Jan Örberg

Bone mineral density in male Baltic grey seal (Halichoerus grypus).

Abstract Bone mineral density (mg cm−3) was studied in male Baltic grey seals (4–23 years of age) by noninvasive computed tomography (pQCT). The material was grouped according to year of collection. Group A: 1850–1955, a period before the main introduction of organochlorines (OCs); Group B: 1965–1985, a period with very high OC contamination; and Group C: 1986–1997, a period with decreasing concentrations of OCs. The reproducibility of the measurements was good with a Coefficient of Variation (CV) ranging from 0.1% to 2.1%. Trabecular bone mineral density of the radius was significantly higher in specimens collected 1986–1997 than in those collected 1965–1985 (p < 0.05). Cortical bone mineral density of the mandible was significantly lower in specimens collected 1986–1997 compared with those collected 1850–1955 (p < 0.05). These results indicate different responses over time in trabecular and cortical bone. During the period of very high OC contamination (1965–1985), trabecular bone density was lowest, whereas cortical bone density was lowest in specimens collected 1985–1997, representing a period of fairly low OC contamination. The mechanisms behind these effects are not known. However, it can be assumed that OCs are involved. Information about residue levels of OCs in the studied individuals is lacking and, therefore, it was not possible to evaluate the impact of OCs in this respect.

Torsional Testing and Peripheral Quantitative Computed Tomography in Rat Humerus

Peripheral quantitative computed tomography (pQCT) is a noninvasive method mainly used to evaluate the densitometric and geometric properties of bone. In the present study, we evaluate the different variables provided by pQCT examination and their ability to predict the mechanical strength properties of the rat humerus. Humeri from 68 female rats were utilized. These humeri represented bone with a wide range of mechanical and densitometric properties as well as geometric dimensions. Various characteristics, such as volumetric cortical density, total mineral content, cortical thickness, total cross-sectional area, cortical area, and polar strength strain index (SSI), were measured by pQCT. The reproducibility of these measurements was good, with a coefficient of variation (CV) ranging from 0.8% to 4.9%. Bone composition (e.g., ash weight, water content, and inorganic content) and bone dimensions (e.g., length, waist, and volume) were also determined. The mechanical properties (maximum torque, torsion at failure, and stiffness) were measured by torsional testing. Stepwise multiple linear regression was performed to identify the best explanatory variables for each mechanical parameter. Total cross-sectional area and polar SSI were equally well correlated to stiffness (r = 0.57, p < 0.001), whereas ash weight was superior to the pQCT variables to explain maximum torque (r = 0.42, p < 0.001). No other independent pQCT variable entered the two models in the stepwise regression analysis. It was found to be feasible to measure properties of the rat humerus with pQCT. Cross-sectional area and the polar SSI were shown to be the best explanatory variables for stiffness, whereas ash weight was the best predictor for maximum torque.

Bisphenol A exposure increases liver fat in juvenile fructose-fed Fischer 344 rats

BACKGROUND Prenatal exposure to bisphenol A (BPA) has been shown to induce obesity in rodents. To evaluate if exposure also later in life could induce obesity or liver damage we investigated these hypothesises in an experimental rat model. METHODS From five to fifteen weeks of age, female Fischer 344 rats were exposed to BPA via drinking water (0.025, 0.25 or 2.5 mg BPA/L) containing 5% fructose. Two control groups were given either water or 5% fructose solution. Individual weight of the rats was determined once a week. At termination magnetic resonance imaging was used to assess adipose tissue amount and distribution, and liver fat content. After sacrifice the left perirenal fat pad and the liver were dissected and weighed. Apolipoprotein A-I in plasma was analyzed by western blot. RESULTS No significant effects on body weight or the weight of the dissected fad pad were seen in rats exposed to BPA, and MRI showed no differences in total or visceral adipose tissue volumes between the groups. However, MRI showed that liver fat content was significantly higher in BPA-exposed rats than in fructose controls (p=0.04). BPA exposure also increased the apolipoprotein A-I levels in plasma (p<0.0001). CONCLUSION We found no evidence that BPA exposure affects fat mass in juvenile fructose-fed rats. However, the finding that BPA in combination with fructose induced fat infiltration in the liver at dosages close to the current tolerable daily intake (TDI) might be of concern given the widespread use of this compound in our environment.

Exposure to pastures fertilised with sewage sludge disrupts bone tissue homeostasis in sheep

The femurs of male and female sheep (Ovis aries), aged 18 months, bred on pastures fertilized twice annually with sewage sludge (2.25 tonnes dry matter/ha; Treated; T)) or on pastures treated with inorganic fertilizer (Control; C) were studied, using peripheral Quantitative Computed Tomography (pQCT) and the three-point bending test. Males were maintained on the respective treatments from conception to weaning and then maintained on control pastures while the females were maintained on the respective treatments until slaughter. T rams exhibited increased total bone mineral density (BMD) at the metaphyseal part of femur (+10.5%, p<0.01) compared with C rams but had a reduced total cross sectional area (CSA, -11.5%, p<0.001), trabecular CSA (-17.1%, p<0.01) and periosteal circumference (-5.7%, p<0.001). In the mid-diaphyseal part, T rams had an increased total BMD (+13.8%, p<0.0001) and stiffness (+6.4%, p<0.01) but reduced total CSA (-12.1%, p<0.0001) and marrow cavity (-25.8%, p<0.0001), relative to C rams. In ewes although pQCT analysis of neither the metaphyseal nor the mid-diaphyseal part of the female femur bones showed any significant differences with treatment, the biomechanical method revealed a reduction in load at failure (-17.3%, p<0.01) and stiffness (-10.7%, p<0.05) amongst T ewes. It is concluded that exposure to pollutants present in sewage sludge can perturb bone tissue homeostasis in sheep, but particularly in males.

Pregnant ewes exposed to multiple endocrine disrupting pollutants through sewage sludge-fertilized pasture show an anti-estrogenic effect in their trabecular bone.

Pregnant ewes were maintained on pastures fertilized, twice yearly, with either sewage sludge (2.25tonnes dry matter/ha; Treated; T) or inorganic fertilizer containing equivalent amounts of nitrogen (Control; C), to determine effects on maternal and fetal bone structures, density and mechanical properties of exposure to environmental concentrations of multiple endocrine disrupting compounds (EDCs) and heavy metal pollutants. The ewes were maintained on the respective pastures from the age of about 8months until they were 4-6years of age and they were slaughtered at 110d gestation. Metaphyseal parts of adult ewe femurs exhibited a significantly reduced mean, total cross sectional area (CSA, -4%; p<0.05), lower trabecular bone mineral content (BMC, mg/mm; -18%; p<0.05), trabecular bone mineral density (BMD, mg/cm(3), -8.0%; p<0.05) and trabecular CSA, mm(2), -11.1%; p<0.05) in T compared with C animals. Femurs of T ewes were stronger than those of C ewes but this may reflect greater body weights. At the mid-diaphyseal part of the fetal bones, there was a reduction in endosteal circumference (-6.7%, p<0.05) and marrow cavity area (-13.8%, p<0.05) in the female T fetuses compared with female C fetuses. In the male fetuses the mid-diaphyseal part total bone mineral content was higher (+3.0%, p<0.05) in T than in C animals. No treatment difference in biomechanical bending was detected in the fetuses. It is concluded that ewes grazing pasture fertilized with sewage sludge exhibited an anti-estrogenic effect on their trabecular bone in the form of reduced mineral content and density, despite increased body weight. It is suggested that human exposure to low levels of multiple EDCs may have implications for bone structure and human health.

Bisphenol A is related to circulating levels of adiponectin, leptin and ghrelin, but not to fat mass or fat distribution in humans

OBJECTIVE Since bisphenol A (BPA) has been shown to induce obesity in experimental studies, we explored the associations between BPA and fat mass, fat distribution and circulating levels of adiponectin, leptin and ghrelin in humans. METHODS In the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS), fat mass and fat distribution were determined in 70-year-old men and women (n=890) by dual-energy X-ray absorptiometry (DXA) and magnetic resonance imaging (MRI) (n=287). Serum levels of BPA were analyzed using isotope liquid chromatography/tandem mass spectrometer (API4000LC-MS/MS). Hormone levels were analyzed with radioimmunoassays (RIA) or enzyme-linked immunosorbent assay (ELISA). Imaging was performed approximately two years following collection of other data. RESULTS Serum concentrations of BPA were not related to adipose tissue measurements by DXA or MRI. BPA associated positively with adiponectin and leptin, but negatively with ghrelin, following adjustments for sex, height, fat mass, lean mass, smoking, alcohol consumption, physical activity, energy intake, and educational levels (p<0.001, p=0.009, p<0.001, respectively). The relationship between BPA and ghrelin was stronger in women than in men. CONCLUSION Although no relationships between BPA levels and measures of fat mass were seen, BPA associated strongly with the adipokines adiponectin and leptin and with the gut-hormone ghrelin suggesting that BPA may interfere with hormonal control of hunger and satiety.

Effects on bone tissue in ewes (Ovies aries) and their foetuses exposed to PCB 118 and PCB 153

The aim of the present study was to investigate whether low levels of mono-ortho PCB 118 and di-ortho PCB 153, affect bone composition and strength in ewes (Dala breed) and their foetuses following exposure starting at conception and ending a week before expected delivery. In male foetuses, trabecular bone mineral content at the metaphysis was almost 30% lower in the PCB 118 (49 microg/kg body wt/day) group compared to the control group (corn oil) (ANCOVA, P<0.05). In female foetuses of the PCB 153 (98 microg/kg body wt/day) group trabecular cross-sectional area at the metaphysis was 19% smaller than in the controls (ANCOVA, P<0.05). At the diaphysis a smaller marrow cavity area (up to 24% reduction) was observed in female and male foetuses exposed to PCB 153 compared with controls (ANCOVA, P<0.05). There were also significant differences at the mid diaphyseal measure point between the PCB 153 and the control group females (ANCOVA, P<0.05). Cortical and total bone mineral density, cortical thickness were significantly higher, endosteal circumference shorter and marrow cavity significantly smaller in the PCB 153 group (ANCOVA, P<0.05). In conclusion there were gender dependent effects on bone tissue and cortical bone was more affected than trabecular bone.

Effects of Short-term Exposure to the DDT Metabolite p,p′-DDE on Bone Tissue in Male Common Frog (Rana temporaria)

Experimental studies as well as studies in free-ranging animals have shown that endocrine-disrupting chemicals (EDCs) impair bone tissue composition and strength. The aim of the present study was to expand our studies on bone tissue in a new group of animals by investigating whether bone tissue in frogs is an additional potential target of EDCs. Adult male European common frogs (Rana temporaria) were divided into 5 groups (n = 20) and injected (sc, single injection) with p,p′-DDE, a total dose of 0.01, 0.1, 1, or 10 mg of p,p′-DDE/kg body weight, respectively. A control group was treated with the vehicle (corn oil). Two weeks after injection the frogs were euthanized and samples taken. The diaphysis of the excised left femur was scanned using peripheral quantitative computed tomography (pQCT) and cortical variables, such as cortical bone mineral density (BMD), cortical cross-sectional area (CSA), and periosteal circumference, were determined. In addition, biomechanical three-point bending of the bones was conducted, with the load being applied to the same point as where the pQCT measurement was performed. The results from the pQCT measurements show that bone tissue in male frogs exposed to p,p′-DDE is negatively affected. A significant decrease in cortical BMD at the diaphysis was observed in frogs exposed to 1 mg p,p′-DDE. However, the biomechanical testing of the bones showed no significant differences between exposed and control group. Although this is the only study performed to date examining the possible relationships between EDCs and negative effects on frog bones, it supports both previous experimental findings in rodents and findings in free-ranging animals.

Health of Herring Gulls (Larus argentatus) in Relation to Breeding Location in the Early 1990s. III. Effects on the Bone Tissue

Health effects associated with the Great Lakes environment were assessed in adult herring gulls (Larus argentatus) in the early 1990s, including the size and quality of their bones. Femurs were excised from 140 individuals from 10 colonies distributed throughout the Great Lakes and 2 reference colonies in Lake Winnipeg (freshwater) and the Bay of Fundy (marine). Femurs of gulls from the Great Lakes differed from the freshwater or marine reference for 9 of 12 variables of size, composition, and strength assessed using peripheral quantitative computed tomography (pQCT) and biomechanical testing. Femurs of Great Lakes gulls were significantly smaller in length (−2.9%), periosteal circumference (−2.4%), and cross-sectional area (−5.4%) than freshwater reference birds. Femurs of the Great Lakes gulls had a lower significant cortical bone mineral content (−8.1%) and density (−2%) than the marine reference. A significant increase in the amount the bone could bend before it broke (+34%) and the energy required to break it (+44%) and a significant decrease (−16.3%) in stiffness during three-point biomechanical bending test were also detected in Great Lakes versus the freshwater gulls. These differences are indicative of impaired mineralization. When divided into high and low 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity equivalent (TCDD-TEQ) colonies, the amount the bone could bend before it broke and the energy required to break it were significantly higher in the high TEQ colonies, but not high polychlorinated biphenyl (PCB) colonies. Breeding location and dietary choices of Great Lakes herring gulls in the early 1990s resulted in modulations of physiological processes that affected the size, mineralization, and biomechanical properties of bone.

Bone mineral density in Swedish otters (Lutra lutra) in relation to PCB and DDE concentrations

The aim of this study is to elucidate if DDE (dichlorodiphenyldichloroethylene) or PCB (polybrominated diphenyls), are responsible for the pathological alterations observed in Swedish otter bone tissues. Femurs from 86 male otters collected between 1832 and 2004 were measured using peripheral quantitative computed tomography (pQCT). Some otters had very high and others fairly low concentrations of OCs (ranging between 1.4-970 mg SigmaPCB/kg l.w. and 0.0-24 mg DDE/kg l.w. in muscle tissue). Positive relationships were found between three of the four cortical bone variables analysed (area, content and thickness) and SigmaPCB concentration, while no significant relationships with DDE concentration were found. None of the trabecular variables were significantly related to PCB or DDE concentration. Three of the four trabecular bone variables showed decreasing values in the beginning and increasing values at the end of period 1974-2004. No temporal trends were found for cortical bone variables. OC concentrations decreased between 1974 and 2004.