Low-dose developmental exposure to bisphenol A alters the femoral bone geometry in wistar rats.

BACKGROUND: Bisphenol A (BPA) is a chemical produced in large volumes for use in manufacturing of consumer products and industrial applications, and an endocrine disruptor known to affect several hormonal systems. Bone produces hormones and is additionally a sensitive hormone target tissue, and is thus potentially sensitive to low doses of endocrine disruptors such as BPA, especially during development. METHODS: 110 pregnant Wistar rats were gavaged with 0; 25 µg; 250 µg; 5000 µg or 50,000 µg BPA/kg bodyweight (bw)/day from gestational day 7 until weaning at postnatal day 22. The three-month-old offspring were sacrificed and right femurs collected for length measurements, geometrical measurements by peripheral quantitative computed tomography (pQCT), as well as for analyses of biomechanical properties using the three-point-bending method. RESULTS: The femur was elongated in female offspring of dams exposed to 25 or 5000 µg BPA/kg bw/day (1.8% and 2.1%, respectively), and increased cortical thickness (4.7%) was observed in male offspring of dams exposed to 25 µg BPA/kg bw/day, compared to controls (p < 0.005). The biomechanical properties of the bone were not significantly altered. CONCLUSIONS: In utero and lactational exposure to the lowest BPA dose used in this study altered femoral geometry in both male and female offspring. This was observed at 25 µg BPA/kg bw/day, a dose lower than the Human Equivalent Dose (HED) applied by EFSA to set a temporary TDI (609 µg BPA/kg bw/day), and far lower than the No-Observed-Adverse-Effect-Level (NOAEL) (5000 µg BPA/kg bw/day) on which the US FDA TDI is based.

Subclinical hypervitaminosis A causes fragile bones in rats.

Excessive intake of vitamin A has been associated with an increased risk of hip fracture in humans. This finding has raised the question of whether long-term intake of relatively moderate doses ("subclinical" hypervitaminosis A) contributes to fracture risk. Although it has been known for more than half a century that toxic doses of vitamin A lead to spontaneous fractures in rats, the lowest intake that induces adverse effects is not known, and the result of exposure to excessive doses that do not cause general toxicity has been rarely investigated. In this study, mature female rats were fed a standard diet with 12 IU vitamin A/g pellet (control, C), or standard diet supplemented with either 120 IU ("10 x C") or 600 IU ("50 x C") vitamin A/g pellet for 12 weeks. Fifteen animals were included in each group. The supplemented diets correspond to a vitamin A intake of approximately 1800 IU/day and 9000 IU/day, respectively. The latter dose is about one third of that previously reported to cause skeletal lesions. At the end of the study, serum retinyl esters were elevated 4- (p < 0.01) and 20-fold (p < 0.001) and the total amount of liver retinoid had increased 3- (p < 0.001) and 7-fold (p < 0.001) in the 10 x C and 50 x C group, respectively. The animals showed no clinical signs of general toxicity, and there were no significant bone changes in the 10 x C group. However, in the 50 x C group, a characteristic thinning of the cortex (cortical area -6.5% [p < 0.001]) and reduction of the diameter of the long bones were evident (bone cross-sectional area -7.2% [p < 0.01] at the midshaft and -11.0% [p < 0.01] at the metaphysis), as measured by peripheral quantitative computed tomography. In agreement with these data and a decreased polar strength strain index (-14.0%, p < 0.01), the three-point bending breaking force of the femur was reduced by 10.3% (p < 0.01) in the 50 x C group. These data indicate that the negative skeletal effects appear at a subchronic vitamin A intake of somewhere between 10 and 50 times the standard diet. This level is considerably lower than previously reported. Our results suggest that long-term ingestion of modest excesses of vitamin A may contribute to fracture risk.

Reproductive toxicity in mink (Mustela vison) chronically exposed to environmentally relevant polychlorinated biphenyl concentrations.

Female mink were exposed to a technical polychlorinated biphenyl (PCB) preparation (Clophen A50 [A50]; 0.1 or 0.3 mg/animal/d), one fraction of A50 containing the non- and mono-ortho-chlorinated congeners (0-1-ortho-chlorobiphenyls [CBs]), another fraction of A50 containing the congeners with two to four ortho-chlorines (2-4-ortho-CBs), or an organic extract from Baltic gray seal blubber. The animals were exposed for 18 months, including two reproduction seasons. Among the animals given the highest dose of A50, the whelping frequency was reduced in the second reproductive season, and all kits died within 24 h of birth. Reproduction was also impaired by the lower dose of A50. Daily exposure to the 0-1-ortho-CBs separated from 0.3 mg A50 severely reduced kit survival. Reproduction was not significantly impaired by daily exposure to the 2-4-ortho-CBs separated from 0.3 mg A50 or by exposure to the blubber extract. We conclude that the reproductive toxicity in chronically PCB-exposed mink is caused by the aryl hydrocarbon (Ah) receptor agonists. The lowest-observed-effect level for reproductive impairment was 2.4 ng 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (TEQs) per kilogram body weight and day (22 pg TEQs/g feed). Ethoxyresorufin-O-dealkylase (EROD) was strongly induced by the 0-1-ortho-CBs and pentoxyresorufin-O-dealkylase by the 2-4-ortho-CBs. High EROD activity was correlated with low kit production, and consequently EROD may serve as a marker for reproductive toxicity by Ah receptor agonists in mink.

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.

Bone tissue composition, dimensions and strength in female rats given an increased dietary level of vitamin A or exposed to 3,3',4, 4',5-pentachlorobiphenyl (PCB126) alone or in combination with vitamin C.

In previous studies we have described structural and functional changes in rat bone tissue caused by 3,3',4,4',5-pentachlorobiphenyl (PCB126). Some of the effects caused by PCB126 resemble those found in vitamin C-deficient rats, as well as those found in rats with a high dietary intake of vitamin A. The present investigation was designed to determine if these PCB126-induced changes could be inhibited by addition of vitamin C to the drinking water and if they could be evoked by vitamin A administration. Five groups of female rats were used in this study, which lasted for 12 weeks. Three of the groups were exposed to PCB126 (total dose 320 microgram/kg, bw), either alone or in combination with vitamin C added to the drinking water (1 and 10 g/l, respectively). One group was given feed with increased level of vitamin A (600000 U/kg pellet) and the fifth group served as controls. Using peripheral quantitative computed tomography (pQCT), it was found that PCB126 increased trabecular density and cortical thickness, but reduced the trabecular area. Furthermore, maximum torque and stiffness of the humerus during torsional testing and serum osteocalcin levels were reduced by PCB126. Of the PCB126 induced effects observed, addition of vitamin C only inhibited the reduction of serum osteocalcin. Like PCB126 vitamin A supplementation increased the inorganic content and the bone density and also reduced the trabecular area and polar moment of inertia but did not increase the cortical thickness or reduce maximum torque, stiffness or serum osteocalcin level. Apparently, the effects induced by PCB126 are not mediated either via decreased vitamin C level or increased vitamin A level.

Change of bone tissue composition and impaired bone strength in rats exposed to 3,3',4,4',5-pentachlorobiphenyl (PCB126).

The aim of this study was to compare effects of estrogen depletion (ovariectomy) and exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB126) on bone strength and bone tissue composition in the rat. Half of the rats were ovariectomized (n=20) and the remainder were sham-operated. Ten of the ovariectomized rats and ten of the sham operated were exposed to PCB126 (ip injections) for 3 months (total dose, 384 microgram/kg bodyweight), while those remaining received the vehicle. The humerus and femur were used for analysis of torsional strength and biochemical studies, respectively. Both sham-operated and ovariectomized animals showed a significantly shorter bone length, lower water content and a decreased torsional stiffness when exposed to PCB126. Sham-operated rats exposed to PCB126 had lower maximum torque when compared with sham operated controls. The PCB126-exposed rats also exhibited a significantly lower collagen concentration, but showed a higher pyridinoline concentration of cortical bone. PCB126 exposure decreased the hepatic level of vitamin A but increased vitamin A levels in serum and kidneys. Ovariectomy per se increased bone length and organic content and decreased the inorganic content significantly, but did not affect any of the tested biomechanical parameters. In conclusion, this study showed that the common environmental pollutant PCB126 impaired bone strength and altered bone composition. It is hypothesized that these effects might partly be explained by PCB-induced retinoid disturbances.

Effects of the antiestrogenic environmental pollutant 3,3',4,4', 5-pentachlorobiphenyl (PCB #126) in rat bone and uterus: diverging effects in ovariectomized and intact animals.

The purpose of this study was to compare effects on rat bone and uterus of estrogen depletion and exposure to the coplanar PCB-congener 3,3',4,4',5-pentachlorobiphenyl (PCB #126) which exhibits anti-estrogenic properties. Half of the rats were ovariectomized (n = 20) and the other half were sham-operated. Ten of the ovariectomized rats and ten of the sham operated were exposed to PCB #126 (ip injections) for 3 months (total dose: 384 microgram/kg body wt). The remaining control rats were injected with corn oil (vehicle). The rats were killed and the tibiae and uteri were dissected. The left tibia was used for measurements of weight, length, and bone mineral density and the right for histomorphometrical analysis. The uteri were analyzed with respect to estrogen receptor content. PCB #126 exposure did not affect bone mineral density or trabecular bone volume of tibia in sham-operated rats. In ovariectomized rats PCB #126 exposure resulted in a decreased length and an increased bone mineral density of tibia. An obvious PCB #126 induced increase in osteoid surface was observed in sham-operated rats. The cortical thickness and the organic content of the tibia were also increased in these rats. In estrogen deprived tissue like the uteri of ovariectomized rats, PCB #126 showed weak estrogen agonistic activity. The observed effects of PCB #126 on bone and uterine tissues differed between ovariectomized and sham-operated rats.

Selection for egg shell strength in laying hens using shell membrane characteristics.

1. Divergent selection for attachment strength between the shell membrane and the calcium shell was performed in a White Leghorn strain. Multivariate analysis was used to estimate genetic parameters for shell membrane measurements and shell thickness. The aim was to investigate the possibility of improving shell strength in laying hens by selecting for increased attachment strength. 2. A significant direct selection response for attachment strength was achieved. There was a favourable correlated selection differential in the frequency of cracked eggs which resulted in a nonsignificant but favourable, correlated response. Selection for a strong attachment resulted in a thinner egg shell, and vice versa. Heritabilities were all relatively high (0.30 to 0.70). Most of the genetic correlations were in agreement with the achieved correlated responses. 3. Two factors that probably contributed to the responses in egg shell thickness were an unfavourable genetic correlation between attachment strength and shell thickness, and natural selection against changes in attachment strength, in the form of increased mortality during incubation and hatching. Problems connected with breeding for increased shell strength are discussed.

Effects of gastrin on calcium homeostasis in chickens.

As in the rat, gastrin and an extract of the acid-producing part of the stomach (proventriculus) were found to lower the blood Ca2+ concentration in the chicken. Furthermore, gastrin enhanced the uptake of 45Ca into the femur. It has been suggested previously that gastrin causes hypocalcemia in the rat by releasing gastrocalcin, a hypothetical hormone thought to reside in the acid-producing part of the stomach. The results of the present study in the chicken are in agreement with this concept. Not only exogenous, but also endogenous gastrin lowered blood calcium levels. Thus, the serum gastrin concentration was increased in response to ranitidine-evoked blockade of the gastric acid output; the rise in gastrin was associated with a transient drop in blood calcium. Also, food intake produced a rise in the serum gastrin concentration and a transient drop in blood calcium. However, injection of ranitidine or food intake in proventriclectomized (acid-producing part of the stomach extirpated) chickens failed to lower blood calcium, supporting the view that the gastrin-evoked hypocalcemia depends upon an agent in the gastric (proventriculus) mucosa. We suggest that endogenous and exogenous gastrin evoke hypocalcemia in the chicken by the same mechanism as that which has been postulated in the rat, i.e. by mobilization of the candidate hormone gastrocalcin from endocrine cells in the acid-producing gastric mucosa.

Organization of collagen fibers in the intestine.

The characteristic extinction pattern which is observed when the submucosa is viewed in the optical polarizing microscope has been analyzed in terms of the configuration and orientation of the 4 micron diameter collagen fibers. It is shown that the observed polarization effects are produced by periodic variations in orientation of fully birefringent fibers. The fiber configuration required to produce the observed polarization effects is a tilted wave configuration with a crimp period of approximately 20 micron. In the model, the tilted waveform fibers are crimped in register and form parallel arrays. The arrays are oriented in layers at approximately +30 degrees and -30 degrees to the longitudinal direction and are mirror images of each other. Analysis of the extinction pattern shows that the model satisfactorily accounts for the observed polarization effects at several different angles of the crossed polaroids. The calculated strain necessary to straighten the wavy fibers of the model correlates well with the observed strain to uncrimp the collagen fibers in the intestine. This suggests that the initial response to stress is gradual uncrimping of the collagen fibers, and concurrently, a decrease in the angle between biaxially oriented fibers, rather than extension of the straight fibers.

A serial study of fluid balance during pregnancy, lactation and anestrus in goats.

Water and salt balance was studied in the same goats during pregnancy, lactation and anestrus. All goats increased their water intake during the course of pregnancy, but individual differences were large. In general, twin pregnant (TP) goats drank more water than single pregnant (SP) animals (3.4 +/- 0.4; N = 11 as compared to 2.5 +/- 0.3 litres/day; N = 5) during the last weeks of pregnancy. During lactation the high water intake TP goats persisted and the SP animals increased their intake to the same level as the TP goats. The water intake was reduced to about 2 1/day in both categories of animals during anestrus. Urine volume largely followed the changes in water intake in the individual animal. A continuous decrease in urine osmolality during the course of pregnancy occurred, but during lactation urine osmolality increased towards anestrus levels. TP goats generally retained more sodium than SP animals during pregnancy and during anestrus, whereas the figures were similar during lactation. Plasma Na, K and osmolality remained unchanged during pregnancy, lactation and anestrus, but a large fall in total plasma proteins and a moderate fall in hematocrit were observed during the course of pregnancy. Glomerular filtration rate of TP goats was elevated by about 35% during the 4th month of pregnancy, but did not differ from anestrus levels during the 3rd and 5th month or during lactation. Effective renal plasma flow was highest during the 3rd pregnancy month and then fell to reach lactation and anestrus levels during the 4th month of pregnancy. A few hours before parturition the animals became markedly dehydrated as shown by sudden increases in plasma Na, K, osmolality, total proteins and hematocrit. This water deficit was replenished within 26 h post-partum.

Scanning electron microscopy of collagen fibers in intestine.

Collagen fibers of rat intestine were observed with and without mechanical stress in the scanning electron microscope. Observations were correlated with the previous results obtained by optical polarized microscopy to provide further insight into the organization and morphology of intestinal collagen. Larger fibers, approximately 4 micrometers in diameter, are densely packed in parallel undulating arrays. The initial response to stress is straightening of the original fibers. The extended fibers are biaxially oriented at +30 degrees and -30 degrees to the longitudinal direction. These large fibers appear as assemblies of subfibers. At higher magnifications, the larger fibers appear to be enmeshed in a network of small randomly oriented fibers approximately 0.2 micrometer in diameter. Study of the effect of age on fiber morphology showed that the length of the large fiber undulations increases during maturation but remains constant during aging. The diameters of large and small fibers appear not to change with age, but more of the 4 micrometers fibers are loosely associated into larger fibers which can be observed at both the optical microscope and scanning electron microscope levels. A hierarchical organization of intestinal collagen is proposed.

Effects of low protein consumption on the renal clearance of 2,4-dichlorophenoxyacetic acid (2,4-D) in goats.

Effects of low protein consumption on the renal excretion of 2,4-dichlorophenoxyacetic acid (2,4-D) in four female goats were studied by means of clearance methods. During periods of low protein consumption the 2,4-D clearance values are lower than during periods of normal protein consumption. The average reduction of 2,4-D clearance varies between 20 and 50 per cent.

Observations on the 2,4-dichlorophenoxyacetic acid (2,4-D) excretion in the goat.

The protein binding of 2,4-D in goat plasma was studied by means of equilibrium dialysis and the renal excretion of 2,4-D in three female goats by means of clearance methods. At low plasma levels of 2,4-D (less than 20 microgram/ml) about 97% of 2,4-D is bound to plasma proteins. The fraction of 2,4-D bound to plasma proteins decreases with rising plasma 2,4-D concentrations. A tubular secretion mechanism for 2,4-D, with a Tm value of about 9 mg (40 mumol)/min., is demonstrated. The contribution from the tubules to the 2,4-D excreted per minute is greater than 97% at a plasma 2,4-D level of 10 microgram/ml. At moderate and high plasma 2,4-D concentrations (greater than 40 microgram/ml) the inulin clearance decreases with rising plasma 2,4-D concentrations.

Effects of pure chlorobiphenyls (2,4',5-trichlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl) on the reproductive capacity in female mice.

The effects of two pure chlorobiphenyls (2,4',5-trichlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl) on the reproductive capacity in female mice have been studied. The results obtained show that females given 0.5 mg 2,5',5-trichlorobiphenyl (TCB)/day or 0.5 mg 2,2',4,4',5,5'-hexachlorobiphenyl (HCB)/day for six consecutive days, starting on the first day of pregnancy, have significantly lower frequencies of implanted ova than the control females, whereas the frequency of implanted ova does not differ either between females given 0.05 mg/day of TCB or HCB and the control females, or between females given TCT 0.05 mg/day or 0.5 mg/day and those given the corresponding dose of HCB. Females given 0.5 mg/day of TCB or HCB, respectively, also have significantly higher contents in the livers of cytochrome P-450, on Day four of pregnancy, than the control females. The results also show that, under the experimental conditions used, neither the frequency of pregnancies nor the number of implanted ova per pregnant female differ significantly between the different groups.

Effects of pure chlorobiphenyls (2,4',5-trichlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl) on the post-natal growth in mice.

The post-natal growth rate of young mice, pre- and post-natally exposed to either 2,4',5-trichlorobiphenyl or 2,2',4,4',5,5',-hexachlorobiphenyl has been studied. The results show that both the chlorobiphenyls have the ability toincrease the post-natal growth rate in mice and they indicate that this ability of 2,4',5-trichlorobiphenyl is most pronounced in females whereas that of 2,2',4,4',5,5',-hexachlorobiphenyl is most pronounced in males.

Presence of oestradiol-17beta in the rabbit ovum and its investments.

Untreated rabbit eggs contained 1-2 pg oestradiol-17beta/egg. Treatment with hyaluronidase for 20 min, but not for 10-13 min, removed the cumulus and coronra cells and all oestradiol.

Effects of pure chlorobiphenyls (2,4',5-trichlorobiphenyl or 2,2',4,4',5,5'-hexachlorobiphenyl) on the disappearance of 14C from the blood plasma after intravenous injection of 4-14C-progesterone and on the hepatic drug metabolizing system in the female rat.

Effects of two pure chlorobiphenyls (2,4',5-trichlorobiphenyl or 2,2',4,4',5,5'-hexachlorobiphenyl) on the hepatic drug metabolizing system and on the elimination rate of 14C from the blood after intravenous injection of 4(-14)C- progesterone have been studied in the female rat. The results obtained show that females pre-treated with small amounts of 2,2',4,4'5,5'-hexachlorobiphenyl (o.5 mg/day for 14 consecutive days) have significantly higher elimination rates of 14C (originating from intravenous injection of 4(-14C-progesterone) from the blood plasma, significantly higher content of hepatic cytochrome P-450 and significantly heavier livers than control females. On the other hand, trichlorobiphenyl (0.5 mg/day for 14 consecutive days) do not differ significantly from the results obtained from control females.