/cJ P2X7(-/-) showed reduced levels of serum C-telopeptide fragment (s-CTX), higher bone mineral density, and increased bone strength compared to the wild type littermates. In conclusion, we have shown that the genetic background of P2X7(-/-) mice strongly influences the bone phenotype of the P2X7(-/-) mice and that P2X7 has a more significant regulatory role in bone remodeling than found in previous studies.

Mineral density and biomechanical properties of bone tissue from male Arctic foxes (Vulpes lagopus) exposed to organochlorine contaminants and emaciation.

We investigated the impact from dietary OC (organochlorine) exposure and restricted feeding (emaciation) on bone mineral density (BMD; g hydroxy-apatite cm(-2)) in femoral, vertebrate, skull and baculum osteoid tissue from farmed Arctic blue foxes (Vulpes lagopus). For femur, also biomechanical properties during bending (displacement [mm], load [N], energy absorption [J] and stiffness [N/mm]) were measured. Sixteen foxes (EXP) were fed a wet food containing 7.7% OC-polluted minke whale (Balaenoptera acutorostrata) blubber in two periods of body fat deposition (Aug-Dec) and two periods of body fat mobilisation (Jan-July) in which the food contained less energy and only 2% blubber. SigmaOC food concentration in the food containing 7.7% whale blubber was 309 ng/g wet mass. This corresponded to a SigmaOC exposure of ca. 17 microg/kg body mass/d and a responding SigmaOC residue in subcutaneous adipose tissue of ca. 1700 ng/g live mass in the 8 EXP fat foxes euthanized after 16 months. A control group (CON) composed of 15 foxes were fed equal daily caloric amounts of clean pork (Sus scrofa) fat. After 16 months, 8 EXP and 7 CON foxes were euthanized (mean body mass=9.25 kg) while the remaining 8 EXP and 8 CON foxes were given restricted food rations for 6 months resulting in a body weight reduction (mean body mass=5.46 kg). The results showed that only BMD(skull) vs. BMD(vertebrae) were significantly correlated (R=0.68; p=0.03; n=10) probably due to a similar composition of trabecular and cortical osteoid tissue. No difference in any of the BMD measurements or femoral biomechanical properties was found between EXP and CON foxes although BMD baculum was 1.6-folds lower in the EXP group. However, lean summer foxes had significantly lower femoral biomechanical properties measured as displacement (mm), energy absorption (J) and time (s) biomechanical properties than fat winter foxes (all p<0.004). This indicates lower stiffness and softer bones from fasting which is in agreement with previous studies. Further, it should be kept in mind when studying bone tissues in Arctic mammals also in order to avoid confounding effects from body condition.

Does the nutrition profile of vitamins, fatty acids and microelements counteract the negative impact from organohalogen pollutants on bone mineral density in Greenland sledge dogs (Canis familiaris)?

There is a great need for understanding the impact from dietary OHCs (organohalogen compounds) on bone mineral composition - and thereby osteoporosis - in especially arctic wildlife such as polar bears (Ursus maritimus) as well as humans. For that purpose, we measured BMD (bone mineral density) by DXA scanning (g/cm(-2)) in 15 age and weight normalized sledge dog (Canis familiaris) bitches and their 26 pups divided into a control group (n=26) given 50-200 g/day clean pork (Suis scrofa) fat and a treated group (n=15) given 50-200 g/day OHC polluted minke whale (Balaenoptera acutorostrata) blubber as main lipid sources. The results showed that BMD increased significantly with age (linear regression: p<0.0001, r(2)=0.83, n=41) while no sex difference was found in the F-generation (two-way ANOVA: all p>0.3). No differences in BMD(femur) or BMD(vertebrae) between exposed and control individuals in the bitch generation were found (linear mixed effect model: both p>0.38). Likewise, no difference between exposed and control subadults and juveniles in the F-generation was found (two-way ANOVA: all p>0.33). Correlation analyses between BMD(femur), BMD(vertebrae) and groups of OHCs, respectively, did not show any statistically significant relationships nor a clear or decreasing trend (Pearson's: p: 0.07-0.78; r: -0.2-0.59; n: 10-18). As the groups were similar regarding genetics, age and sex are the only factors that can explain this observation. Either the pollutants did not have an impact on BMD using the present time frame and OHC concentrations (threshold levels not reached), or the difference in food composition (mainly vitamins and n3 fatty acids) conceal the potential OHC impact on BMD. Such information is important when evaluating the positive and negative health consequences from eating polluted marine species.