Effects of in utero and lactational exposure to bisphenol A on bone tissue in rats

Ling Shen

3

SUMMARY

Bisphenol A (BPA) is an industrial chemical that is used in the production of polycarbonate

plastics and epoxy resins. The applications result in consumer exposure to BPA via the diet.

The toxicity and hormonal activity of BPA in laboratory animals have been studied and

hormone related cancers and metabolic disorders have been observed in rodents. The

objective of this project was to study effects on bone tissues of rats exposed to low doses of

BPA in utero and during lactation. 110 pregnant Wistar rats were randomly distributed into

five groups and gavaged with (0, 0.025, 0.25, 5 or 50 mg BPA/kg body weight (b.w.)/day)

from gestation day 7 until weaning at pup day 22, offspring were exposed in utero and

during lactation. Body weights of the offspring were recorded and right femurs were

collected for geometry and densitometry measurements using peripheral quantitative

computed tomography and also for analysis of biomechanical properties via three-point

bending. The major findings of the study: Increased femur length in female offspring of dams

exposed to 0.025 mg BPA/kg b.w./day or 5 mg BPA/kg b.w./day; Increased cortical thickness

of male offspring of dams exposed to 0.025 mg BPA/kg b.w./day. No effects of

biomechanical properties were seen on the bones of either sex offspring.

In conclusion, this study demonstrates that in utero and lactational exposure to BPA at levels

relevant to current human exposure alters femoral geometry in rat offspring. BPA showed

endocrine disruptive effects on both female and male offspring bone tissue. The

mechanisms behind these differences are unknown.

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Table of Contents

SUMMARY ............................................................................................................................................... 3

1. INTRODUCTION ............................................................................................................................... 7

1.1. Bone ......................................................................................................................................... 7

1.1.1. Bone development and growth ....................................................................................... 8

1.1.2. Hormonal regulation of bone growth ............................................................................. 9

1.2. Endocrine disruptors ............................................................................................................... 9

1.2.1. Bisphenol A .................................................................................................................... 10

1.2.2. Effects of bisphenol A on bone ...................................................................................... 11

1.3. Objective of this study ........................................................................................................... 13

2. MATERIAL AND METHOD .............................................................................................................. 14

2.1. Materials ................................................................................................................................ 14

2.2. Animals .................................................................................................................................. 14

2.3. Experimental design .............................................................................................................. 14

2.4. Preparation of bones ............................................................................................................. 15

2.5. pQCT (peripheral Quantitative Computed Tomography) ..................................................... 15

2.5.1. Metaphysis measurement ............................................................................................. 16

2.5.2. Diaphysis measurement ................................................................................................ 16

2.5.3. Reproducibility .............................................................................................................. 17

2.6. Three-point bending test ....................................................................................................... 17

2.7. Statistics................................................................................................................................. 18

3. RESULTS ......................................................................................................................................... 19

3.1. pQCT measurements ............................................................................................................. 19

3.1.1. Reproducibility .............................................................................................................. 19

3.1.2. Measurements .............................................................................................................. 20

3.2. Three- point bending test ...................................................................................................... 26

4. DISCUSSION ................................................................................................................................... 28

REFERENCE ............................................................................................................................................ 31

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1. INTRODUCTION

1.1. Bone

Bone is a living mineralized tissue with vital functions in the body. It forms the skeletal

system and supports the body weight and movement; bone protects the organs that it

surrounds; it stores minerals (e.g. calcium and phosphorus) and produces blood cells in the

red bone marrow. As a tissue, mature bone consists of extracellular matrix with

approximately 35% organic and 65% inorganic material and bone cells (Seeley et al. 2003).

The shaft of a long bone, for example a femur, is called diaphysis, which mainly consists of

compact bone (cortical bone) and the ends of a long bone are called epiphyses which consist

primarily of cancellous bone (trabecular bone) (Figure 1). The area where the diaphysis

meets the epiphysis is metaphysis. It includes the epiphyseal plate in growing bones and red

marrow, the site of blood cell formation. The open space within the diaphysis is medullary

cavity (or marrow cavity) that is filled with yellow marrow containing adipose tissue (Seeley

et al. 2003).

Figure 1. The structure of a femur.

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1.1.1. Bone development and growth

In the mammalian embryo and fetus, the skeleton is principally made up of cartilage, a tough

and flexible connective tissue that contains no minerals. The formation of cartilage in

humans begins at approximately the fourth week of development. As the fetus grows,

osteoblasts and osteoclasts slowly replace cartilage cells and bone formation (ossification)

begins (Seeley et al. 2003). Osteoclasts are multinucleated cells that dissolve both the

inorganic and the protein portions of the bone matrix. Osteoblasts differentiate from

mesenchymal stem cells and are responsible for the production of new bone matrix. There

are mainly two kinds of ossification, intramembranous and endochondral ossification.

Intramembranous ossification is a process in which the mesenchymal tissue is converted

directly into bone tissue. In humans this occurs primarily in the skull bones and begins at

approximately the eighth week of development and is completed by two years of age.

In other cases, mesenchymal cells differentiate into chondrocytes which later stop dividing

and increase their volume dramatically, becoming hypertrophic chondrocytes. The

hypertrophic chondrocytes die by apoptosis and this space will become bone marrow. As the

cartilage cells die, a group of cells that have surrounded the cartilage model differentiate

into osteoblasts. The osteoblasts begin forming bone matrix on the partially degraded

cartilage. Eventually, all the cartilage is replaced by bone. This process is called endochondral

ossification (Gilbert 2006).

During infancy and youth, interstitial growth of the epiphyseal plate cartilage and its

replacement by bone enable long bone to lengthen and appositional growth makes all bones

grow in width. Long bones increase in width (diameter) when osteoblasts beneath the

periosteum secrete bone matrix on the external bone surface and osteoclasts on the

endosteal surface of the diaphysis remove bone. Normally, the amount of bone that is

removed is slightly less than that is formed so a thicker and stronger bone is produced but

the bone is also prevented from becoming too heavy.

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Longitudinal bone growth ends when the bone of the epiphysis and diaphysis fuses, which is

called epiphyseal plate closure. In humans this happens at about 18 years of age for girls and

at about 21 years of age for boys (Marieb et al. 2010). In rats the bones continue to grow

throughout life, although at a slower rate in adult animals.

1.1.2. Hormonal regulation of bone growth

The growth of bones that occurs until young adulthood is extensively controlled by a

symphony of hormones including growth hormone, thyroid hormones and sex hormones

(testosterone and estrogen). At the later stage of puberty, estrogen induces epiphyseal plate

closure and ends longitudinal bone growth (Marieb et al. 2010).

During the second half of the 20th century, estrogen therapy was administered to prevent

otherwise healthy girls with tall stature from becoming tall adults by inhibiting further linear

growth (Lee et al. 2006).

1.2. Endocrine disruptors

Endocrine disruptors (EDs) are exogenous chemicals that interfere with the normal function

of hormones by mimicking effects of natural hormones, blocking the synthesis of a hormone,

the binding to its receptor and/or interfering with the transport or elimination of a hormone.

EDs are highly heterogeneous molecules and include synthetic chemicals and their

byproducts e.g. polychlorinated biphenyls (PCBs), dioxins, bisphenol A (BPA), phthalates,

dichlorodiphenyltrichloroethane (DDT), vinclozolin and diethylstilbestrol (DES) as well as

natural chemicals such as phytoestrogens found in human and animal food like soybeans

and alfalfa (WHO/UNEP 2013).

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1.2.1. Bisphenol A

Bisphenol A (BPA) (CAS Number: 80-05-7, Chemical name: 4. 4’-isopropylidenediphenol) is a

synthetic organic chemical largely produced for polycarbonate compound and epoxy resins

(Figure 5). Plastics made with polycarbonates containing BPA monomers have a high impact

strength, increased hardness and transparency. Polycarbonate plastic is resistant to a wide

range of temperatures (between about –40˚C and 145˚C), many acids, greases and oils.

Some flame retardants and rubber chemicals also contain BPA (KEMI 2013).

Figure 5. Chemical Structure of bisphenol A (CAS Number: 80-05-7).

BPA has a water solubility of 120 to 300 mg/l at 20 to 25 ˚C (pH 7) and the log octanol-water

partition coefficient (Kow) value is between 2.2 and 3.8. The vapor pressure of BPA is 5.32 ×

10-7 Pa at 25˚C. Following an oral administration in rodents and humans, BPA is extensively

absorbed from the gastrointestinal tract and metabolized in the gut and liver into its primary

metabolite monoglucoronide conjugate and other minor metabolites (BPA sulfate and

BPA-3,4-quinone) which are excreted via urine and feces (Doerge et al. 2010). New findings

suggest that BPA is excreted much more via sweat than in urine. In this study, human serum

BPA level was found to be 70% lower than it was in sweat samples among the 20

participants (Genuis et al. 2012). In the rat, both BPA and the inactive metabolite of BPA

(BPA-glucuronide (BPA-GA)) are proved to pass through placenta to the fetus. Furthermore,

the rat fetus has the ability to deconjugate BPA-GA into free BPA which causes toxicities. The

fetal liver however, has very low capacity to conjugate BPA into its inactive form again

(Nishikawa et al.2010).

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BPA was discovered to be an artificial estrogen already in the 1930s (Dodds et al. 1936), long

before it was used in production of plastics and resins in 1950s. BPA is used in a multitude of

applications and they result in ubiquitous exposure to the general public. Food and beverage

packaging lined with epoxy resins containing BPA are considered to be the greatest potential

sources. Many reusable plastic bottles and food containers, including infant bottles, are

made from BPA-containing polycarbonate plastic. Even though the use of BPA in baby

bottles has been banned in some countries, we are still chronically exposed to BPA in the

environment through food and drinking water. European Food Safety Authority (EFSA)

established the present tolerable daily intake (TDI) of 50 µg/kg body weight (b.w.) (EFSA

2008), which was based on a NOEAL of 5 mg/kg b.w./day in rats (Tyl et al. 2002). However, a

re-evaluation for a new TDI was first suggested in 2010 after the EFSA panel meeting and

then in 2012 after a review on low dose effects of BPA done by the Swedish Chemicals

Agency in 2012 (Beronius et al. 2012).

Some known effects of BPA as an endocrine disruptor are mainly shown as hormone-related

cancers or metabolic disorders. For example in rodents, early exposure to BPA during

organogenesis causes alterations in the mammary gland, which renders it more sensitive to

estradiol at later developmental stages and more susceptible to neoplasia. BPA disturbs the

regulation of adipose tissue deposition and food intake by acting via estrogen receptors in

fat cells and brain cells, and further, it also results in increased insulin resistance and

secretion as well as decreased glucose tolerance in adult male rodents. BPA exposure to

pregnant women decreases glucose tolerance during pregnancy and increased insulin

resistance is seen both during pregnancy and four months post-partum. In amphibians BPA

has been shown to block thyroid hormone induced metamorphosis (WHO/UNEP 2013).

1.2.2. Effects of bisphenol A on bone

1.2.2.1. Experimental animals

Loss of bone tissue in aromatase knock-out mice (ArKO mice) was prevented in individuals

fed a diet supplemented with 0.1% or 1% (w/w) BPA for 5 months (Toda et al. 2002). This

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study showed that the BMD in BPA exposed individuals increased in a dose-dependent

manner. An in utero and lactational exposure to 0.01 mg/kg/day BPA caused a 10.3%

decrease of femur mechanical strength in female mice (13 weeks of age), but not in males

(Pelch et al. 2012). In another study, the tibia BMD of OVX rats decreased after a three

month dietary exposure to BPA (0.037 mg/kg b.w. /day or 0.37 mg/kg b.w. /day) compared

to the unexposed controls (Seidlova-Wuttke et al. 2004). No vertebral malformations or

adverse effects on skeletal development were observed in fathead minnows (Pimephales

promelas) exposed to BPA (0.1 to 1.0 µg/L) from egg stage to 25 to 26 day after hatching

(Warner et al. 2007).

1.2.2.2. Epidemiological studies

The number of studies on effects of BPA on bone tissues in humans is very limited. Urinary

BPA levels were not related to bone mineral densities, bone resorption marker N-

telopeptide or bone formation parameter osteocalcin in premenopausal women (Zhao et al.

2012).

1.2.2.3. In vitro effects

The Tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) were used as

markers for osteoclast and osteoblast activities, respectively and were both found to be

suppressed significantly by BPA (10-5 M) in the cultured scales of goldfish (Suzuki et al. 2003).

In another study, BPA (10-10- 10-6 M) increased ALP activity and enhanced bone

mineralization in MC3T3-E1 cells (osteoblast-like cell line) (Kanno et al. 2004).

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1.3. Objective of this study

This study is a part of a collaboration project between Division of Toxicology and Risk

Assessment, National Food Institute in Denmark, Monica Lind at the Department of Medical

Sciences-Occupational and Environmental Medicine, Uppsala University, Sweden and Jan

Örberg at the Department of Environmental Toxicology, Uppsala University, Sweden. The

aim of the original project was to study endocrine disrupting properties and effects of

bisphenol A on reproductive parameters in rats. The objective of this part is to study effects

on bone tissues of rats exposed to low doses of BPA in utero and during lactation.

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2. MATERIAL AND METHOD

2.1. Materials

Bisphenol A (purity > 99.5%, CAS no. 80-05-7) and corn oil were purchased from Sigma-

Aldrich (Brøndby, Denmark). Corn oil was provided in glass bottles. The solutions were kept

in the dark at room temperature and continuously stirred during the dosing period.

2.2. Animals

110 pregnant Wistar rats were housed in pairs until gestation day (GD) 17 and thereafter

individually under standard conditions in polysulfone cages with Aspen wood chip bedding

(Tapvei, Gentofte, Denmark), Enviro Dri nesting material (Brogaarden, Denmark) and Tapvei

Aspen wood shelters (Brogaarden, Denmark). The animal room was maintained with a 12

hour light-dark cycle (light intensity 500 lux, starting at 9 pm), humidity 55% ± 5,

temperature at 21 ˚C ± 1˚C and ventilation changing air 10 times per hour. All rats were fed

on a standard diet with soy and alfalfa free ALROMIN 1314 (ALTROMIN GmbH, Lage,

Germany). Daily individual body weight was recorded from GD 7 for dams and pups were

weighed on pup day (PD) 7 and 14. Tap water was provided in polysulfone bottles ad libitum.

2.3. Experimental design

On GD 4, the dams were randomly distributed into five groups (0, 0.025, 0.25, 5 and 50 mg

BPA/kg b.w.) with 22 rats of similar body weight per group. BPA was dissolved in corn oil and

the rats were gavaged with a stainless probe 1.2 × 80 mm once daily from GD 7 until

weaning at PD 22, at a constant volume of 2 ml/kg b.w. The control group was given corn oil.

Maximum numbers of pups from each dam included in the study were one female and one

male.

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2.4. Preparation of bones

At three months of age, the pups were sacrificed and right femurs from 89 male and 93

female were dissected, cleaned and placed in 10 ml centrifuge tubes with Ringer solution

(pH 7.4, Tris 0.3 g/l, NaCl 9 g/l, CaCl2·2H2O 0.24 g/l, KCl 0.4 g/l, 2.05 × 10 -3 M HCl) and frozen

at -18˚C pending peripheral quantitative computed tomography (pQCT) and biomechanical

testing. Bones were placed in a refrigerator (+8˚C) approximately 24 hours before the

analyses. For each pQCT analysis, the femur lengths were measured using a slide caliper

(accuracy of 0.1 mm) from the proximal end of the femur to the distal end, with the bone

parallel to the ruler. The bone was then covered with a piece of 5×5 cm highly pure non-

woven compress (OneMed Sverige AB, Spånga, Sweden) moistened with Ringer solution and

then placed in a 10 ml centrifuge tube to prevent the femur from drying.

2.5. pQCT (peripheral Quantitative Computed Tomography)

Femur dimension and composition were examined with an X-ray based density

measurement machine, pQCT (Stratec XCT Research SA+, Stratec Medizintechnik, Pforzheim,

Germany, software version 6.00) at Uppsala University Hospital, Uppsala, Sweden (Figure 6).

The machine was calibrated every day before the measurement started with the

manufacturer specified reference object (phantom).

Figure 6. Stratec XCT Reserch SA+ peripheral quantitative computed tomograph (pQCT).

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According to the experiment design, the bone was re-positioned between measurements on

the pQCT holder so that the scanner could scan across more than 50% of the whole bone

length from the distal end of the epiphysis. After a contrast enhancement was generated on

the computer screen, the reference line and measurement line(s) were displayed on the

scout view (SV) image. A scout view in a CT study is a radiographic image used to plot the

locations where the subsequent slice images will be obtained. When the reference line was

adjusted to be at the exact edge of the distal epiphysis on the SV, the measurement line(s)

were placed accordingly.

2.5.1. Metaphysis measurement

The metaphysis of the femur was analyzed by examining one 1 mm thick slice at 20% of the

bone length from the reference line. The following settings were used: voxel size 0.070 mm.

peel mode 20, threshold 280 mg/cm3, contour mode 1 and the measures obtained from the

analysis were total bone mineral content (Total BMC, mg/mm), total bone mineral density

(Total BMD, mg/mm3), trabecular bone mineral content (Trabecular BMC, mg/mm),

trabecular bone mineral density (Trabecular BMD, mg/mm3), total cross sectional area (Total

CSA, mm2), trabecular cross sectional area (Trabecular CSA, mm2) and Periosteal

circumference (mm).

2.5.2. Diaphysis measurement

The diaphysis of the femur was analyzed by examining one 1 mm thick slice at 50 % of the

bone length from the reference line. The following settings were used: contour mode 1 and

threshold 710 mg/ cm3 and the measures obtained from the analysis were total BMC

(mg/mm), total BMD (mg/mm3), total CSA (mm2), cortical BMC (mg/mm), cortical BMD

(mg/mm3), cortical CSA (mm2), cortical thickness (mm), periosteal circumference (mm),

endocortical circumference (mm) and marrow cavity (mm2).

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2.5.3. Reproducibility

To create a valid method, the coefficient of variation (CV= Standard deviation × 100 /Mean)

was calculated from 10 repeated pQCT measurements performed on a single rat femur. For

each measurement, one 1 mm thick slice from the metaphysis and one 1 mm thick from the

diaphysis were analyzed.

2.6. Three-point bending test

A three-point bending test was performed to measure the bone strength using an

electromechanical material testing machine (Avalon technologies, MN, USA) with a span

length of 13 mm and a loading speed of 0.2 mm/sec. The load was applied to the anterior

surface of the mid part of the diaphysis with the intention to apply load at the same site as

where the pQCT scan had been performed. The load and deformation data were recorded

and sampled with 50 Hz. Load at failure (N), displacement at failure (mm), stiffness (slope of

the load-deflection curve. representing the elastic deformation, N/mm) and energy

absorption until failure (surface under the curve, N×mm) were calculated from the load-

deflection curves (Figure 7).

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Figure7. A typical three-point bending test results in a curve. Load (N) is a function of Displacement (mm). Max

stiffness (N/mm) can be calculated from the slope at the steepest part of the curve. Energy to failure (energy

absorption when the bone breaks, N× mm) is the gray area under the curve.

2.7. Statistics

The results were evaluated by analysis of variance (ANOVA) and analysis of covariance

(ANCOVA) with Bonferroni/Dunn as the post-hoc test was used to adjust the continuous

variable of body weight (StatView, Version 5.0.1; SAS Institute Inc., Cary, NC, USA).

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3. RESULTS

3.1. pQCT measurements

3.1.1. Reproducibility

The results from the pQCT reproducibility measurements are presented in tables 1 and 2. 10

repeated pQCT measurements from a single rat femur gave coefficient of variations (CV) that

varied from 0.35% to 3.36% at the 20% metaphyseal measure point and 0.22% to 2.74% at

the 50% diaphyseal measure point.

Table 1. The reproducibility calculated from 10 repeated measurements conducted on a single rat femur using

peripheral quantitative computed tomography (pQCT). The metaphyseal measure point was located at 20% of

the total bone length from the distal epiphyseal tip. BMC = bone mineral content, BMD = bone mineral density,

CSA = cross sectional area.

Metaphyseal bone 20% Mean ± SD CV(%)

Total BMC (mg/mm) 12.0 ± 0.1 0.79

Total BMD (mg/mm3) 625.4 ± 2.2 0.35

Total CSA (mm2) 19.2 ± 0.1 0.73

Trabecular BMC (mg/mm) 1.8 ± 0.1 3.36

Trabecular BMD (mg/mm3) 205.8 ± 5.5 2.68

Trabecular CSA (mm2) 8.6 ± 0.1 0.72

Periosteal circumference (mm) 15.5 ± 0.1 0.37

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Table 2. The reproducibility calculated from 10 repeated measurements conducted on a single rat femur using

peripheral quantitative computed tomography (pQCT). The diaphyseal measure point was located at 50% of

the total bone length from the distal epiphyseal tip. BMC = bone mineral content, BMD = bone mineral density,

CSA = cross sectional area.

Diaphyseal bone 50% Mean ± SD CV(%)

Total BMC (mg/mm)

13.1 ± 0.1 0.43

Total BMD (mg/mm3)

925.0 ± 8.1 0.88

Total CSA (mm2)

14.1 ± 0.1 0.97

Cortical BMC (mg/mm)

11.7 ± 0.1 0.47

Cortical BMD (mg/mm3)

1440.9 ± 3.2 0.22

Cortical CSA(mm2)

8.1 ± 0.0 0.62

Cortical thinkness (mm) 0.7 ± 0.0 1.32

Periosteal circumference (mm)

13.3 ± 0.1 0.49

Endocortical circumference (mm)

8.7 ± 0.1 1.38

Marrow cavity (mm2)

6.0 ± 0.2 2.74

3.1.2. Measurements

3.1.2.1. Body weight

The mean body weight of the female offspring in the different groups varied between 208.0

and 218.1 g but did not differ significantly (Table 3).

The mean body weight of the male offspring varied between 330.9 and 366.3 g with a

significant difference between the treated groups 0.025 and 0.25 mg BPA/kg b.w./day

(p<0.005) (Table 3). The mean body weight of the male offspring of dams exposed to 0.025

mg BPA/kg b.w./day was 10.7% higher than that of dams exposed to 0.25 mg BPA/kg

b.w./day.

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3.1.2.2. Bone length

Compared with the control group, the femurs were significantly longer in female offspring of

dams exposed to 0.025 or 5 mg BPA/kg b.w./day (p<0.005, Figure 8).

Female Bone Length

0

0.02

50.

25 5 50

2.6101

2.8101

3.0101

3.2101

3.4101

3.6101

* *

Bisphenol A exposure (mg/kg bw/day)

Bo

ne

le

ng

th(m

m)

Figure 8. Femur length (mm, mean ± SD, n= 14 to 20) of female Wistar offspring from dams exposed to 0, 0.025,

0.5, 5 or 50 mg BPA/kg b.w./day from gestational day 7 until postnatal day 21. Values with * are statistically

significant compared with the control group (p<0.005, ANCOVA with Bonferroni post-hoc).

No significant differences were found in femur length between male offspring of dams

exposed to BPA and control group. However, the mean femur length from the male offspring

of dams exposed to 0.025 mg BPA/kg b.w./day was 3.3% longer (p<0.005) compared to

those of dams exposed to 0.25 mg BPA/kg b.w./day (Table 3).

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Table 3. Body weight (g, mean ± SD) and femur length (mm, mean ± SD) of Wistar offspring from dams exposed

to 0, 0.025, 0.5, 5 or 50 mg BPA/kg b.w./day from gestational day 7 until postnatal day 21. The value with * is

statistically significant compared with the one of the control group and values with the same letter (a or b) are

significantly different from each other (p<0.005). n= number of individuals, ANOVA was used for body weight

and ANCOVA with Bonferroni post-hoc was used for femur length.

Bisphenol A (mg/kg body weight/day)

0 (n=19) 0.025 (n=21) 0.25 (n=15) 5 (n=18) 50 (n=15)

Female Body weight (g) 212.7 ± 14.2 218.1 ± 11.8 208.0 ± 14.5 213.9 ± 16.2 219.3 ± 36.9

Bone length (mm) 32.3 ± 0.8 32.9 ± 0.8 * 32.5 ± 0.5 33.0 ± 0.8 * 32.5 ± 0.9

0 (n=20) 0.025 (n=20) 0.25 (n=18) 5 (n=20) 50 (n=14)

Male Body weight (g) 349.5 ± 39.6 366.3 ± 30.7 a 330.9 ± 28.8 a 340.7 ± 32.5 343.3 ± 35.4

Bone length (mm) 35.7 ± 1.5 36.5 ± 0.9 b 35.3 ± 1.3 b 35.9 ± 1.1 36.2 ± 0.9

3.1.2.3. Metaphyseal measurements

The results obtained from the metaphyseal measurements on bones from female offspring

are presented in table 4. No significant differences were found in the bone geometry and

densities between female offspring of dams exposed to BPA and female offspring of control

dams.

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Table 4. Results (mean ± SD) from peripheral quantitative computed tomography of femur metaphysis

variables of female Wistar offspring from dams exposed to 0, 0.025, 0.5, 5 or 50 mg BPA/kg b.w./day from

gestational day 7 until postnatal day 21. The metaphyseal measure point was located at 20% of the total bone

length from the distal epiphyseal tip. n= number of individuals, ANCOVA with Bonferroni post-hoc was used for

analyzing. BMC= bone mineral content, BMD = bone mineral density, CSA =cross sectional area.

Female Bisphenol A (mg/kg body weight/day)

0 (n=20) 0.025 (n=20) 0.25 (n=18) 5 (n=20) 50 (n=14)

Total BMC (mg/mm) 11.4 ± 1.4 11.1 ± 0.9 10.8 ± 1.1 11.2 ± 1.1 11.8 ± 1

Total BMD (mg/mm3) 816.5 ± 62.8 808.7 ± 51.3 803.7 ± 58.3 825.5 ± 52.4 837.8 ± 49.3

Trabecular BMC (mg/mm) 3.3 ± 0.8 3.1 ± 0.6 3.0 ± 0.6 3.1 ± 0.6 3.5 ± 0.5

Trabecular BMD (mg/mm3) 523.4 ± 99.1 492.5 ± 86.5 492.4 ± 91.6 514.4 ± 78.9 543.5 ± 71.9

Total CSA (mm2) 13.9 ± 1 13.8 ± 1.1 13.5 ± 0.9 13.5 ± 0.9 14.2 ± 1.2

Trabecular CSA (mm2) 6.3 ± 0.4 6.2 ± 0.5 6.1 ± 0.4 6.1 ± 0.4 6.4 ± 0.5

Periosteal circumference (mm) 13.2 ± 0.5 13.2 ± 0.5 13 ± 0.4 13 ± 0.4 13.3 ± 0.6

The results obtained from the metaphyseal measurements on bones from male offspring are

presented in table 5. No significant differences were found in the bone geometry and

densities between male offspring of dams exposed to BPA and male offspring of control

dams.

Table 5. Results (mean ± SD) from peripheral quantitative computed tomography of femur metaphysis

variables of male Wistar offspring from dams exposed to 0, 0.025, 0.5, 5 or 50 mg BPA/kg b.w./day from

gestational day 7 until postnatal day 21. The metaphyseal measure point was located at 20% of the total bone

length from the distal epiphyseal tip. n= number of individuals, ANCOVA with Bonferroni post-hoc was used for

analyzing. BMC= bone mineral content, BMD = bone mineral density, CSA =cross sectional area.

Male

Bisphenol A (mg/kg body weight/day)

0 (n=19) 0.025 (n=21) 0.25 (n=15) 5 (n=18) 50 (n=15)

Total BMC (mg/mm) 12.4 ± 1.5 12.3 ± 1.3 12 ± 1 12.4 ± 1.1 11.9 ± 1.5

Total BMD (mg/mm3) 705.1 ± 60.5 677 ± 33.9 686 ± 50 690.7 ± 50 667.1 ± 39.8

Trabecular BMC (mg/mm) 3.1 ± 0.9 2.7 ± 0.7 2.7 ± 0.6 2.9 ± 0.6 2.6 ± 0.7

Trabecular BMD (mg/mm3) 389.4 ± 101.1 328.4 ± 60.1 348.1 ± 80.5 358.6 ± 74.8 315.3 ± 60

Total CSA (mm2) 17.6 ± 1.3 18.1 ± 1.5 17.6 ± 1.2 18 ± 0.8 17.8 ± 1.7

Trabecular CSA (mm2) 7.9 ± 0.6 8.1 ± 0.7 7.9 ± 0.5 8.1 ± 0.3 8 ± 0.8

Periosteal circumference (mm) 14.9 ± 0.5 15.1 ± 0.6 14.9 ± 0.5 15 ± 0.3 14.9 ± 0.7

24

3.1.2.4. Diaphyseal measurements

The results obtained from the diaphyseal measurements on bones from female offspring are

presented in table 6. Total BMC was observed to be significantly lower in offspring of dams

exposed to 0.25 mg BPA/kg b.w./day than those of dams exposed to 50 mg BPA/kg b.w./day.

Table 6. Results (mean ± SD) from peripheral quantitative computed tomography of femur diaphysis variables

of female Wistar offspring from dams exposed to 0, 0.025, 0.5, 5 or 50 mg BPA/kg b.w./day from gestational

day 7 until postnatal day 21. The diaphyseal measure point was located at 50% of the total bone length from

the distal epiphyseal tip. Values with the same letter ‘a’ are significantly different from each other (p<0.005,

ANCOVA with Bonferroni post-hoc). n= number of individuals. BMC= bone mineral content, BMD = bone

mineral density, CSA =cross sectional area, marrow cavity = total CSA – cortical CSA.

Female

Bisphenol A (mg/kg body weight/day)

0 (n=19) 0.025 (n=21) 0.25 (n=15) 5 (n=18) 50 (n=15)

Total BMC (mg/mm) 7.6 ± 0.5 7.7 ± 0.4 7.4 ± 0.4 a 7.6 ± 0.5 7.8 ± 0.5 a

Total BMD (mg/mm3) 922.4 ± 45.9 926.8 ± 41.5 922.0 ± 36.2 932.0 ± 32.9 918.3 ± 40.1

Total CSA (mm) 8.3 ± 0.7 8.3 ± 0.6 8.0 ± 0.6 8.2 ± 0.6 8.5 ± 0.8

Cotical BMC (mg/mm) 6.8 ± 0.4 6.8 ± 0.4 6.6 ± 0.4 6.8 ± 0.5 6.9 ± 0.4

Cortical BMD (mg/mm3) 1385.9 ± 16.3 1389.3 ± 16.3 1380.6 ± 15.9 1393.1 ± 9.7 1382.8 ± 15.0

Cortical CSA (mm) 4.9 ± 0.3 4.9 ± 0.3 4.8 ± 0.3 4.9 ± 0.3 5.0 ± 0.3

Cortical thickness (mm) 0.59 ± 0.03 0.59 ± 0.02 0.58 ± 0.02 0.59 ± 0.03 0.59 ± 0.03

Periosteal circumference (mm) 10.2 ± 0.4 10.2 ± 0.4 10.0 ± 0.4 10.1 ± 0.4 10.3 ± 0.5

Endocortical circumference (mm) 6.5 ± 0.5 6.5 ± 0.4 6.4 ± 0.4 6.4 ± 0.4 6.7 ± 0.5

Marrow Cavity (mm2) 3.4 ± 0.5 3.4 ± 0.5 3.3 ± 0.4 3.3 ± 0.4 3.5 ± 0.5

25

The results obtained from the diaphyseal measurements on bones from male offspring are

presented in table 7. Cortical thickness was found to be significantly higher in the offspring

of dams exposed to 0.025 mg BPA/kg b.w./day compared with those from the control group.

Significant differences were observed in total BMC, cortical BMC, cortical CSA and cortical

thickness between offspring of dams exposed to 0.025 mg BPA/kg b.w./day and those of

dams exposed to 0.25 mg BPA/kg b.w./day.

Table 7. Results (mean ± SD) from peripheral quantitative computed tomography of femur diaphysis variables

of male Wistar offspring from dams exposed to 0, 0.025, 0.5, 5 or 50 mg BPA/kg b.w./day from gestational day

7 until postnatal day 21. The diaphyseal measure point was located at 50% of the total bone length from the

distal epiphyseal tip. The value with * is statistically significant compared with the one of the control group and

values with the same letter (a, b, c or d) are significantly different from each other (p<0.005, ANCOVA with

Bonferroni post-hoc). n= number of individuals. BMC= bone mineral content, BMD = bone mineral density, CSA

=cross sectional area, marrow cavity = total CSA- cortical CSA.

Male

Bisphenol A (mg/kg body weight/day)

0 (n=19) 0.025 (n=21) 0.25 ( n=16) 5 (n=18) 50 (n=15)

Total BMC (mg/mm) 9.5 ± 0.9 9.9 ± 0.7 a 9.2 ± 0.7a 9.7 ± 0.6 9.5 ± 0.7

Total BMD (mg/mm3) 888.9 ± 43.0 918.3 ± 35.5 887.6 ± 24.5 906.4 ± 28.8 893.9 ± 45.8

Total CSA (mm) 10.7 ± 1.0 10.7 ± 0.7 10.4 ± 0.9 10.7 ± 0.8 10.7 ± 1.0

Cotical BMC (mg/mm) 8.4 ± 0.8 8.8 ± 0.6 b 8.2 ± 0.7 b 8.7 ± 0.5 8.5 ± 0.6

Cortical BMD (mg/mm3) 1372.3 ± 10.0 1380.6 ± 12.4 1372.7 ± 9.6 1376.7 ± 11.2 1376.1 ± 12.7

Cortical CSA (mm) 6.1 ± 0.6 6.4 ± 0.4 c 6.0 ± 0.5 c 6.3 ± 0.4 6.1 ± 0.4

Cortical thickness (mm) 0.64 ± 0.05 0.67 ± 0.04* d 0.63 ± 0.03 d 0.66 ± 0.03 0.64 ± 0.04

Periosteal circumference (mm) 11.6 ± 0.5 11.6 ± 0.4 11.4 ± 0.5 11.6 ± 0.4 11.6 ± 0.5

Endocortical circumference (mm) 7.5 ± 0.5 7.4 ± 0.4 7.5 ± 0.4 7.5 ± 0.4 7.5 ± 0.6

Marrow Cavity (mm2) 4.5 ± 0.6 4.4 ± 0.5 4.4 ± 0.5 4.4 ± 0.5 4.5 ± 0.7

26

3.2. Three- point bending test

The results from the three- point bending test are presented in tables 8 and 9. No significant

differences in bone strength were observed between control and treated groups in neither

female nor male offspring.

Table 8. Results (mean ± SD) from three-point bending test performed on femurs of female Wistar offspring

from dams exposed to 0, 0.025, 0.5, 5 or 50 mg BPA/kg b.w./day from gestational day 7 until postnatal day 21.

The test was performed using an electromechanical material testing machine (Avalon technologies, MN, USA).

The load was applied at the mid-diaphyseal pQCT measure point (at about 50% of the total bone length from

the distal tip) with a loading speed of 0.2mm/sec. Analysis was done by using ANCOVA with Bonferroni post-

hoc. No significant differences in bone strength were found unless p-value is less than 0.005. n= number of

individuals.

Female

Bisphenol A (mg/kg body weight/day)

0 (n=20) 0.025 (n=20) 0.25 (n=18) 5 (n=20) 50 (n=14)

Displacement (mm) 1.1 ± 0.2 1.1 ± 0.2 1.1 ± 0.2 1.1 ± 0.2 1.1 ± 0.2

Load at failure (N) 106.1 ± 8.4 108.2 ± 10.2 103.8 ± 9.0 108.1 ± 12.1 111.0 ± 12.5

Max Stiffness (N/mm) 182.1 ± 23.6 176.9 ± 29.2 162.6 ± 28.1 176.2 ± 28.5 185.8 ± 28.1

Energy (N×mm) 75.1 ± 17.2 79.6 ± 19.8 70.9 ± 11.0 72.1 ± 11.1 78.6 ± 19.3

27

Table 9. Results (mean ± SD) from three-point bending test performed on femurs of male Wistar offspring from

dams exposed to 0, 0.025, 0.5, 5 or 50 mg BPA/kg b.w./day from gestational day 7 until postnatal day 21. The

test was performed using an electromechanical material testing machine (Avalon technologies, MN, USA). The

load was applied at the mid-diaphyseal pQCT measure point (at about 50% of the total bone length from the

distal tip) with a loading speed of 0.2mm/sec. Analysis was done by using ANCOVA with Bonferroni post-hoc.

No significant differences in bone strength were found unless p-value is less than 0.005. n= number of

individuals.

Male

Bisphenol A (mg/kg body weight/day)

0(n=19) 0.025 (n=21) 0.25(n=15) 5(n=18) 50 (n=15)

Displacement (mm) 1.6 ± 0.3 1.5 ± 0.2 1.5 ± 0.2 1.4 ± 0.2 1.6 ± 0.3

Load at failure (N) 131.6 ± 14.0 141.9 ± 13.8 130.9 ± 15.9 137.2 ± 13.5 133.9 ± 13.5

Max Stiffness (N/mm) 220.4 ± 23.2 227.8 ± 29.7 211.8 ± 36.4 222.0 ± 18.8 223.3 ± 29.2

Engergy (N×mm) 147.9 ± 39.0 151.0 ± 24.6 144.2 ± 31.5 139.3 ± 27.6 148.6 ± 40.2

28

4. DISCUSSION

The most interesting result of this study was that the femur length of female offspring of

dams exposed to 0.025 mg BPA/kg b.w./day or 5 mg BPA/kg b.w./day was significantly

longer compared to that of dams from the control group. The exposure period was chosen

to cover the most sensitive periods of the development of the rat reproductive system. The

BPA doses used, with the lowest observed adverse effect level (50 mg/kg b.w./day) in rats

(Tyl et al. 2002) as the highest exposure level, are hence considered as low doses. The

reproducibility test provided coefficient of variations between 0.22% and 3.36% for the

pQCT methodology which ensured the quality of the test.

When the offspring was evaluated in adulthood, no differences in body weight were found in

females. However, the femur length of female offspring of dams exposed to 0.025 mg

BPA/kg b.w./day or 5 mg BPA/kg b.w./day was significantly longer compared to that of dams

from the control group with a 1.9% and 2.2% increase, respectively (p<0.005). As estrogens

promote the fusion of the growth plate, this finding might indicate an anti-estrogenic effect

of BPA on bone tissue in female offspring (Kennedy et al.1999; Nilsson et al. 2001; Weise

2001).

The cortical thickness was significantly greater (4.7%) in male offspring of dams exposed to

0.025 mg BPA/kg b.w./day compared to those of dams from the control group. A possible

explanation to this effect is the slight decrease of endocortical circumference. The femur

periosteal circumferences (mean ± SD) in offspring of control dams and in offspring of dams

exposed to 0.025 mg BPA/kg b.w./day were 11.6 ± 0.5 and 11.6 ± 0.4 mm, respectively, and

the endocortical circumference (mean ± SD) were 7.5 ± 0.5 and 7.4 ± 0.4 mm, respectively

(Figure 9). In both sexes, estrogen decreases the endocortical resorption of the diaphysis and

thereby causing an increased cortical thickness (Almeida et al. 2013).

29

Figure 9. Cross sectional dimension of the femur showing the increase in cortical thickness of male Wistar

offspring of dams exposed to 0.025 mg BPA/kg b.w./day from gestational day 7 until postnatal day compared

to those of control. The diameters of periosteal circumferences were the same in both groups of femurs

whereas the diameter of the endocortical circumference was smaller in the treated femurs compared to that in

the controls (The illustration depicts the cortical thickness increase and does not represent the actual sizes of

circumferences).

The body weight was 10.7% lower and the bone length was 3.3% shorter in male offspring of

dams exposed to 0.25 mg BPA/kg b.w./day compared to those of dams exposed to 0.025 mg

BPA/kg b.w./day (Table 3). From the pQCT results, the variables total BMC, cortical BMC,

cortical CSA and cortical thickness from the male offspring of dams exposed to 0.25 mg

BPA/kg b.w./day were all seen to be significantly lower than the male offspring of dams

exposed to 0.025 mg BPA/kg b.w./day. The mechanisms behind all these differences are not

known.

No change in the bone strength could be seen from the biomechanical test of this study.

However, a torsional loading test by Pelch et al. (2012) proved that a developmental

30

exposure to BPA (0.01 mg/kg b.w./day) resulted in a decrease of femur energy to failure in

female mice.

In conclusion, this study demonstrates that in utero and lactational exposure to BPA at levels

relevant to current human exposure alters femoral geometry in rat offspring. BPA showed

endocrine disruptive effects on both female and male offspring bone tissue.

31

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Ekotoxikologiska avdelningenUppsala universitet

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Projektrapport från utbildningen iEKOTOXIKOLOGI

Ekotoxikologiska avdelningenUppsala universitet

20. Karin Hanze (1990) Inverkan av fungicider på tillväxt och toxinbildning hos Fusarium spp. 21. Thomas Wenell (1991) Metallundersökning i kallfl öden, Degerforsområdet 1990. 22. Anita Gustavsson och Anna Myrin (1991) Ett förslag till förlängning av Kemikaliein- spektionens 40-lista. En prioriteringsmetod.23. Anders Sellner och Eva Steiner (1991) Tillfriskningsstudier i gruvindustriella reci- pienter. Exempel från Laisvallområdet. 24. Anders Johansson och Anders Normann (1993) Koppar och zink i grönslick (Cladophora glomerata) från Stockholmsområdet - halter och effekter.25. Jonas Falck och Erik Gravenfors (1993) Metodutveckling för studier av tungmetallupptag i växter.26. Sara Gunnare och Anneli Sandberg (1993) Toxicity Tests with the Macroalga Gracilaria Tenuistipitata.27. Andrea Brewer (1994) The Toxic Potency of Organic Compounds Extracted From Sediment and Seston Samples From Sundsvall Bay.28. Mikael Wennström (1994) Förekomst och effekt av oxolinsyra i miljön runt fi sk odlingar. 29. Ulrika Lindbäck (1994) Ekotoxikologisk utvärdering av fungiciden fenpiklonil. 30. Elisabeth Dryselius (1994) Ekotoxikologisk utvärdering av herbiciden propaklor.31. Helena Nerell (1994) Exposure to 2,2',5,5'-tetrachlorobiphenyl during a defi ned period in neonatal life induces permanent changes in behaviour in adult mice. 32. Monica Lind (1994) Sediment från Delångersån. Toxicitet och effect på kvicksil- verupptag.33. Linda Avatare (1995) Slam en resurs för det Estniska jordbruket.34. Päivi Palokangas och Sylvia Karlsson (1995) Ecophysiological Effects of Cad- mium in Relation to Salinity and Temperature Variation in the Tropical Mussel Perna viridis. A Pollution Study in the Gulf of Thailand.35. Anne-Marie von Hofsten (1995) Fjärrspridda tungmetallers påverkan på markbio- logiska i skogsmark.36. Cecilia Berg (1995) Ecotoxicological Evaluation of the Fungicide Ampropylfos.37. Magnus Forsberg (1995) Vägtrafi ken som miljöproblem.38. Jörgen Henriksson (1995) Effects of organoarsenic-based warfare agents on human leucocytes and on three-spined stickleback (Gasterosteus aculeatus L.).39. Torbjörn Johansson (1995) CO2-utsöndringens betydelse för pH-beroende upptag av svaga organiska syror genom perfunderade regnbågsgälar (Oncorhynchus mykiss).40. Olof Holmer (1995) Effektivitetsstudie av fi berfl ock. Ett nytt påväxtskydd för båtar.41. Karin Hellström (1995) Några terpeners antimutagena effekt.42. Malin Ahlgren (1996) Undersökning i Falun av markblyets biotillgänglighet för

bedömning av

hälsoriskerna som blyet utgör för små barn.

Projektrapport från utbildningen iEKOTOXIKOLOGI

Ekotoxikologiska avdelningenUppsala universitet

43. Patric Amcoff (1996) Thiamine (Vitamin B1) Concentrations in Baltic Salmon (Salmosalar) Suffering from the Reproduction Disturbance M74.44. Johan Broman (1996) Effekter på binjuren hos mink (Mustela vison) efter kronisk exponering för o,p'-DDD, MeSO2-PCB och Clophen A50.45. Johan Persson (1996) PCB i fogmassa en studie av förekomst, läckage och toxici

tet vid Rasslebygds avfallsupplag.46. Marie-Louise Nilsson (1996) Irgarol - aktiv substans i båtbottenfärg. Utveckling av analysmetoder (SPE, SFE, GC-MS), mätning av läckage samt toxicitetsstudier

med en marin rödalg som testorganism.47. Thomas Jågas (1996) Lead levels and lead poisoning in Swedish Anseriformes birds.48. Uno Strömberg (1996) Form och mobilitet för koppar och kadmium i sjösediment

nedströms två gruvområden i Dalarna.49. Krister Halldin (1996) EROD Induction in Chick Embryo Liver and Precision Cut Chick Liver Slices: A Rapid Bioassay for Dioxine-like Compounds.50. Petra Lindgren (1996) Fungal Degradation of Organic Pollutants. Infl uence of Different Parameters On the Degradation of SomePesticides In Solid Substrate Cultures of the White Rot Fungus Phanerochaete chrysosporium.51. Malena Granbom (1997) Comparison Between Four Different Biotests, Using Three Waste Waters as Toxicants.52. Annmari Blom (1997) The Use of Human Breast Cancer Cell Line (MCF–7) in Combination with Flow Cytometric Analysis for the Detection of Xenoestrogenic Environmental Pollutants.53. Maria Muribi (1997) Toxicity of mustard gas and two arsenic based chemical warfare agents on Daphnia magna. For evaluation of the ecotoxicological risk of

the dumped chemical warfare agents in the Baltic Sea.54. Niclas Lindqvist (1997) Utvecklingsneurotoxikologiska effekter av två herbicider

hos mus: paraquat och roundup.55. Maria Johansson (1997) An estimation of the glycogen storage in Cletocamptus confl uens and Nitocra spinipes (Copepoda, Harpacticoida) inhabiting brackish shallow water lagoons in the south west Baltic Sea.56. Anneli Schön (1997) Infl uence of Humic Substances and Ionic Strength on the Photo chemical Degradation of Naphthalene, Phenanthrene and Anthracene in Aquatic Media, and an Estimate of their Sunlight Induced Toxicity to Aquatic Bacteria.

57. Erik Beckman (1997) Fjädermygglarvers (Chironomus riparius) överlevnad och tillväxt i bottensediment. En ekotoxikologisk och kemisk karaktärisering av sedi- ment.58. Catharina Knutsson (1997) Effekter av nitrit på regnbåge. Samt en jämförelse med effekter på abborre i Möckeln utanför Björkborns industriområde.

Projektrapport från utbildningen iEKOTOXIKOLOGI

Ekotoxikologiska avdelningenUppsala universitet

59. Emma Ankarberg (1997) Developmental Neurotoxicity of Nicotine.60. Kerstin Gustafsson (1998) Bioaccumulation and Depuration of Polychlorinated Biphenyls (PCBs) and Polybrominated Diphenyl Ethers (PBDEs) by Baltic Sea Blue Mussels, Mytilus edulis.61. William Strandberg (1998) Induction of Alkaline Phosphatase in Ishikawa Cells:

A Bioassay for Screening of Estrogenic Contaminants.62. Leif Olofsson (1998) Comparative study of SemiPermeable Membrane Devices (SPMDs), Poly Urethane Foam (PUF) plugs, Pikes and Seston in regard to uptake characteristics of PCBs. 63. Sanna Eriksson (1998) Toxicitetstest med fotosyntetisk syrgasutveckling som testparameter och med mikroalgen selenastrum capricornutum som testorganism. 64. Henrik Sundberg (1999) Toxicity of Complex Mixtures of Polychlorinated bip henyls (PCBs) in Rat and Seal. With special emphasis on aryl hydrocarbon recep tor (AhR) mediated toxicity.65. David Wästlund (1999) The role of sediment characteristics and food regimen in a toxicity test with Chironomus riparius.66. Sara Malmheden (1999) Organometallic compounds in the Environment.67. Magnus Breitholtz (1999) In Situ Hybridization of steroidogenic cytochromes P- 450: Sites of expression correlated to sites of covalently bound 3-MeSO2-DDE in the adrenal cortex in mice.68. Anders Borgegren (1999) Värdering av metallers bidrag till toxicitet och kombina tionseffekter av metaller i industriavloppsvatten.69. Henrik Viberg (1999) Developmental neurotoxicity of 2,2',4,4',5- pentabromo dip henylether in the neonatal mouse.70. Anna-Karin Johansson (1999) Sammanställning och utvärdering av fi skundersök- ningar i recipienter till cellulosaindustrier längs Gävles läns kust.71. Emma Johansson (1999) In Vitro Assays for Detection of Endocrine Disrupting Compounds Estrogenic Activity in Recombiant Yeast Cells and Aromatase Inhibi- tion in Chicken Embryo Ovaries.72 Per Lind (1999) En livscykelinventeringsmodell för hantering av fast hushållsav fall på nationell nivå.73. Anna Kieffer (1999) VA-mykorrhizans etablering och effekt på skottlängd och sjukdomsangrepp i stråsäd under inverkan av bekämpningsmedel.

74. Per Claesson(2000)UndersökningavmetallsituationeniKolbäcksånstillflödeniFagersta.75. Monika Wikström (2001) Kan C-vitamin motverka effekter av PCB126 på skelettet?76. Sofi Nordfeldt (2001) Destruktion av sprängkapslar.77. Anna Nylander(2001)Syntetiskamyskföreningar.78. Dayteg Joachim och Nohrstedt Lena(2001)Undersökningavvattendirektivets prioriteradeämnen:SituationiStockholmsakvatiskamiljö.79. Elin Wallin (2001) Vittelogenininduktion i juvenil regnbåge (Oncorhynchus mykiss) ochbraxen(Abramisbrama)exponeradeförrenatavloppsvatten.

80. Karolina Stark (2001) Uppskattning av stråldos till grodor från cesium-137 i en våt mark norr om Gävle.81. Jenny Rönngren and Eva-Lotta Ödin (2001) Effects of the cyanotoxin nodularin on early life-stages of Baltic Sea cod, Gadus morhua.82. Ankie Sundberg (2001)Detection and localization of multixenobiotic transport prote ins in blue mussel (Mytilus edulis), rainbow trout (Oncorhynchus mykiss) and common breem (Abramis brama).83. Kim Kultima (2003) Metal levels in Swedish moose (Alces alces)84. Maria Aronsson (2003) Riktvärden för bekämpningsmedel i ytvatten.85. Alexandra Abrahamson (2003) The gill erod biomarker: Establishment in different fish species and use in marine environmental monitoring. 86. Mikaela Gönczi (2003) Discharges of PCBs and organotin compounds from the Muskö naval base.87. Charlotte Holmstrand (2003) Utvärdering av instrumentet XRF: Utredning om vilka faktorer som påverkar instrumentets analysförmåga samt en jämförelse mellan analys metoden XRF och ICP-AES (plasma-emissionsspektrometri).88. Annika Perhans (2003) Utlakning av polycykliska aromatiska kolväten (PAH) ur asfalt och förorenad mark: En litteraturstudie över vilka faktorer som styr utlakningen.89. Barbro Waldenström (2003) Undersökning av lekfrekvens och steroidhormonkoncen trationer i blodplasma hos abborrhonor (Perca fluviatilis) fångade i Stockholms skär- gård och Mälaren.90. Stina Klasson (2004) Dioxins and PCBs in fish oil.91. Erik Gustavsson (2004) Metals in sediment and organisms – A comparison of two catchments on Mauritius.92. Anna Lundqvist (2004) Metodutveckling av gaskromatografisk bestämning av oljehalt i processvatten från stålindustrin SSAB Oxelösund.93. Daniel Karlsson (2004) Interaction study of Bromkal 70-5 DE and Cereclor 70L in the rat.94. Johanna Thyselius (2004) Xenopus tropicalis som testorganism för att studera effekter av hormonstörande kemikalier på grodor. En studie i hur etynylöstradiol påverkar gonaddifferentieringen samt en avels- och utvecklingsstudie.95. Irina Pettersson (2004) Effekter i benvävnaden hos råtta efter tidig exponering av TCDD. Sockerdiet mildrar effekterna av TCDD.96. Veronica Ulfves (2004) Validation of two soil toxicity tests using a soil invertebrate Enchytraeus albidus.97. Eva-Maria Staaf (2004) Orsakar bekämpningsmedlet metribuzin feminisering av hanarna hos vanlig groda?98. Fredrik Svanberg (2004) Lead poisoning in the Marbled Teal (Marmaronetta angusti- rostris) and White-headed Duck (Oxyura leucocephala). A Study of Tissue Concentra- tions and Source of Lead.99. Adrian Sandin Sokolik (2004) Chemical Analysis and Histopathological Effects of Lead and Cadmium in Two Different Populations of the Greylag Goose (Anser anser)

Projektrapport från utbildningen iEKOTOXIKOLOGI

Ekotoxikologiska avdelningenUppsala universitet

Projektrapport från utbildningen iEKOTOXIKOLOGI

Ekotoxikologiska avdelningenUppsala universitet

100. Sofia Sjöholm (2005) Anti-estrogenicity and anti-androgenicity in leachates from Swedish solid waste deposition sites 101. Johanna Dahlberg (2005) Gentoxiciteten av HMF: s metabolit SMF studerad med det   flödescytometerbaserade mikrokärntestet in vivo102. Björn Santesson (2005) Metallers förekomst och läckage i mark vid en träimpregne- ringsanläggning i Halland 103. Anna Maria Svensson (2005) Neurobehavioural disturbances in adult mice neonatally exposed to pyrethriods and organophosphates alone or combined104. Oscar Fogelberg (2005) Dioxinlika ämnen i Stockholms ytvatten. En exponerings- studie med EROD som biomarkör105. Delila Gasi (2005) Coxackie virus infection influences the effects of brominated flame  retardants on cytochrome P450 and thyroid hormones in mice106. Disa Stolt (2006) Dioxiner och dioxinlika PCBer i ekologiska ägg – Fodrets betydelse107. Margareta Brandhorst Daho (2006) Ecotoxicological evaluation of the herbicide Terbutryn. 108. Carolina Hernhag (2006) Effekt av p,p’-DDE på benvävnad i vanlig groda, Rana temporaria109. Beatrice Jonsson (2006) Risk assessment on butylphenol, octylphenol and nonylphenol,   and estimated human exposure of alkylphenols from Swedish fish110. Emma Colleen (2006) Effects of in utero and lactational exposure to PCB 118 and PCB 153 on bone tissue in sheep 111. Frida Broman (2006) Effects on bone tissue in sheep reared on pasture treated with sewage sludge112. Maria Söderqvist (2007) Risk för effekter av tungmetaller i Stockholmsmiljön113. Gustav Montelius (2007) Bone characteristics in Swedish male white-tailed eagle (Haliaeetus albicilla) collected between 1956–2006 and in Swedish male goshawk (Accipiter gentilis) collected between 1848–2005114. Rosita Eklund (2007) Spermiekvalitet hos Xenopus tropicalis (västafrikansk klogroda) efter exponering för EE2 under yngelperioden115. Linnéa Vemhäll (2007) Läckage av etinylestradiol till omgivande vatten från det anti- konceptionella depotplåstret Evra® 116. Kristina Ahlford (2007) Environmental Risk Assessment of Selective Serotonin Reuptake Inhibitors (SSRIs) Fluoxetine, Citalopram, Sertraline, Paroxetine and the Benzodiazepine Oxazepam117. Anna-Karin Persson (2007) Ekologisk riskbedömning av emissioner från hyttsten118. Farzaneh Maddah (2007) Kontaminering av ytvatten via vägdagvatten 119. Anna Stjernström (2008) Miljöriskbedömning av molybden. Utsläpp av molybden från Sandvik AB – effekter på Storsjön120. Hanna Markström (2008) 2,2’,4,4’,5-pentabromodiphenyl ether (PBDE 99), a polybrominated flame retardant, can interact with DDT in Enhancing Developmental Neurotoxic Defects