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ISBN 978-952-62-0508-3 (Paperback)ISBN 978-952-62-0509-0 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)

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Mikko A

. J. Finnilä

OULU 2014

D 1254

Mikko A. J. Finnilä

BONE TOXICITY OF PERSISTENT ORGANIC POLLUTANTS

UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU, FACULTY OF MEDICINE,INSTITUTE OF BIOMEDICINE,DEPARTMENT OF ANATOMY AND CELL BIOLOGY;DEPARTMENT OF MEDICAL TECHNOLOGY;NATIONAL INSTITUTE FOR HEALTH AND WELFARE,DEPARTMENT OF ENVIRONMENTAL HEALTH;UNIVERSITY OF EASTERN FINLAND, FACULTY OF SCIENCE AND FORESTRY,DEPARTMENT OF ENVIRONMENTAL SCIENCE;CRANFIELD UNIVERSITY, CRANFIELD DEFENCE AND SECURITY,CRANFIELD FORENSIC INSTITUTE

A C T A U N I V E R S I T A T I S O U L U E N S I SD M e d i c a 1 2 5 4

MIKKO A. J. FINNILÄ

BONE TOXICITY OF PERSISTENT ORGANIC POLLUTANTS

Academic dissertation to be presented with the assent ofthe Doctora l Train ing Committee of Health andBiosciences of the University of Oulu for public defence inAuditorium A101 of the Department of Anatomy andCell Biology (Aapistie 7 A), on 8 August 2014, at 12 noon

UNIVERSITY OF OULU, OULU 2014

Copyright © 2014Acta Univ. Oul. D 1254, 2014

Supervised byProfessor Juha TuukkanenProfessor Timo JämsäProfessor Matti Viluksela

Reviewed byProfessor Yrjö T. KonttinenProfessor Reijo Lappalainen

ISBN 978-952-62-0508-3 (Paperback)ISBN 978-952-62-0509-0 (PDF)

ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)

Cover DesignRaimo Ahonen

JUVENES PRINTTAMPERE 2014

OpponentAssociate Professor Monica Lind

Finnilä, Mikko A. J., Bone toxicity of persistent organic pollutants. University of Oulu Graduate School; University of Oulu, Faculty of Medicine, Institute ofBiomedicine, Department of Anatomy and Cell Biology; Department of Medical Technology;National Institute for Health and Welfare, Department of Environmental Health; University ofEastern Finland, Faculty of Science and Forestry, Department of Environmental Science;Cranfield University, Cranfield Defence and Security, Cranfield Forensic InstituteActa Univ. Oul. D 1254, 2014University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland

Abstract

Persistent organic pollutants (POPs), especially dioxin-like chemicals, have been shown to haveadverse effects on skeleton and these effects are likely to be mediated via the aryl hydrocarbonreceptor (AHR). In spite of the extensive research, the characteristics of developmental effects ofPOPs are poorly known and the role of AHR in POP bone toxicity and skeletal development ingeneral.

In this project changes in bone morphology and strength as well as tissue matrix mechanics arestudied by applying state of the art biomedical engineering methods. This allows understanding ofthe effects of dioxins exposure and AHR activity on the development and maturation ofextracellular matrix in musculoskeletal tissues from a completely new perspective, and therebyimproving the health risk assessment of POPs.

In the present study skeletal properties of rats exposed maternally to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), Northern Contaminant Mixture (NCM) and Aroclor 1254(A1254) were studied for cross-sectional morphometric and biomechanical properties, and datawere analysed with benchmark dose modelling. In addition, extracellular matrix properties wereanalysed using nanoindentation. Similar measurements were performed for adult wild-type andAHR-null mice after TCDD exposure. The same animals were also analysed for microstructuralchanges using micro-computed tomography and their bone cell activity was estimated from serummarkers and gene expression.

Analyses show decreased bone length and cross-sectional properties with consequentlydecreased bone strength. On the other hand, an increased trabecular bone mineral density inresponse to NCM and A1254 was observed. In addition, bone matrix properties indicated delayedmaturation or early senescence after maternal or adult exposure, respectively. The AHR is mainlyresponsible for bone toxicity of dioxin-like compounds and plays a role in bone development. Thisis likely due to disturbed bone remodeling as indicated by altered serum markers and geneexpression. Overall these results indicate that POPs decrease bone strength, but the interpretationis difficult as there is more trabecular bone within cortical bone with compromised quality andincreased porosity.

Keywords: aryl hydrocarbon receptor, biomechanics, bone growth, dioxins,microstructure, persistent organic pollutants, remodeling

Finnilä, Mikko A. J., Pysyvien orgaanisten yhdisteiden luutoksisuus. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta, Biolääketieteenlaitos, Anatomia ja solubiologia; Lääketieteen tekniikka; Terveyden ja hyvinvoinninlaitos,Ympäristöterveyden osasto; Itä-Suomen yliopisto, Luonnontieteiden ja metsätieteidentiedekunta, Ympäristötieteen laitos; Cranfield University, Cranfield Defence and Security,Cranfield Forensic InstituteActa Univ. Oul. D 1254, 2014Oulun yliopisto, PL 8000, 90014 Oulun yliopisto

Tiivistelmä

Altistumisen pysyville orgaanisille ympäristökemikaaleille on todettu heikentävän luustoa.Dioksiinien ja dioksiininkaltaisten yhdisteiden vaikutusten on havaittu välittyvän aryylihiilivety-reseptorin (AHR) välityksellä. Huolimatta pitkään kestäneestä tutkimuksesta POP-yhdisteidensikiönkehityksen aikaisen altistuksen vaikutukset ja etenkin niiden mekanismit ovat edelleenhuonosti tunnettuja, samoin kuin AHR:n osuus POP-yhdisteiden luutoksisuudessa ja luustonkehityksessä ylipäätään.

Tässä työssä tutkittiin luuston rakenteellisia ja mekaanisia ominaisuuksia niin perinteisilläkuin uusimmilla biolääketieteen tekniikan menetelmillä. Tutkimuksen tavoitteena on saada uut-ta tietoa POP-altistuksen ja AHR-aktiivisuuden vaikutuksista luuston kehitykseen ja luukudok-sen ikääntymisprosesseihin, mikä edesauttaa kyseisten yhdisteiden riskinarviointia.

Tutkimuksissa altistettiin kantavia rottaemoja 2,3,7,8-tetraklooridibenzo-p-dioksiinille(TCDD), pohjoiselle saasteseokselle ja kaupalliselle Arokloori 1254 PCB-seokselle. Sikiönkehi-tyksen aikana altistuneiden jälkeläisten luuston poikkileikkauksen morfologia ja biomekaanisetominaisuudet mitattiin ja tulokset mallinnettiin vertailuannoksen määrittämiseksi. LisäksiTCDD-altistettujen rottien luustomatriisin ominaisuuksia selvitettiin nanoindentaatiomenetel-mällä. Samaa menetelmää käytettiin myös aikuisiässä TCDD:lle altistettujen villityypin hiirtenja AHR-poistogeenisiten hiirten tutkimiseen. Näiden hiirten luuston hienorakennetta mitattiinmyös korkean resoluution mikro-tietokonetomografialla ja niiden luusolujen aktiivisuutta tutkit-tiin seerumin biomarkkerien ja luun muodostumiseen osallistuvien geenien ekspressiotasojenavulla.

Sikiönkehityksen aikainen altistuminen pohjoiselle saasteseokselle ja Arokloori 1254:llehidasti luiden pituuskasvua. Lisäksi luiden poikkileikkauspinta-alat olivat pienentyneet jamekaaniset ominaisuudet heikentyneet. Toisaalta hohkaluun määrä oli lisääntynyt altistumisenseurauksena.

Myös sikiönkehityksen aikainen altistuminen TCDD:lle hidasti luukudoksen kypsymistä jajohti aikuisiällä luukudoksen ennenaikaiseen vanhenemiseen. AHR:llä oli päärooli ainakinaikuisiän vaikutusten ilmenemiselle ja reseptorilla vaikutti olevan rooli luuston kehityksessä yli-päätään. Seerumin biomarkkereiden ja geeniekspression muutosten perusteella nämä vaikutuk-set johtuvat todennäköisesti luuston uusiutumisen häiriöistä. Yhteenvetona voidaan todeta, ettäPOP-yhdisteet heikentävät luustoa, mutta tämän ilmiön diagnosoiminen on hankalaa, koska huo-nolaatuisen kuoriluun sisällä hohkaluun määrä on lisääntynyt.

Asiasanat: aryylihiilivetyreseptori, biomekaniikka, dioksiinit, hienorakenne, luunkasvu, luun uudismuodostus, pysyvät orgaaniset yhdisteet

7

Acknowledgements

The study was conducted in close collaboration between Institute of Biomedicine

(Department of Anatomy and Cell Biology as well as Department of Medical

Technology), University of Oulu; National Institute for Health and Welfare

(Department of Environmental Health), Kuopio; Cranfield Forensic Institute

(Biomechanics Laboratories) Cranfield University (UK) and Institute of

Environmental Medicine, Karolinska Institutet, Sweden. However, this work

would not have been possible without the funding I have been lucky to enjoy

from Yrjö Jahnsson Foundation, Tekniikan edistämissäätiö, Graduate School of

Musculoskeletal Disorders and Biomaterials and University of Oulu Research

council.

I want thank all my supervisors Professors Juha Tuukkanen, Timo Jämsä and

Matti Viluksela. Juha you have shown the way of the bone head and it has been

pleasure to work in your research group all these years. Timo I wish to thank you

for pushing me forward to continue for doctoral studies and being a role model

for successful researcher. Matti although located in Savo you have always had

time to discuss and share the knowledge from your bottomless expertise within

the field of toxicology. Language was revised by Professor Elizabeth Tanner and

final criticism was collected from reviewers Professor Yrjö T. Konttinen and

Professor Reijo Lappalainen, who did excellent work at final stages of this book

and prepared me to defend this work.

Months of the time I spent in Dr. Peter Zioupos’ biomechanics lab in

Cranfield UK. This time has been extremely valuable since you have taught me

(most of the tissue biomechanics I know) a lot and it has been pleasure to share

ideas with you. Especial thanks for my Swedish colleagues Dr. Maria Herlin and

Dr. Lubna Elabbas as well as Prof. Helen Håkansson in Karolinska Institute, it has

been pleasure to host you in Oulu and visit at your site and to rest of the co-

authors for their contribution Dr. Hanna Miettinen, Dr. Ulla Simanainen, Dr.

Natalia Stern, Christina Trossvik, Dr. Rachel Heimeier, Dr. Agneta Åkesson, Filip

Rendela, Dr. Wayne Bowers, Dr. Jamie Nakai, Dr. Antti Aula, Prof. Juha Risteli,

Dr. Merja Korkalainen.

I’m extremely thankful for my supervisory board members Professors Petri

Lehenkari and Miika Nieminen as well as Associate Professor Simo Saarakkala

for encouragement and advices that have been simultaneously valuable and

critical and support to carry these studies to finalization.

8

I also would like to thank the numerous colleagues at both Department of

Anatomy and Cell Biology and Department of Medical Technology as well as

around Institute of Biomedicine, University of Oulu and at the Department of

Engineering and Applied Science (Biomechanics Laboratories) Cranfield

University (UK). I’m not going to even try to list all the names since I’m sure that

I would forget few names from this list. Especially thanks for the Shrivenham

crew, with whom I was originally able to practice my rally Finglish so that it has

matured to a decent level. Same applies for my mates during the studies in HyTe

and TBDP programs, you were helpful bunch and really good company.

Finally thanks to my parents who have invested in their son’s (future) studies,

hopefully it will pay off someday and for Riina for all love and caring regardless

endless travelling, my willingness to rather stay over hours without being paid

than helping at home; and during the little free time too often forced to listen

work related gossip.

9

Abbreviations and symbols

1,25(OH)2D3 1,25-dihydroxyvitamin D3

µCT Micro-computed tomography

τ Loading interval

υ Poisson’s ratio

ψp Plastic (absorbed) energy

ψe Elastic (stored) energy

A1254 Aroclor 1254

Acreep Creep amplitude

AGEs Advanced glycation end products

AHR Aryl hydrocarbon receptor

ALP Alkaline phosphatase

Ap Projected contact area

Bcreep Initial creep rate

BD Benchmark Dose

BDL Benchmark Dose lower bound of the 95% confidence

BMD Bone mineral density

BMC Bone mineral content

BV/TV Trabecular bone volume fraction

Cat K Cathepsin K

CA-AHR Constitutively active AHR

Ct.A Cortical bone area

Ct.Po Cortical porosity

Ct.TMD Cortical tissue mineral density

CTX C-terminal telopeptides of type I collagen levels

DDT Dichlorodiphenyltrichloroethane

DMA Dynamic mechanical analysis

DXA Dual-energy X-ray absorptiometry

E* Complex modulus

EIT Indentation modulus

Elos Loss modulus

ER Estrogen receptor

ERα Estrogen receptor type α

ERβ Estrogen receptor type β

Er Reduced (effective) modulus

Estor Storage modulus

10

Fmax Peak load

GD Gestational day

HCB Hexachlorobenzene

hm Peak displacement

hc Contact depth

HIT Universal (instrumented) hardness

Hyl Hydroxylysine

Ip Plasticity index

KO Knock-out

K Elimination constant

Lys Lysine

M-CSF Macrophage colony-stimulating factor

Msx2 Msh homeobox 2

NCM Northern Contaminant Mixture

Ocn Osteocalcin

OPG Osteoprogerin

OPN Osteopontin

OVX Ovariectomy

PBB Polybrominated biphenyls

PBDD Polybrominated dibenzo-p-dioxins

PBDF Polybrominated dibenzofurans

PCB Polychlorinated biphenyls

PCDD Polychlorinated dibenzo-p-dioxins

PCDF Polychlorinated dibenzofurans

PFOA Perfluorooctanesulfonic acid

PINP N-terminal propeptide of type I collagen levels

PND Postnatal day

Po.Th Pore thickness

PPARγ Proliferator activating receptor subtype gamma

PTH Parathyroid hormone

S Contact stiffness

SMI Structural model index

SOST Sclerostin

t1/2 Elimination half-life

TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin

TEQ Toxic equivalent

TMD Tissue mineral density

11

TNAP Tissue non-specific alkaline phosphatase

TRACP-5b Tartrate resistant acid phosphatase

Tb.N Trabecular number

Tb.Sp Trabecular separation

Tb.Pf Trabecular pattern factor

Trab.BMD Trabecular bone mineral density

VDR Vitamin D reseptor

WT Wild type *0x Loading dose

x0 Maintenance dose

12

13

List of original publications

The thesis consists of four original publications that are referred to in the text by

their Roman numerals (I-IV). Additional yield and trabecular bone mineral

density data related to studies II and III are also included.

I Finnilä MA, Zioupos P, Herlin M, Miettinen HM, Simanainen U, Håkansson H, Tuukkanen J, Viluksela M & Jämsä T (2010) Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on bone material properties. J Biomech 43: 1097–1103.

II Elabbas LE, Finnilä MA, Herlin M, Stern N, Trossvik C, Bowers WJ, Nakai J, Tuukkanen J, Heimeier RA, Åkesson A & Håkansson H (2011) Perinatal exposure to environmental contaminants detected in Canadian Arctic human populations changes bone geometry and biomechanical properties in rat offspring. J Toxicol Environ Health A 74: 1304–1318.

III Elabbas LE, Herlin M, Finnilä MA, Rendel F, Stern N, Trossvik C, Bowers WJ, Nakai J, Tuukkanen J, Viluksela M, Heimeier RA, Akesson A & Håkansson H (2011) In utero and lactational exposure to Aroclor 1254 affects bone geometry, mineral density and biomechanical properties of rat offspring. Toxicol Lett 207: 82–88.

IV Herlin M*, Finnilä MAJ*, Zioupos P, Aula A, Risteli J, Miettinen HM, Jämsä T, Korkalainen M, Tuukkanen J, Håkansson H & Viluksela M (2013) New insights to the role of aryl hydrocarbon receptor in bone phenotype and in dioxin-induced modulation of bone microarchitecture and material properties. Toxicol Appl Pharmacol 273(1): 219–226.

*Equal contribution

14

15

Contents

Abstract

Tiivstelmä

Acknowledgements 7 

Abbreviations and symbols 7 

List of original publications 13 

Contents 15 

1  Introduction 17 

2  Review of the literature 19 

2.1  Bone ........................................................................................................ 19 

2.2  Bone tissue formation and (re)modeling ................................................. 19 

2.3  Bone composition and extracellular matrix physics ................................ 23 

2.4  Bone morphology .................................................................................... 25 

2.5  Persistent organic pollutants (POP) ......................................................... 27 

2.6  POP effects on bone tissue ...................................................................... 29 

2.7  Nuclear receptors and bone toxicity of POPs .......................................... 31 

2.7.1  Aryl Hydrocarbon receptor ........................................................... 32 

2.7.2  Estrogen Receptors (ER) .............................................................. 33 

2.7.3  Vitamin D Receptor ...................................................................... 34 

3  Aims of the study 37 

4  Materials and methods 39 

4.1  Chemicals ................................................................................................ 39 

4.2  Study permissions ................................................................................... 39 

4.3  Experimental designs .............................................................................. 39 

4.3.1  Maternal TCDD exposure ............................................................ 39 

4.3.2  Maternal NCM exposure .............................................................. 40 

4.3.3  Maternal A1254 exposure ............................................................. 40 

4.3.4  Role of AHR in skeletal development and TCDD toxicity .......... 40 

4.4  Bone sample preparation ......................................................................... 41 

4.5  Peripheral Quantitative Computed Tomography (pQCT) ....................... 42 

4.6  Microtomography (μCT) ......................................................................... 42 

4.7  Whole bone biomechanical testing ......................................................... 43 

4.8  Nanoindentation of bone tissue ............................................................... 44 

4.9  Serum markers for bone remodeling ....................................................... 47 

4.10 Osteogenesis PCR array .......................................................................... 47 

4.11 Statistical analyses and dose response modelling ................................... 47 

16

5  Results 51 

5.1  Bone size ................................................................................................. 51 

5.2  Bone morphology and mineralization ..................................................... 51 

5.2.1  Cortical bone ................................................................................ 51 

5.2.2  Trabecular bone ............................................................................ 54 

5.3  Mechanical properties ............................................................................. 57 

5.4  Intrinsic matrix properties ....................................................................... 58 

5.5  Serum markers ........................................................................................ 60 

5.6  Gene expression ...................................................................................... 60 

5.7  Dose modelling ....................................................................................... 61 

6  Discussion 63 

6.1  Novel methods in bone toxicology .......................................................... 63 

6.2  Model compound and genetic model studies .......................................... 65 

6.3  Towards studies with naturally occurring POP mixtures ........................ 66 

6.4  Translation of findings to human health .................................................. 67 

7  Conclusions 71 

References 73 

Original publications 89 

17

1 Introduction

There is a huge range of different persistent organic pollutants (POP) including

dichlorodiphenyltrichloroethane (DDT), polychlorinated and polybrominated

biphenyls (PCB, PBB), dibenzo-p-dioxins (PCDD, PBDD) and dibenzofurans

(PCDF, PBDF) present in nature. Although their production has been tightly

regulated, due to their stability and long elimination half-life they are still present

in biota and humans. PCDDs alone have 210 congeners of which 17 are

extremely toxic. These compounds have spread around the world (Tuomisto et al.

1999). Humans are exposed to POP’s mainly via diet, especially meat, milk and

fish products.

In particular fish consumption is problematic as fish is good source of

vitamin D and omega-3-fatty acids. Sufficient vitamin D levels can prevent

osteoporosis (Aro 2005) and decrease cancer incidence (Veldhuis et al. 2010),

while omega-3-fatty acids may have a role in the prevention of cardiovascular

diseases (Kris-Etherton et al. 2002). Thus, it would be tempting to recommend

increased fish consumptions. However, such recommendations could potentially

lead to developmental defects and cancer, as the margin of exposure between

tolerable daily intake (TDI) and found concentrations of POP’s is very narrow in

humans (European Commission 2000, WHO 2000). Therefore, better

understanding of toxicity on the development and maturation of skeletal system is

needed for more accurate risk assessment.

POP’s share some chemical characteristics as they are lipophilic and

extremely persistent. Due to lipophilicity they accumulate in adipose tissue and

can be transferred to mother’s milk. Therefore, alone for dioxins a child can get a

significant proportion of mother’s body burden, about 20–25% in the case of

dioxins (Tuomisto 2001). Moreover, only for this single group of chemicals, a

huge spectrum of toxic effects has been recognized from mild biochemical

changes to lethal wasting syndrome (Pohjanvirta & Tuomisto 1994). Most of the

dioxin-induced effects are mediated via the aryl hydrocarbon receptor (AHR), a

ligand-activated transcription factor. Osteoclasts (Ilvesaro et al. 2005), osteoblasts

(Gierthy et al. 1994, Ilvesaro et al. 2005, Ryan et al. 2007), ameloblasts and

odontoblasts (Sahlberg et al. 2002), and chondrocytes (Yang & Lee 2010) express

AHR indicating potential for adverse effects of dioxin compounds in

musculoskeletal tissues. Indeed, hypomineralization of molars has been observed

in Finnish children exposed to background levels of POP’s via their mother’s milk

(Alaluusua et al. 1999). This has been confirmed in animal models, where dioxin

18

disrupts the molar development in rats (Kattainen et al. 2001, Miettinen et al.

2002). In addition, dioxin accelerates eruption of the lower incisors and retards

the eruption of molars (Miettinen et al. 2002). Furthermore, dioxin exposure

decreases bone strength and bone geometry in adult rats (Jämsä et al. 2001). Later

it was discovered that combined in utero and lactational exposure caused similar

effects with decreased mineralization but with a dose less than one tenth

compared to adults (Jämsä et al. 2001, Miettinen et al. 2005). Moreover, similar

effects had been discovered on rats exposed to hexachlorobenzene (HCB)

(Andrews et al. 1989, Andrews et al. 1990) and PCBs (Lind et al. 1999, Lind et al.

2000a, Lind et al. 2000b, Lind et al. 2004).

Developmental defects are potentially the most significant toxic effects of

dioxins for human health, because they have been observed at very low exposure

levels and they are potentially permanent (Alaluusua et al. 1999, Alaluusua &

Lukinmaa 2006, European Commission 2000, Gray et al. 1997, Kattainen et al.

2001, Lukinmaa et al. 2001, Mably et al. 1992, Miettinen et al. 2002, Miettinen et

al. 2005, WHO 2000). Teeth (Alaluusua et al. 1999, Kattainen et al. 2001,

Lukinmaa et al. 2001, Miettinen et al. 2002), bones (Miettinen et al. 2005) and

the reproductive system (Gray et al. 1997, Mably et al. 1992) have already been

shown to be sensitive to dioxin exposure especially during embryonic

development.

In spite of extensive research the characteristics and mechanisms of the

developmental effects of dioxins are still insufficiently known. This project

studies changes in bone morphology and strength as well as tissue matrix

mechanics by applying state of the art biomedical engineering methodologies.

This will allow us to understand the effects of dioxins exposure and AHR activity

on the development and maturation of the extracellular matrix in musculoskeletal

tissues from a completely new perspective, and thereby improve the health risk

assessment of dioxin-like compounds.

19

2 Review of the literature

2.1 Bone

Bone is highly specialized mineralized connective tissue, which is mainly

composed of hydroxyapatite, collagen and water contributing to hardness (Currey

& Brear 1990, Zioupos 2005), toughness (Wang et al. 2001) and viscosity (Pathak

et al. 2011), respectively. Besides varying matrix composition and material

properties, bones come with multiple shapes and have locally varying

ultrastructure both contributing to the overall strength of the bone (Chappard et al.

2011, Rho et al. 1999).

These properties are maintained in constant remodeling process (Robling et

al. 2006), where osteoclasts resorb old bone matrix and osteoblasts lay new

osteoid, which becomes more mineralized with a time. Additionally, if bone shape

does not correspond to its mechanical demands, it is modelled to have optimal

shape (Biewener & Bertram 1994, Plochocki et al. 2008, Sharir et al. 2011).

Healthy bone is hard and tough, but changes at any hierarchical level can lead to

decreased bone strength, fracture and loss of function (Rho et al. 1998).

Thus it is important to understand the link between modifications in matrix

properties due to changes in cellular activity and overall strength. In this work the

focus is in the effects of persistent organic pollutants on bone structure and

strength at various hierarchical levels and the role of the AHR in these effects in

vivo.

2.2 Bone tissue formation and (re)modeling

Bone development starts with condensation of mesenchymal stem cells followed

by either of the two distinct ossification pathways. Most of the flat bones are

formed by intramembranous ossification and rest by endochondral bone

formation (Zelzer & Olsen 2003). In intramembranous ossification, cells

differentiate directly to osteoblasts and start producing bony structures. The

endochondral bone formation begins with the condensation of mesenchymal stem

cells to cartilougous anlagen. Primary ossification center will appear in the

diaphyseal part of anlagen, where a collar of bone is formed. Later secondary

ossification centers are formed inside the epiphyseal parts of bone surrounded by

cartilaginous structure growth plate and articular cartilages.

20

Thereafter bone is modelled (growth and shape adaptation) and remodelled to

maintain tissue quality (Fig. 1). These processes are regulated by various peptides

and cytokines reflecting bone health, and they can be analyzed from blood

samples. Due to slow turnover of bone tissue the changes in remodeling process

have to be long lasting in order to ultimately cause alterations in physical

properties of bone that could have any clinical significance.

In order to produce bone, mesenchymal stem cells need to differentiate

towards osteoblastic phenotype as indicated by expression of Runt-related

transcription factor 2, distal-less homeobox 5 and msh homeobox 2 (Msx2).

Further dedication is indicated by expression of osterix, and β-catenin, T cell

factor/lymphoid enhancer-binding factor 1 (Robling et al. 2006). Once the

osteoblastic phenotype has been reached, cells start to produce type I pro-collagen,

(Fig 1E), the main component of organic matrix, and tissue non-specific alkaline

phosphatase (TNAP), a mineralization catalyst. Besides producing mineralization

regulators (TNAP, Phospho1, Progressive ankylosis protein, nucleotide

pyrophosphatase/phosphodiesterase, Fetuin-A) (Harmey et al. 2004, Szweras et al.

2002), osteoblasts produce paracrine regulators of remodeling (FasL,

osteoprotegerin) and even endocrine regulators osteocalcin (Ocn), osteopontin

(OPN) (Karsenty & Oury 2012) demonstating that skeleton is part of endocrine

system and endocrine disturbing chemicals (EDC) have potential skeletal

manifestations.

To achieve amazing mechanical properties, possessed by bones, both collagen

synthesis and matrix mineralization are orchestrated in a complex way. Collagen

has to be assembled in triple helical form and packed into fibrils. Assembly of

pro-collagen to triple helical form requires post-translation modification by

collagen prolyl 4-hydroxylase (C-P4H) to result in a biopolymer stable at body

temperature (Myllyharju & Kivirikko 2004). Upon the release respective

proteinases cleave N and C terminal propeptides, while rest assembles as part of

fiber in extracellular space (Fig 1F). Cleaved propeptides especially procollagen

type I N-terminal peptide (PINP) measured from serum and urine are used as

markers of bone formation (Suvanto-Luukkonen et al. 1997). Finally, to ensure

optimal (post-yield) mechanical properties of collagen molecules, covalent

intermolecular crosslinks are formed (Saito & Marumo 2010), while

mineralization gives strength and stiffness to bone matrix (Fig 1G and Fig 1H).

Optimal mineralization requires action of various mineralization promoters, and

inhibitors to give hardness required from load bearing skeletal structures and to

protect cells from being petrified with mineral dissipates as well as other organs

21

from ectopic calcification (Kornak 2011). Most important ones for mineralization

are TNAP and Phospho1 (Huesa et al. 2011, Yadav et al. 2011), regulating

pyrophosphate/phosphate ratio and hydroxyapatite nucleation.

Some of the matrix producing osteoblasts undergo apoptosis (Karsdal et al.

2002), while some are trapped inside the matrix and start maturing to osteocytes

(Fig 1E, G, A). Although bone matrix has good mechanical properties it will

suffer from loading induced micro-cracking, which have to be remodelled

(rebuild) (Fig 1B) in order to avoid complete fracture of whole bone. Both

remodeling and modeling are most likely controlled by osteocytes, cells that are

mechano-sensors as well as markers of bone quality. Osteocytes will increase

production of nitric oxides (Zaman et al. 1999) and prostaglandins (Rawlinson et

al. 1991) and decrease sclerostin (SOST) (Robling et al. 2008) as response to

increased loading enhancing bone formation. In cases where loading causes

damage bone osteocytes have been reported to secrete Macrophage colony-

stimulating factor (M-CSF), and Receptor Activator for Nuclear Factor κ B

Ligand (RANK-L) (Kurata et al. 2006), a surprisingly similar process as observed

in unloading of a bone (O'Brien et al. 2013). Microfractures in the bone matrix as

such do not probably play a role in this process (Rumpler et al. 2012). Osteocyte

apoptosis leads to recruitment of pre-osteoclasts and enhancement of

osteoclastogenesis, under a canopy (Fig 1C) that covers whole remodeling site,

coupling bone resorption and formation (Andersen et al. 2009).

Osteoclastogenesis requires presence of M-CSF and RANK-L to drive

differentiation of pre-osteoclasts to a more mature cellular phenotype (Teitelbaum

2000). Osteoblasts participate to osteoclastogenesis through their membrane

bound RANK-L, that can be blocked by osteoprogerin (OPG), hence a direct

contact between osteoblastic/stromal cells with pre-osteoclastic cells is required

for osteoclastogenesis in vivo. Mature osteoclasts bind to bony surface by forming

a sealing zone via αvβ3 integrin and start releasing bone matrix degrading

Cathepsin K (Cat K) in acidic medium (Boyle et al. 2003). Degradation is further

continued in intracellular vesicles containing tartrate resistant acid phosphatase

(TRACP 5b) in addition to Cat K (Vääräniemi et al. 2004). These transcytotic

vesicles are transported to the functional secretory domain in the basolateral

membrane (Salo et al. 1997), where degradation products are released and can

enter circulation and urine where they can be detected.

22

Fig. 1. Bone remodeling process. A) In a steady state the osteocytic network is active

and SOST signals other bone cells to remain inactive. B) Damage causes local

apoptosis of osteocytes, C) leading to formation of a canopy recruitment site for both

hematopoetic and mesenchymal stem. Concurrent presence of both stem cells types

is required for osteoclast activation, since osteoblastic RANKL binds to the RANK

receptor of pre-osteoclasts activating a fusion process resulting in mature

multinuclear osteoclast. D) Mature osteoclast secretes TRACP-5b and during

resorption collagen telopeptides, both are released to circulation. When the damaged

site is removed pre-osteoblasts start producing RANKL inhibiting and OPG stopping

further fusion of osteoclasts. Then pre-osteoblasts E) start proliferating and further

differentiating to osteoblasts that fill the resorption cavity F) with collagenous matrix

by producing type I pro-collagen that is delivered to extracellular space, where

propeptides are cleaved and remaining polymer is added to the fiber. G) Some

osteoblasts are trapped inside unmineralized matrix called, osteoid, which is later

mineralized H) with crystals growing around collagen fibers.

23

2.3 Bone composition and extracellular matrix physics

Although the skeleton appears to be inert organ, the well-controlled remodeling is

the key for maintaining tissue quality at the level required to survive daily

activities without plastic deformation or even fracturing.

The backbone of all the skeletal matrices is type I collagen. This

heterodimeric fibrillar collagen is composed of two α1(I) chains and one α2(I)

chain. During the processing in the endoplamic reticulum various post-

translational modifications takes a place. Upon the release collagen fibrils line up

with 40 nm spacing and overlap 27 nm with fibril layers above giving the familiar

periodic 67 nm cross-striated pattern. Here lysyl oxidase forms reactive aldehydes

from lysine (Lys) and hydroxylysine (Hyl) residues in telopeptides (Gelse et al.

2003). These hydroxy-allysines and allysines bind to helical Lys and Hyl resulting

in reducible immature crosslinks. Mature pyridinoline and pyrrole cross-links are

formed when second telopeptic aldehyde binds to divalent complex (Eyre & Weis

2013). At this point collagen starts to mineralize. Senescence of bone introduces

another type of crosslinks, non-enzymatic AGEs (advanced glycation end

products). Generally the collagen content (Saito & Marumo 2010) seems to

follow a similar pattern of overall bone mass (Haapasalo et al. 1996, Isaksson et

al. 2010). As could be expected the amount of immature collagen cross-links

decline rapidly while the mature cross-links and especially AGEs increase with

age (Saito & Marumo 2010). Accumulated evidence until now suggests that the

enzymatic cross-links improve the mechanical competence of collagen network

while AGEs reduce it (Nalla et al. 2006, Nyman et al. 2007, Oxlund et al. 1995,

Saito & Marumo 2010, Vashishth et al. 2001, Zimmermann et al. 2011) by

increasing bone plasticity and microcracking (Zimmermann et al. 2011).

The early form of bone is called osteoid and is composed mainly of collagen.

The positively charged regions in collagen fibers provide optimal sites for

nucleation of the plate-like mineral crystals (2–5 nm thick, 5–25 nm wide and 15–

55 nm long) (Nudelman et al. 2010), but the activity of TNAP is required for

sufficient extravesicular mineralization (Yadav et al. 2011). Intrafibrillar crystals

account only about 20% of bone mineral, while the majority lies in interfibrillar

space (McNally et al. 2012). These interfibrillar crystals are about 5 nm thick, 50

nm wide and 40–200 nm long having most likely plate-like shape (McNally et al.

2012). Bone mineral has been shown to grow becoming more elongated with age

(Su et al. 2003, Tong et al. 2003). The main mineral in bone tissue is structurally

similar to the naturally occurring hydroxyapatite (HA), however due to extensive

24

specific surface (100–200 m2/g) (Boivin & Meunier 2003) apatite in bone is

unstable and often substituted with available impurities (Boskey 2003). The

mineralization increases as function of tissue age (Turunen et al. 2011, Turunen et

al. 2013) while the water content decreases (Mueller et al. 1966). This also leads

to fundamental idea between the relationship of material properties and

mineralization level, which should follow each other, where an increase in the

mineral content increases both the modulus and hardness (Currey & Brear 1990,

Riggs et al. 1993b, Zioupos 2005, Zioupos & Rogers 2006), decreases the

plasticity index and reduces both viscoelasticity (i.e. decreased storage modulus /

increased loss modulus), and the overall creep strain magnitude, last with a

concomitant increase in the early creep strain rate (Zioupos 2005, Zioupos &

Rogers 2006). As bone is formed in physiological fluid and phosphate is a strong

acid, a part of it is transformed to hydrogen phosphate, which is then detectable in

young bone tissue. However, with maturing bone tissue the quantity of hydrogen

phosphate decreases and in tissue senescence natural physiological buffer,

carbonate, is substituted in hydroxyapatite (Legros et al. 1987).

The final major component in this three-phase composite is water. It is well

known that water is critical for mechanical performance of bone and thus

hydration state should been hold close to native during all material testing.

However, only recently it was suggested that water participates to organization of

mineral crystals (Wang et al. 2003) and in amorphous layers could act as re-

usable glue that allows some strain between crystals and even when broken could

be easily re-formed (Duer & Veis 2013, Wang et al. 2013). Indeed a presence of

sacrificable bonds in bone has been suggested (Thompson et al. 2001) and those

could be the water between the plates.

Since collagen has Young’s modulus of 5GPa (Strasser et al. 2007, van der

Rijt et al. 2006, Wenger et al. 2007, Yadavalli et al. 2010) or possibly much lower

(Grant et al. 2008), is too flexible to maintain shape under daily compressive

loading. On other hand Young’s modulus of pure mineral is about 150 GPa

(Vanleene et al. 2012) and would fracture at low energies. For example the

fracture toughness of enamel (Imbeni et al. 2005) (the most mineralized tissue in

body) is about a quarter of bone (Nalla et al. 2005). In the healthy bone these

material types are present in volume fractions of 62.6%, 28.3% and 11.3%, for

mineral, organic and water respectively (Ding et al. 1997, Ding et al. 2012, Li &

Aspden 1997), this combination results to unique mechanical properties of bone,

where collagen conducts most of strain energy (Gupta et al. 2006) and mineral

provides hardness. Such material possesses minimal weight (density) and still

25

provides a sufficient strength and toughness for skeletal structures (Ritchie 2011).

Bone material is also heterogeneous increasing its energy dissipation capability

(Tai et al. 2007). Although the matrix composition and material behaviour plays a

role in overall mechanical performance of bone these changes are often buried

under overall quantity of the various micro- and macrostructure.

2.4 Bone morphology

When moving from nanostructure towards higher hierarchical structural levels

first there are lamellae, where collagen fibrils run in parallel to each other within

the sheet. They contribute to mechanical properties of bone trough their

organization. For example in race horse radius, lamellae (collagen) are organized

according to loading environment, where longitudinal orientation is found at

cranial segment and oblique to transverse orientation in the caudal segment of

cortical bone(Riggs et al. 1993a, Riggs et al. 1993b). Longitudinally orientated

collagen can better resist tension while the transverse orientation is designed for

compression (Riggs et al. 1993b) matching the strain during the pacing (Riggs et

al. 1993a).

The lamellae run as sheets on periosteal as well endosteal bone envelopes.

These parallel sheets form individual trabeculae but when wrapped around each

other they form circular osteons, where the trabeculae and the osteons provide the

next higher structural level. Furthermore the osteons buried between endosteal

and periosteal bone borders form the first macrostructure, cortical bone, which

forms surrounding layer over all the bones. The osteons form either Haversian or

Volkmann’s canals depending whether they run along (Haversian) or across

(Volkmann) the bone axis. These canals contain blood vessels and form vascular

network of bone, a critical transportation system. However, they also increase the

porosity of bone and as such reduce bone strength (Riggs et al. 1993b,

Zimmermann et al. 2011). Osteons are remodeled by bone multicellular units

where osteoclasts resorb some of the primary osteon making space for secondary

osteons. These primary osteon remnants have become more mineralized and thus

if their amount increases due to reduced remodeling or insufficient filling of

cavity the whole bone may become brittle (Akkus et al. 2003). If the resoption

surpass the bone formation the cortical porosity increases (Idelevich et al. 2011),

as happens with aging (Chen et al. 2010, Zebaze et al. 2010, Zimmermann et al.

2011), even to such an extent, where endocortical bone starts to resemble

trabecular bone (Zebaze et al. 2010). This reduces bone toughness by limiting

26

capacity of the crack-bridging, one of the bone toughening mechanisms

(Zimmermann et al. 2011). One possible mechanism for increased porosity is

decline in osteocyte density with age (Busse et al. 2010) reducing bone quality

control. Furthermore observation of hypermineralized occlusion within these

lacunas (Busse et al. 2010) could be indication of cell death that could increase

the bone resorption as described previously.

Another microstructure is the individual trabeculae. Generally the trabeculae

have lower modulus and hardness compared to cortical bone (Lewis & Nyman

2008, Szabo et al. 2011). Same applies for their mineralization (Tjhia et al. 2012)

and mineral:matrix ratios (Szabo et al. 2011), while the link to material properties

has been difficult to show (Szabo et al. 2011, Tjhia et al. 2012, Zebaze et al.

2011). This could indicate that the material properties are more related to

orientation at lower structural levels and collagen properties (Szabo et al. 2011) or

alternatively it could be that nanoindentation results are affected by osteocyte

lacunas under the measurement point. The trabeculae can take various shapes

from spheres to plates. The dominant shape on the other hand depends on

anatomical location (Turunen et al. 2013) and possibly prevailing pathology

(Salmon 2004). The study of individual trabeculae is not simple and thus most of

the data arises from studying the second macrostructure trabecular bone. When

studying trabecular bone specimens, it has been possible to show that mechanical

properties follow mostly logical pattern where thicker trabeculae with shorter

separation and higher number of trabeculae increase trabecular bone volume

fraction, often giving a plate-like structure with low fragmentation gives high load

capacity and stiffness (Mittra et al. 2005, Teo et al. 2006). This is further

supported by studying fracture regions showing absence of plate-like structures,

trabecular orientation parallel to loading direction or high trabecular bone specific

surface or thickness are not present in fractured areas (Tassani & Matsopoulos

2014). Also increased microdamage accumulation in rod-like trabeculae has been

reported (Karim & Vashishth 2011). Somewhat surprisingly connectivity has low

correlation to mechanical properties (Teo et al. 2006) and seems not to provide

any resistance against fractures (Karim & Vashishth 2011).

Bones come in variety of shapes and the relative amounts of trabecular and

cortical bone varies between bones. Even though much effort in this review has

been spent on bone “quality” aspect and its relation to bone strength, the overall

quantity has huge impact on strength. Maybe for the idea that “the bigger the

stronger“ has shifted the diagnostic focus on the bone quantity meters as they are

relatively simple and still clinically relevant. On the other hand, dual energy x-ray

27

based bone mineral density (BMD) may not be sufficient to predict fracture risk

(Pulkkinen et al. 2011) and for this reason the bone qualitative properties are

clinically relevant.

Fig. 2. Finite element models of tube-like cortical structures with constant volume and

varying structure. The endosteal perimeter is defined as the periosteal diameter on

left. This decreases cortical thickness but still increases bending resistance. For

example 17% increase in diameter increases inertia by 52% and stiffness by 30%.

Also the gain of strength only by increasing size would require more energy from

movement. Most likely for this reason, most of the long bones are built according

to principle that same amount of material, when placed further away of mass

center gives most resistance against bending and torsion through increased inertia

(Fig. 2). For this reason many morphological variables have good correlation to

mechanical properties (Jämsä et al. 1998, Lind et al. 2001). However, the bones

are not perfect tubes and their shape is fine tuned to correspond to loading

activities already during embryogenesis (Sharir et al. 2011).

2.5 Persistent organic pollutants (POP)

Criteria for classification of chemicals as POPs they have to be (1) highly toxic to

humans and the environment, (2) persistent in the environment, resisting bio-

degradation, (3) taken up and bio-accumulated in terrestrial and aquatic

ecosystems and (4) capable of long-range, transboundary atmospheric transport

and deposition. Table 1 presents different POPs with their sources and possible

28

indication of skeletal effects. In the Stockholm convention there are 12

compounds (including their congeners) listed, whose usage were agreed to be

limited. More recently this list was extended in 2009.

It has been approximated that due to industrialization only the level of dioxin-

like compounds were tripled in the nature from 1920 to 1970. Fortunately their

levels seem to decline slowly. However, recently a wider nominator endocrine

disturbing chemical (EDC) is used for these chemicals. It includes POPs and

many more and potentially involves 800 chemicals (WHO 2013), many of them

are potentially toxic to bone.

Humans are exposed to POPs mainly via diet, especially meat, fatty milk and

fish products. Due to lipophilicity of these compounds they are distributed to

tissues with high lipid content, they are able to cross the placenta and during

lactation they mobilize readily into mother’s milk that has high lipid

concentration. For dioxin-like compounds lactational exposure is quantitatively

more significant than placental transfer (Hurst et al. 2000, Li et al. 1995,

Miettinen et al. 2005). This results in relatively high exposures to infants, who in

the case of dioxins can be exposed to 20–25% of the mother’s body burden

(Tuomisto 2001). High prenatal and neonatal exposure during critical windows of

sensitivity may lead to potentially permanent developmental defects (Tuomisto &

Vartiainen 2004, WHO 2013).

The governmental actions for risk management of these chemicals have

shown to be effective and the levels of several POPs have turned to slow decline.

However, they still are significant global pollutants due their persistency. While

the regulation of these chemicals seems to have achieved the wanted effects on

their existence, the chemicals designed to replace them are on rise and often

possess endocrine disturbing chemical-type activity. (WHO 2013)

29

Table 1. List of POPs and their usage (or source). Indication if compounds were used

in A1254 or Northern Contaminant Mixture (NCM) and bone toxicity are given.

Chemical Source A1254(2) NCM(3) Bone effects

Aldrin Pesticide to treat seed and soil. 3 [1]

(Oxy)Chlordane Pesticide and as an additive in

plywood adhesives

3 [2]

Dichlorodiphenyltrichloroethane (DDT) Pesticide p,p’-DDE3

p,p’-DDT3

[3-4]

Dieldrin Pesticide (agriculture) 3

Heptachlor Pesticide (building materials) 3

Hexachlorobenzene (HCB) A solvent for pesticides (e.g. in Ky-5) 3 [5-6]

Mirex Pesticide 3

Polychlorinated biphenyls (PCB) Dielectric, coolant, lubricant,

plasticizers, pesticide extender, flame

retardants, de-dusting agents, water-

proofing, microscopy fixative, surgical

implants.

PCB283 [7-10]

PCB772 PCB523

PCB812 PCB993

PCB1052 PCB1013

PCB1142 PCB1053

PCB1182 PCB1183

PCB1232 PCB1283

PCB1262 PCB1383

PCB1533

PCB1562 PCB1563

PCB1572 PCB1703

PCB1672 PCB1803

PCB1592 PCB1833

PCB1892 PCB1873

Polychlorinated dibenzodioxins (PCDDs1) Waste incineration, by-product in

chemical process

[11-13]

Polychlorinated dibenzofurans (PCDF) 2,3,7,8-TCDF2

1,2,3,7,8-PCDF2

2,3,4,7,8-PCDF2

1,2,3,4,7,8-HexaCDF2

1,2,3,6,7,8-HexaCDF2

2,3,4,6,7,8-HexaCDF2

1,2,3,4,6,7,8-HCDF2

1,2,3,4,7,8,9-HCDF2

1,2,3,4,6,7,8,9-OCDF2

Toxaphene Pesticides 3

Added in 2009

α, β and γ-Hexachloro-cyclohexane γ-HCH (lindane) to control of head

lice and scabies.

β-HCH3 [14]

Chlordecone

Hexabromobiphenyl

Hepta and heptabromobiphenyl ethers Recycling and reuse of articles

containing these compounds

Pentachlorobenzene

Tetra- and pentabromodiphenyl ethers

Perfluorooctanesulfonic acid (PFOA), its

salts

Various mainly for surfactant

properties

Perfluorooctanesulfonyl fluoride

Reported skeletal effects [1] (Singh & Jha 1982); [2] (Stranger & Kerridge 1968); [3,4](Beard et al. 2000, Lundberg et al.

2007); [5,6] (Andrews et al. 1989, Andrews et al. 1990); [7-10] (Lind et al. 1999, Lind et al. 2000a, Lind et al. 2000b, Lind et

al. 2004) [11-13] [(Jämsä et al. 2001, Lind et al. 2009, Miettinen et al. 2005) and [14] (Andrews & Gray 1990)

30

2.6 POP effects on bone tissue

The adverse effects of POPs on the skeleton have been observed in humans

(Alveblom et al. 2003, Beard et al. 2000, Cho et al. 2011, Cote et al. 2006, Wallin

et al. 2005), in wild life (Lind et al. 2003, Lind et al. 2004, Roos et al. 2010,

Sonne et al. 2004) and in laboratory animals (Alvarez-Lloret et al. 2009, Andrews

1989, Andrews et al. 1989, Andrews et al. 1990, Herlin et al. 2010, Jämsä et al.

2001, Lind et al. 1999, Lind et al. 2000a, Lind et al. 2000b, Lind et al. 2004, Lind

et al. 2009, Lundberg et al. 2006, Miettinen et al. 2005, Nishimura et al. 2009).

Fig. 3. A schematic summary on skeletal toxicity of POPs constructed according to

average results of animal experiments reported. The left image presents cortical

effects and right trabecular. Green and grey colors present controls and exposed

animals respectively.

Maybe the most important report so far on skeletal toxicity of POPs in humans

has been the increased vertebral fracture incidence in women living in

contaminated areas (Alveblom et al. 2003). Later a few studies have associated

bone properties and POP burden (Beard et al. 2000, Cote et al. 2006, Eskenazi et

al. 2014, Glynn et al. 2000, Hodgson et al. 2008, Paunescu et al. 2013b, Rignell-

Hydbom et al. 2009, Wallin et al. 2005), however the statistical significance often

disappeared after normalization with possible confounding factors (Cote et al.

2006, Glynn et al. 2000, Rignell-Hydbom et al. 2009, Wallin et al. 2005).

HCB was the first POP shown to affect skeletal properties of adult laboratory

rats (Andrews et al. 1988, Andrews et al. 1989, Andrews et al. 1990). Later

similar effects have been observed in adult rats after exposure to A1254

(commercial PCB mixture) (Andrews 1989) and individual PCB congeners (Lind

et al. 1999, Lind et al. 2000a, Lind et al. 2000b, Lind et al. 2004). These studies

report a similar general pattern of decreased bone length, periosteal and endosteal

perimeter with decreased cortical bone area (Ct.A.), but increased cortical

thickness (Fig. 3) with consequently decreased bone strength.

31

Also the most toxic POP, TCDD, reduces bone size and strength (Jämsä et al.

2001). Somewhat surprisingly, TCDD can have some short term effects on

trabecular bone with reduced bone remodeling only 5 days after exposure to a

single high dose (Lind et al. 2009). A similar phenotype was present in cortical

bones of maternally exposed rat offspring exposed in utero / lactationally to very

low dose of TCDD including reduced BMD (Miettinen et al. 2005). The

amplitude of the effect was dependent on the AHR genotype of rats (Miettinen et

al. 2005).

In both TCDD and PCB exposed rats a mild decrease in trabecular bone

volume fraction has been observed histologically (Jämsä et al. 2001, Lind et al.

1999, Lind et al. 2004). On the other hand, when studied with pQCT increased

trabecular BMD has been reported with PCB153 (Lundberg et al. 2006) and

PCB126 (Alvarez-Lloret et al. 2009, Lind et al. 2000a), the later with decreased

trabecular area. The increased trabecular BMD has been observed in tibias from

American alligators (Alligator mississippiensis) (Lind et al. 2004).

Histologically PCB 126 has been shown to increase osteoid (Lind et al. 1999).

Interestingly overall collagen and hydroxyproline contents were decreased with

increased number of mature cross-links (Lind et al. 2000b). Compositionally this

is reflected as increased organic and inorganic contents, but reduced water content

(Lind et al. 1999) (Lind et al. 2000b). More recently PCB126 exposure in adult

rats has been reported to reduce matrix mineralization, crystal size and

crystallinity (Alvarez-Lloret et al. 2009). In acute TCDD toxicity decreased

monohydrogen phosphate levels have been reported indicating more mature bone

tissue (Lind et al. 2009).

2.7 Nuclear receptors and bone toxicity of POPs

POPs are considered to be endocrine disrupting chemicals and some of them have

been shown to be able to interfere with nuclear receptors. It has been shown that

dioxin-like chemicals can bind to AHR (Okey et al. 2005); Tributyltin with

retinoid-X-receptor and peroxisome proliferator activating receptor subtype

gamma (PPARγ) (Grun & Blumberg 2006); and PFOA only with PPARγ and

PPARα (Vanden Heuvel et al. 2006). Due the focus on mainly dioxin-like

compounds brief explanations of AHR, estrogen and vitamin D signaling will

follow focusing bone cell biology and skeletal phenotypes during the absence of

ligand or receptor.

32

2.7.1 Aryl Hydrocarbon receptor

The most studied group of POPs, dioxins mainly mediated their effects via AHR.

This member of the basic helix-loop-helix Per-Amt-Sim family of proteins binds

specifically the “classical” AHR ligands dioxin-like compounds, polyaromatic

hydrocarbons as well as a variety of structurally diverse “nonclassical” AHR

ligands, such as benzimidazole drugs (e.g. omeprazole), indoles, flavones and

midazoles. In addition to regulating xenobiotic metabolism and mediating toxic

effects AHR has ubiquitous physiological functions and is involved at least in

organogenesis, reproduction, neuronal differentiation, vascular development,

cardiac function, cell cycle control, immunity and regulation in circadian rhytms

(Pohjanvirta 2012).

As mentioned previously, the lipophilicity of these chemicals allows them to

penetrate cell membranes and in cytoplasm these chemicals can bind to AHR.

This causes release of receptor bound to hsp90 and other possible chaperons such

as p60s-src. Then the new complex travels to nucleus and binds with aryl

hydrocarbon nuclear translocator protein (ARNT) and starts reading the specific

regulatory aryl hydrocarbon response elements (AHRE), which is also referred as

dioxin-response element (DRE) (Lukinmaa et al. 2007, Okey et al. 2005, Okey

2007). Han/Wistar rats have been found to be a dioxin resistive rat line for their

point mutation in AHR (Pohjanvirta et al. 1998).

As a curiosity it could be mentioned, that human AHR is highly homologous

to guinea pigs, a species very sensitive to dioxin (Korkalainen et al. 2001) with

lethal dose 50% (LD50) of 0.6–2.1 μg/kg (compared to hamsters and male H/W

rats 3000 μg/kg). Considering varying ligand binding activity to the receptor

between species and the functional responses from altered hormonal activity to

changes in bone morphology after POP exposure are often non-monotonic (WHO

2013), translation of data is often difficult.

The AHR is also found at least in osteoblasts (Ilvesaro et al. 2005, Ryan et al.

2007) and osteoclasts (Ilvesaro et al. 2005, Korkalainen et al. 2009) further

indicating capability of dioxins to disturb well-orchestrated symphony of

osteoblasts and osteoclasts during the remodeling process. Indeed, it can be seen

in cell culture of calvarial osteoblasts that with normal cellular proliferation the

mineral nodule formation is decreased (Gierthy et al. 1994) with reduced levels of

alkaline phosphatase (ALP), collagen type I (Singh et al. 2000) OPN, Ocn and

bone sialoprotein (Gierthy et al. 1994, Koskela et al. 2012, Ryan et al. 2007,

Singh et al. 2000, Wejheden et al. 2006). Osteoblast function was possible to

33

rescue with AHR antagonists MNF (3'4 methoxynitroflavone) (Ryan et al. 2007)

or resveratrol (3,5,4'-trihydroxystilbene) (Singh et al. 2000). Furthermore TCDD

does not have any effects on osteoblasts from AHR knockout mice Ahr-/-

(Korkalainen et al. 2009).

The data on osteoclasts indicates disturbed osteoclastogenesis (Iqbal et al.

2013, Korkalainen et al. 2009), while evidence of weak activation of

differentiated osteoclasts exists (Ilvesaro et al. 2005). Also the genetically

modified mice with constitutively active AHR (CA-AHR) present a skeletal

phenotype with increased osteoclastic activity. In the same model collagen

synthesis was increased, while the ALP levels were decreased, all together

causing reduced cortical BMD on larger cross-section (Wejheden et al. 2010).

CA-AHR also caused reduction of trabecular area (with consequently decrease in

trabecular BMC) in females while it increased trabecular BMD in males.

2.7.2 Estrogen Receptors (ER)

Some of the skeletal effects of POPs (Kuiper et al. 1998) or AHR binding

chemicals (Ohtake et al. 2003) might be mediated by disrupted estrogen signaling.

The effects of estrogens (and androgens) on the skeleton have been well studied

and it is known that AHR signaling could interfere with ER signaling for which

the following four possible mechanisms have been suggested: (1) increased

metabolism of estradiol (E2), (2) reduced amount of ER, (3) suppressed E2

induced transcription and (4) competition of shared cofactors (Nilsson et al. 2001).

Estrogen affects bone remodeling by inducing osteoblastic production of Fas

ligand (FasL) (Krum et al. 2008) that activates apoptosis in osteoclasts

(Nakamura et al. 2007). Recently estrogen ablation has also been reported to

increase osteocytic apoptosis in ovariectomy (OVX) models (Emerton et al. 2010,

Huber et al. 2007). It is well known that OVX increases body mass (Banu et al.

2012, Tivesten et al. 2004), reduces trabecular bone fraction (Banu et al. 2012,

Maimoun et al. 2012, Tivesten et al. 2004) and cortical area and thickness (Banu

et al. 2012) with consequently bone strength reducing effect (Brennan et al. 2009,

Väänänen & Härkönen 1996). At bone matrix level, the lack of estrogen decreases

hardness of both cortical and trabecular compartments as well as elastic modulus

and energy dissipation in trabecular bone (Brennan et al. 2009, Maimoun et al.

2012).

These OVX induced skeletal effects can be compensated with hormone

replacement therapy by administrating selective estrogen receptor modulators

34

(Huber et al. 2007), bisphosphonates, parathyroid hormone (Brennan et al. 2009),

estradiol or estradiols and androgens (Moverare et al. 2003, Tivesten et al. 2004).

ER type α is the main mediator of estradiol effects on bone (Sims et al. 2003).

ERα has been shown to be important for skeletal maturation in both men (Smith

et al. 1994) and women (Quaynor et al. 2013). Increased trabecular bone volume

fraction and decreased cortical thickness has been reported in ERα null mice

(Callewaert et al. 2009, Lindberg et al. 2001, Lindberg et al. 2002, Sims et al.

2002). In males these effects to require orchidectomy to appear (Lindberg et al.

2001, Lindberg et al. 2002). In ERα-/- also increased serum leptin concentration

and body fat percentage has been observed (Lindberg et al. 2001). In ERβ-/- mice

increased cortical bone quantity has been observed in adult females, but the null

genotype is not enough to suppress the catabolic effects of OVX (Windahl et al.

1999) or aging (Ke et al. 2002).

2.7.3 Vitamin D Receptor

Vitamin D has been shown to have multiple health promoting effects from

reducing vascular calcification and osteoporosis in epidemiological studies. 1,25-

dihydroxyvitamin D3 (1,25(OH)2D3) is the active form of the Vitamin D. It

mainly stimulates calcium uptake in the intestine, but can activate bone resorption

and inhibit bone formation, when calcium levels are low (Lieben & Carmeliet

2013a). In bone it has similar effect as parathyroid hormone (PTH), i.e. increased

osteoclastogenesis and regulation of expression of osteoblastic genes. This effect

might be because PTH stimulates CYP27B1 production and thus activates

vitamin D signaling. This, on the other hand, stimulates fibroblast growth factor

23 production that acts as negative feedback for both PTH and CYP27B1 (Lieben

& Carmeliet 2013b).

It has been shown that increased 1,25(OH)2D3 can increase cortical porosity

and decrease bone formation and cortical thickness (Idelevich et al. 2011). On the

other hand, it has been also shown that alfacalcidol, a prodrug metabolized to

1,25(OH)2D3, could prevent bone loss in OVX model by inhibiting

osteoclastogenesis (Shibata et al. 2002).

In a vitamin D receptor (VDR) knock-out model the mice have been shown

to suffer from alopecia, hypercalcaemia, infertility and rickets with epiphyseal

widening and thinner cortex. In the same mice the number of osteoclasts was

similar with wild type mice, while osteoblasts were rather active, but unable to

mineralize (Yoshizawa et al. 1997). However, skeletal phenotype can be rescued

35

by restoring mineral homeostasis (Amling et al. 1999). Indeed recently it has been

shown, that ablation of VDR in the intestine can cause thinner cortex, increase

cortical porosity and lead to decreased trabecular bone volume fraction, while

osteocytic VDR-KO have relatively normal bone phenotype (Lieben et al. 2012)

and osteoblastic VDR-KO even increased bone mass (Yamamoto et al. 2013).

Interestingly, it has been shown that maternal dioxin exposure increases renal

CYP27B1 activity leading to increased active 1,25(OH)2D3 in serum with

decreased BMD, cortical thickness and expression of bone formation marker

genes with increased the amount of osteoid (Nishimura et al. 2009). On other

hand, adult exposure to dioxin-like PCB126 has been reported to decrease 25-OH

vitamin-D levels with altered bone mineralization (Alvarez-Lloret et al. 2009).

Early studies on POPs, have indeed shown elevated serum D3 together with PTH

and cholesterol after HCB exposure in adult rats as well (Andrews et al. 1988).

Two later reports showed increased bone density with decreased bone volume

(ostesclerotic changes) (Andrews et al. 1989) and increased bone strength,

cortical area with decreased medullar volume (Andrews et al. 1990). However,

later only changes in PTH levels were observed in HCB exposed rats (Andrews et

al. 1990).

36

37

3 Aims of the study

The main goal of this thesis is to understand the effects of POPs on bone strength

and morphology, and the role of AHR in bone toxicity and skeletal development.

In this thesis, bone was studied at multiple hierarchical levels to test the

hypothesis that POPs affect not only bone quantity, but also bone quality. This is

important for environmental health risk assessment. Four individual subworks

have been build around the following hypotheses:

1. Developmental TCDD exposure interferes with development of bone material

properties, which causes bones to be weaker.

2. Exposure to NCM, the mixture of chemicals present in the Canadian Arctic

(including heavy metals), decreases bone size and strength at dose levels

detected in the present arctic population.

3. PCB mixture A1254 causes skeletal changes and this response is proportional

to TCDD exposure estimated via chemical TEQ of A1254.

4. The AHR has a major role in TCDD induced bone toxicity and bone

homeostasis.

38

39

4 Materials and methods

4.1 Chemicals

Details of the chemical for studies I and IV as well as II and III have been

described elsewhere by Miettinen et al. (Miettinen et al. 2005) and Chu et al.

(Chu et al. 2008), respectively. Northern Contaminant Mixture (NCM) used in

study II was reconstructed according to the profile of chemicals found in 130

maternal blood samples of Canadian Arctic populations (Butler Walker et al. 2003,

Van Oostdam et al. 1999). It consisted of 14 PCB congeners, 12 organochlorine

pesticides and MeHg. Technical grade A1254 (AccuStandard, New Haven, CT)

was used in study III. Concentrations of individual PCB and PCDF congeners in

dosing solution and the corresponding concentrations of toxic equivalent (TEQ)

was estimated according to van den Berg et al. (Van den Berg et al. 2006).

4.2 Study permissions

Study protocols were reviewed and approved by either the Animal Experiment

Committee of the University of Kuopio (Kuopio, Finland) No. 03–04 in study I

and No. 05–42 for study IV, or the Canadian Council Animal Care guidelines and

Health Canada’s Institutional Animal Care Committee No. 01045 for studies II

and III.

4.3 Experimental designs

4.3.1 Maternal TCDD exposure

A single intragastric dose of 0.01; 0.03; 0.1; 0.3; and 1.0 µg TCDD/kg or corn oil

(vehicle) was administered to pregnant Sprague-Dawley rats at the previously

detected most sensitive time point on gestational day (GD) 11 (Miettinen et al.

2005). This is two days before the condensation of mesenchymal cells starts in the

rat skeleton (Hebel & Stromberg 1986). All the dose groups were used in study III

for dose modelling but only the highest dose was examined in study I. This

section was done based on observation that exposure of pregnant rats to 1.0 µg

TCDD/kg body weight (bw) is the smallest dose able to significantly decrease

bone geometry and mechanical strength without maternal toxicity (Miettinen et al.

40

2005). Also in study I only females were selected since they have been

demonstrated to be more sensitive to dioxin exposure than males. Litters were

weaned on postnatal day (PND) 28 and bones were collected on PND 35 and

PND 70. As the elimination half-life of TCDD in adult female Sprague-Dawley

rats is about 26 days (Li et al. 1995), the lactational exposure continued until

weaning, and TCDD is present in tissues of the offspring on PND 35 and to lesser

extent also on PND 70.

4.3.2 Maternal NCM exposure

In study II in order to evaluate the impact of perinatal NCM exposure on bone

development in rat offspring, pregnant Sprague-Dawley dams were administered

corn oil (as vehicle), or NCM at dose levels of 0.05, 0.5, or 5 mg/kg bw from the

GD 1 until weaning on PND 23 via Teddy Graham cookie (Nabisco Ltd., Toronto,

ON) as previously described (Chu et al. 2008). Animals were sacrificed PNDs 35,

77 and 350 following sample collections.

4.3.3 Maternal A1254 exposure

For study III pregnant Sprague-Dawley dam rats were administered corn oil (as

vehicle), or A1254 (15 mg/kg bw/day, equivalent to 0.123 µg TEQ/kg bw/day)

from the GD 1 until weaning on PND 23 via cookies and bone samples were

collected as in NCM study.

4.3.4 Role of AHR in skeletal development and TCDD toxicity

In study IV Ahr-/- and Ahr+/+ mice in a C57BL/6J background (Schmidt et al.

1996) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and

maintained using heterozygous breeding. At the start of the treatment the mice

were 8–12 weeks old, and randomized by body weight into treatment groups of 6

males and 6 females per genotype.

TCDD was mixed in corn oil using a magnetic stirrer and ultrasonicated for

20 min before administering by oral gavage at 4 ml/kg, whereas controls were

given pure corn oil. To rapidly achieve the kinetic steady state (0), the total dose

of 200 µg/kg was divided into one loading dose ( *0; x 40 µg/kg) and 9 maintenance

doses (x0;18 µg/kg), in weekly intervals (τ), which were calculated according

using first-order kinetics (equation 1) and assuming 100% bioavailability (Gibaldi

41

M. 1975). To calculate elimination constant (K) elimination half-life (t1/2) of 10

days was used (Birnbaum 1986).

*0 0

1

1 Kx x

e

(1)

12

ln 2 /K t (2)

At the end of the treatment period of 10 weeks (one week after the last

maintenance dose) the mice were anesthetized with CO2/O2 (70/30%). Blood

samples were drawn from the left ventricle using Venoject needles (Terumo) and

blood collection tubes, and the mice were killed by exsanguination.

Fig. 4. Estimated TCDD body burden over 10 week experimental period in the study IV.

Body burden is considered to follow first order kinetics with 100% bioavailability and

10 days elimination half-life.

4.4 Bone sample preparation

Bones were dissected and cleaned of soft tissue. Femoral and tibial lengths were

measured from distal point of condylus medialis to caput femoris or from medial

condyle to the medial malleolus, respectively using an electronic sliding caliper

(IP65, Sylvac SA, Crissier, Switzerland). Bones were then stored in Ringers

solution at –20 °C until analysis.

After mechanical testing, bones were air dried in room temperature for 24 h

before embedding in polyester resin (KLEER-SET type FF Resin and hardener kit,

MetPrep Ltd, Coventry, United Kindom) for nanoindentation measurements. A

metallurgical diamond saw was used to cut a 1mm thick slice from the tibial

0

10

20

30

40

50

1 8 15 22 29 36 43 50 57 64Estimated body burden 

(ug/kg)

Treatment days

42

cortical bone close to the fracture surface from mechanical testing (publication I

Figure 1 A.) in study I. In study IV two blocks were prepared. The first block was

prepared by cutting the whole block in two from the middle of diaphysis (about

50% of length). The proximal half was used for cortical bone characterization,

while to study trabecular bone an additional cut was made 15% of length

proximally from femoral condyles. All surfaces were prepared by grinding on

sand papers using water to cool and lubricate the sample. Grit size was

successively decreased and finalized by polishing on cloth with a small amount of

0.05 µm γ-alumina suspension. Before indenting bones were cleaned using an

ultrasonicator.

4.5 Peripheral Quantitative Computed Tomography (pQCT)

All bones were scanned using a pQCT system (Stratec XCT Research SA+ with

software version 5.50R, Norland Stratec Medizintechnik, GmbH, Birkenfeld,

Germany). System stability and calibration was performed with a phantom

provided by the manufacturer. Transverse tomographic slices were collected

perpendicular to the long axis of the bone with a voxel size of 0.07×0.07×0.5

mm3. A scan to represent cortical bone properties was collected at 50% of bone

length. Representative slice from metaphyseal bone was collected at 15% from

condyles.

The manufacturer’s software was used for numerical analysis. To analyze

cortical bone a density value of 710 mg/cm3 was used for thresholding. For

trabecular bone periosteal surface was segmented by thresholding images by 400

mg/cm3. Inside of the segmented bone, trabecular and subcortical bone were

separated at 200 mg/cm3, the lower pixel values representing trabecular bone.

4.6 Microtomography (μCT)

In µCT imaging recent guidelines for the assessment of bone microstructure in

rodents using micro–computed tomography were followed (Bouxsein et al. 2010).

The source voltage was set to 49 kV with 200 µA current in micro computed

tomography (µCT) device (SkyScan 1172, SkyScan, Kontich, Belgium). An

aluminium filter with thickness of 0.5 mm was used to reduce the beam hardening

effect and noise was reduced by using frame averaging of three frames. Tibias

were imaged wrapped in parafilm and vertebras were scanned in 1 ml PCR tubes

using respective isotropic voxel sizes of 4.7 µm and 9.9 µm.

43

Images were reconstructed in NRecon using ring artefact correction of 10 and

beam hardening of 40%. The cross-sectional datasets were analyzed with the

CTAn (version 1.12.0.0+) software provided with the device. Analysis of the

trabecular region of interest (ROI) in tibia was 0.19 mm (40 slices) from the distal

end of the growth plate towards the metaphysis, and extended 1.64 mm (350

slices). In vertebrae, ROI was 0.10 mm (10 slices) after distal growth plate and

extended over the whole medullar space until 0.10 mm (10 slices) to proximal

growth plate. The cortical ROI was 2.44 mm (520 slices) from the growth plate

and extended 0.94 mm (200 slices) further. ROI for trabecular bone was separated

from the cortical bone with a freehand drawing tool while a bitwise operation

ROI-shrinks wrap function was used for cortical bone analyses. Tissue mineral

density was calibrated against hydroxyapatite (HA) phantoms provided by the

manufacturer. A global threshold value was used to separate bone from the

surrounding matter. Thresholded images were despeckled and analyzed. Cortical

bone analyses were performed in three steps. First overall morphology was

analyzed with medullary area included in ROI. For cortical porosity and tissue

mineral density (TMD) analyses, thresholded image was selected as ROI using

bitwise operation and original images were reloaded for density analysis and

thresholded with appropriate threshold revealing pores.

4.7 Whole bone biomechanical testing

The mechanical behavior of bones were evaluated in three-point-bending tests for

femoral (studies II and III) and tibial diaphysis (studies I-IV) or in site specific

axial loading of the femoral neck (studies II and III) using a universal mechanical

testing machine (Instron 3366, Instron Corp., Norwood, MA, USA) as described

previously (Jämsä et al. 1998, Miettinen et al. 2005, Peng et al. 1994). In

diaphyseal tests bones were positioned horizontally with the anterior surface

upwards on the supports with a span length of 13 mm or 6.5 mm for rat and

mouse bone, respectively. For the testing of the femoral neck the proximal half

was placed in a plastic holder with anatomically shaped holes for femurs

changing diameter every 0.1 mm, and load was applied axially on the femoral

head using concave loading cup. In all experiments load was applied with

constant crosshead speed of a 0.155 mm/s until failure, and load-deformation

curves were recorded. Stiffness was calculated as a slope of the linear part of the

force-displacement data and maximal force, deformation and energy absorption

44

were defined. Additionally for study IV, yield parameters were defined from the

point where the fitted slope separated from the measured.

4.8 Nanoindentation of bone tissue

Material properties of bone extracellular matrix were analyzed with

nanoindentation testing using a Berkovich diamond tip, equipped in CSM-

NanoHardnessTester system controlled with Indentation v.3.75 software (CSM

Instruments SA; 2034 Peseux, Switzerland). Two different indentation protocols

were used denoted as quasistatic and dynamic loading modes. Indenter properties

have been presented in more detail earlier (Zioupos 2005, Zioupos & Rogers

2006). Creep and static material properties were analyzed from a quasistatic

loading protocol with 60 s or 15 s loading and unloading phase and 60 s or 30 s

hold phase at peak load of 10 mN for rat (study I) and mice (study IV) bone,

respectively. Viscoelastic properties were analyzed from dynamic loading

protocol consisting of 1.00 mN vibration amplitude at 1.5Hz superimposed on the

loading signal applied with 0.05 N/Ns until depth of 750 nm. The following

parameters were defined from every measurement: peak load Fmax, peak

displacement hm, contact depth hc, projected contact area (Ap) and contact stiffness

(S).

The Ap (equation 3) was defined from area function calibrations by using a

series of 110 indentations at different loads and was approximated by a

polynomial function for the contact depth (hc)

5 1

2

0

24.5n

p c n cn

A h C h

. (3)

Using this relationship as well as the Oliver Pharr method (Oliver & Pharr 1992)

to calculate S, where a power law fitted polynomial between 98% and 40% of

Fmax from unloading phase data was defined, well established parameters were

defined as in equation 4 for universal (instrumented) hardness (HIT, in GPa) and

in equation (5) indentation modulus (EIT) (Oliver & Pharr 1992). HIT was

calculated as peak force ratio to projected area (equation 4):

max

ITp c

FH

A h (4)

45

The reduced (effective) modulus (Er) was calculated according equation (6)

(Oliver & Pharr 1992). The reduced modulus was used to determine the

indentation modulus EIT as

2R

p

SE

A

. (5)

2

2111i

IT sr i

vE v

E E

(6)

where v is the Poisson ratio, i refers to indenter, and s to sample, respectively.

These values used were: vi = 0.07, vs = 0.3 and Ei = 1141 GPa.

The quasistatic protocol employed hold phase necessary for stabilizing

viscosity and presence of “nose” in unloading phase (Ebenstein & Pruitt 2006).

During the hold at maximal load the material creeps and this can be analyzed

from consequent deformation as following:

ln 1dh A Bth (7)

where h is the indentation depth (nm), t is the time (s) and A, B are constants

representing creep amplitude and initial creep rate, respectively. The initial 30 s of

hold phase time-deformation data was fitted to equation (7) (Chudoba & Richter

2001) using a numerical approximation code based on least square fitting in the

SigmaPlot for Windows (v.10) or Matlab (Mathworks) (10,000 iterations,

tolerance 0.0001, starting values A = 0.02; B = 1) (Zioupos & Rogers 2006).

The ‘plasticity index’ (Ip) was defined from the dynamic protocol. The plastic

response of bone was calculated from load penetration data according to Briscoe

et al. (Briscoe et al. 1998) using equation 8 for Ip. Total energy, a sum of energies

absorbed in plastic (ψp) and elastic (ψe) modes, was defined by numerical

integration under the loading trace. The ratio of the energies absorbed plastically

over the total energy input was calculated by:

pp

p e

I

(8)

The viscoelastic behaviour of bones was also analyzed from the dynamic protocol

because both storage (Estor) and loss modulus (Elos) can be calculated within each

cycle in the loading phase. We used the dynamic mechanical analysis (DMA)

option available in v.3.75 of the CSM software. The modulus values from DMA

46

for Estor and Eloss vary with the depth of indentation and came to some stable value

for penetration depths of 100nm (Zioupos 2005, Zioupos & Rogers 2006) or more.

In the study I, approximately seven indentations with both protocols were

performed on anterio-medial section of cortical bone as indicated in Fig. 5.

Fig. 5. Polished bone surface in nanoindentation insturment. White crosses indicate

locations for 16 indentations with quasistatic (1–8) and dynamic (9–16) protocols.

Scale bar shows 200 µm.

Approximately twelve indentations on anterior segment of cortical (Fig. 6) and

eight indentations on trabecular bone (Fig. 6) were performed with both dynamic

and quasistatic loading protocols. Indentations were performed on control and

dioxin exposed Ahr-/- and Ahr+/+ mice of both genders.

Fig. 6. Indentations positioned in A) anterior segment of femoral cortex and B) various

trabeculae over the transverse cross-sections. Specific care was taken to measure

individual trabeculae using both loading protocols. Scale bars shows 50 and 200 µm

in A and B, respectively.

A) B)

47

4.9 Serum markers for bone remodeling

Bone formation and resorption markers were measured in study IV from serum

samples by analyzing N-terminal propeptide of type I collagen levels, (PINP)

(Immunodiagnostic Systems, Boldon, UK) and C-terminal telopeptides of type I

collagen levels (CTX), RatLapsTM (Immunodiagnostic Systems, Boldon, UK) in

enzyme immunoassays following the manufacturers’ introductions.

Measurements were performed in duplicates for both markers. To estimate if bone

remodeling is in balance the PINP:CTX ratio was calculated (Chopin et al. 2008).

4.10 Osteogenesis PCR array

The mouse osteogenesis PCR array (The Mouse Osteogenesis RT2 Profiler PCR

Array, PAMM-026A, SABiosciences, a Qiagen Company) was performed using

pooled samples of three animals per group in study IV. Humeri were

homogenized using Ultra-Turrax T8 homogenizer, followed by RNA extraction

using Trizol (Life Technologies, Carlsbad, CA, USA) and RNeasy Mini kit

(Qiagen, Hilden, Germany). 1 µg of pooled RNA was generated into cDNA using

RT2 First Strand Kit (SABiosciences). cDNA samples were mixed with RT2 qPCR

Master Mix (SABiosciences) and distributed in every well on PCR array plate.

This array profiled the expression of 84 genes related to osteogenic differentiation

(skeletal development, bone mineral metabolism, cell growth and differentiation,

extracellular matrix proteins, cell adhesion proteins, transcription factors and

regulators). The array also contained controls for RT reaction and PCR reaction as

well as a genomic DNA control. Applied Biosystems 7000 Real-Time PCR

System (Applied Biosystems, Foster City, CA, USA) was used to determine Ct-

values of each well. The fold-changes in Ct-values were calculated using

SABiosciences web-based data analysis program.

4.11 Statistical analyses and dose response modelling

Statistical comparison between groups were conducted using SPSS Statistical

Software (SPSS Inc. Chicago, USA) (Study I ver. 16.0 Study II and III and Study

IV PASW18.0). Distributions of variables were studied using Kolmogorov

Smirnov (variations distributed equally around mean) test or by P-P plot and

boxplot methods. In studies II and III variables were log-transformed for

statistical comparison between groups or tested using the Mann-Whitney test. All

48

data were also tested with Levene test for homogeneity (deviations with equal

width). If homogeneity test failed for log-transformed data, Kruskal-Wallis one

way ANOVA rank sum test was used, followed by Mann-Whitney U test, with

compensation for multiple comparisons (p≤0.017) (studies II and III) or

assumption of equal variances for t-test was not used (studies I and IV). In cases

in which all requirements were fulfilled, groups were compared with Student’s t-

test (studies I, IV) or one-way analysis of variance (ANOVA) and Dunnett post

hoc test when ANOVA was significant. Data were evaluated in one-way or three-

way repeated measures ANOVA, if one of the variables had degrees of freedom

two or higher. Repeated measures ANOVA was performed to assess differences

over age, treatment, sex; and interactions “age x treatment”, “age x sex”,

“treatment x sex”, and “age x treatment x sex”.

Relationships between bone mineralization and nanomechanical parameters

were evaluated from Pearson’s correlations, and analysis of covariance

(ANCOVA) was performed with SigmaPlot for Windows (version 10) (SYSTAT

Software, Point Richmond, CA) to evaluate if these relations differ between

groups. SigmaPlot was also used for generating graphs in publication I. Graphs in

this thesis were made with GraphPad Prism version 5.00 (GraphPad Software,

San Diego, CA).

Finally PROAST (version 20.2) plugin in R-software (version 2.7) was used

in dose-response modelling. Dose-response relations were studied by fitting

mathematical models to data using the Benchmark Dose (BD) method (Slob 2002)

allowing more accurate estimation of toxic doses than traditional “no observed

adverse effect level”. A family of nested models was fitted to morphological and

mechanical variable after NCM and TCDD exposures in studies II and III,

respectively. The BD was read from a fitted model at 5% response level in

agreement with European Food Safety Authority’s (EFSA) guidelines for

continuous endpoints (European Food Safety Authority 2009). Modelling was

performed only for all age groups in study II but only for PND 35 in study III.

Both genders were implemented into models simultaneously. The likelihood ratio

test was used as a formal criteria to select one member from a family of nested

models including minimal number of fitted parameters, but still having the ratio

between BD and BD at the lower bound of the 95% confidence interval (BDL)

over 10.

In study III TCDD dose was read from the fitted dose-response curve at

measured effect size for A1254 exposure. These “TCDD equivalents” were

49

compared against chemically calculated toxic equivalent (TEQ) to test if bone

effects of A1254 are mediated by TCDD like compounds.

TEq = Σ (Ci * TEFi) (9)

These theoretical chemically calculated TCDD TEQs (equation 9) for A1254

were based on concentrations (C) of PCBs and PCDFs reported previously by

Rushneck et al. (Rushneck et al. 2004) and Kodavanti et al. (Kodavanti et al.

2001) and their toxic equivalency factor (TEF) (Van den Berg et al. 2006).

50

51

5 Results

5.1 Bone size

Developmental exposure to persistent organic pollutants decreased bone length

(studies I-III). Because no statistically significant gender-by-treatment or gender-

by-treatment-by-age interactions were detected for bone geometry or density data,

male and female offspring were combined for NCM and A1254. TCDD, NCM

and A1254 decreased tibial length by 4.2%, 5.2% and 7.4% in PND 35 rats,

respectively. However, only after TCDD exposure effects remained significant at

PND 70, while bone length was the same or close to controls in rats exposed to

NCM and A1254, although measured at PND 77. Similarly to previous reports in

TCDD, NCM and A1254 effects were not present after one year. On the other

hand, adult exposure to TCDD neither caused changes in tibial length nor

vertebral height or width (study IV).

5.2 Bone morphology and mineralization

5.2.1 Cortical bone

Besides reducing overall bone length, TCDD, NCM and A1254 reduced cross-

sectional cortical bone size. Cortical bone morphology was measured in 2D with

pQCT (studies I - III) and in 3D with µCT (study IV). TCDD, NCM and A1254

all decreased cross-sectional parameters at PND 35 (Fig. 7). A1254 decreased

cross sectional Ct.A. at most by 16% (p < 0.01), while both TCDD and NCM

decreased Ct.A. by 14% (p < 0.01 and p < 0.05, respectively). These changes

were due to decreased periosteal and endosteal circumferences by 7% and 5% for

TCDD; 7% and 7% for NCM; as well as 6% and 5% for A1254 respectively. This

was further seen as decreased cortical thickness (Ct.Th) by 8%, 7% and 14% for

TCDD, NCM and A1254, respectively. The cross-sectional data from pQCT

suggested decreased mechanical strength, with decreased polar moment of inertia

(J) (−27%) and moment of resistance −23%) for TCDD, and decreased J by 31%

and 29% and polar strength strain index by 22% and 23% for NCM and A1254,

respectively.

In developmental studies I-III, only TCDD caused changes in cross-sectional

bone properties at PND 70 (publication I, table I), while no significant treatment-

52

related changes were observed for these parameters at PND77 for NCM or A1254.

There were no detectable changes in any of the studies I-III at PND 350. However,

it should be pointed out that exposure had terminated at PND 28 for TCDD and

terminated PND 23 for NCM and A1254.

In study IV, pQCT derived diaphyseal parameters indicated statistically

significantly decreased Ct.Th in TCDD exposure in Ahr+/+ females as well as

reduced Ct.Th and Ct.A in Ahr-/- females.

Fig. 7. Representative cross-sectional images from PND 35 control, NCM (0.05, 0.5 and

5 mg/kg) and A1254 exposed male and female rats. A1254 caused visually detectable

changes, with similar magnitude compared to 5mg/kg of NCM. These results might be

due to low density areas at endosteal side of NCM exposed rats, which are not

included in the thresholded images.

53

High-resolution µCT showed also decreased Ct.Th in TCDD-treated Ahr+/+

females and control Ahr-/- females (Fig. 8) as well as reduction in Ct.Th cortical

thickness in males. Additionally, cortical volume was decreased in TCDD

exposed Ahr+/+ males and in females with AHR ablation. In TCDD exposed Ahr+/+

males the medullary volume was also smaller. Decreased size of anterior crest

(Fig. 8) and reduced eccentrity in TCDD exposed wild types and control

knockout females were seen.

Fig. 8. The upper four images present variations in cortical bone morphology and

density in tibias of female control and TCDD exposed AHR wild type (Ahr+/+) and

knockout (Ahr-/-). Lower four pictures are 3D representations of cortical pores

extrapolated from the upper pictures.

54

Study I showed observable difference in cortical porosity (Figure 1. in

supplementary material of publication I). A detailed analysis on cortical porosity

was performed on µCT data (study IV) as described in paragraph 4.6. These

results (Fig. 8) indicated that dioxin exposure increases cortical porosity (Ct.Po)

by 13% in TCDD exposed Ahr+/+ females mainly by increasing pore thickness

(Po.Th) of 32 µm in controls to 43 µm in TCDD exposed (p < 0.0001). Po.Th was

also increased by 16% at statistically significant level in control Ahr-/- females but

it was not sufficient to cause statistically significant changes in Ct.Po. However,

TCDD exposed Ahr-/- males had reduced number of pores (23%) with increased

spacing (4 µm) and decreasing Ct.Po by 23%.

It is possible to estimate bone mineralization level from both pQCT and µCT

data, where µCT produces estimation of tissue density (tissue mineral density -

TMD) while pQCT returns volumetric density (bone mineral density - BMD)

including porosity as well as overall content of mineral in bone (bone mineral

content - BMC).

In study II exposure to NCM did not cause any statistically significant

changes in cortical BMD (Ct.BMD). In study III A1254 decreased tibial Ct.BMD

by 3%. Consistent changes were neither observed at PND 77 nor at PND 350.

Both maternal and adulthood exposure to TCDD as well as AHR ablation

decreased Ct.BMD. In study I developmental exposure to TCDD caused

reduction in cortical BMC (Ct.BMC) being 16 % lower at PND 35 and 11 %

lower at PND 70 in the exposed animals compared to controls. Ct.BMD was

reduced by 0.9 % at PND 70 (p < 0.05). In study IV, TCDD exposed Ahr+/+ and

control Ahr-/- females Ct.BMD was decreased by 3% in both. However µCT

derived mineralization parameters showed a trend of increased Ct.TMD in TCDD

exposed Ahr+/+ (not statistically significant). In control Ahr-/- males, Ct.TMD was

decreased by 2% (p < 0.05), while it was increased by 4% (p < 0.005) after

TCDD administration to Ahr-/- males.

5.2.2 Trabecular bone

Trabecular bone mineral density (Trab.BMD) was decreased at the lowest dose of

NCM at PND 35 in males, while there was significant dose dependent increase in

Trab.BMD in males at PND 77 at doses of 0.5mg/kg and 5mg/kg (Fig. 9). An

even higher increase was detected in Trab.BMD in males exposed to A1254 at

this time point. Neither NCM nor A1254 altered trabecular bone parameters in

females at any studied time point.

55

Fig. 9. Summary of trabecular bone mineral density in females and males exposed

maternally to NCM or A1254. ANOVA indicated significant treatment effect in males at

PND 35 and PND 77. The post hoc test level for signicant intra-group difference are

indicated with * p < 0.05 and ** p < 0.005.

Adult exposure to TCDD in Ahr+/+ increased pQCT derived Trab.BMD in both

females and males by 54% and 28%, respectively. Also in AHR ablating females

Trab.BMD was increased by 16%.

Trabecular bone morphology was further studied from proximal tibiae and 4th

lumbar vertebrae (Fig. 10) using µCT. In tibia, TCDD exposure increased the

trabecular bone volume fraction (BV/TV) by 58% and 122%, respectively in both

male and female Ahr+/+ mice. This was mainly caused by increased trabecular

number (Tb.N) (by 56% and 113%, in males and females, respectively) and

decreased trabecular separation (Tb.Sp) (by 17% and 25%, respectively). The

trabecular pattern factor (Tb.Pf) was decreased (by 68% and 58%, respectively),

indicating higher connectivity of trabecular bone. A related parameter, structural

model index (SMI), was decreased (by 45% and 34% in males and females,

respectively), indicating more plate-like trabeculae rather than sphere-like

structure.

Similarly in vertebrae in both genders of Ahr+/+ mice, the BV/TV of lumbar

vertebrae was increased by TCDD-exposure (by 25% and 39%, respectively) (Fig.

56

10). Further, increased SMI and decreased Tb.Pf indicated again trabeculae in

TCDD-exposed mice to be more plate-like trabecular structure and connected in

both genders.

AHR ablation increased BV/TV (23% and 41%, in males and females

respectively) as compared to control Ahr+/+ mice. Again this was mainly due

increased Tb.N (22% and 48% in males and females, respectively), and decreased

Tb.Sp (by 11% and 21%, respectively). Increased SMI and decreased Tb.Pf

suggested again that the trabecular mesh has more plate like structures with

higher connectivity in female Ahr-/- mice.

A similar pattern of increased connectivity and more plate-like trabecular

morphology was observed in the vertebral trabecular bone of female Ahr-/- mice

compared to the Ahr+/+ mice, as indicated by decreased Tb.Pf (by 40%) and SMI

(by 26%). No differences in vertebral trabecular bone structure were seen

between male Ahr-/- and Ahr+/+ mice. TCDD exposure did not affect the tibial or

vertebral trabecular microstructure in Ahr-/- mice.

Fig. 10. A coronal view of vertebrae, where segmented trabecular bone is presented in

green in otherwise grey colored vertebral bodies of control and TCDD exposed Ahr+/+

and Ahr-/- mice. Upper pictures are females and lower males. There is evident increase

in BV/TV in TCDD exposed Ahr+/+ mice causing related changes in trabecular bone

morphology (plate like trabeculae which are more closely packed).

57

5.3 Mechanical properties

Table 2. Mean ± SD of the biomechanical properties in femoral diaphysis of Sprague-

Dawley offspring maternally exposed to NCM and A1254.

Dose Control 0.05 mg/kg 0.5 mg/kg 5 mg/kg A1254

37 days

N 17 17 18 20 16

k (N/mm)*** 57.5 ± 17.3 71.4 ± 18.8 68.4 ± 24.1 43.8 ± 10.9 49.9 ± 14.9

Dmax (µm) 715.9 ± 139.1 695.6 ± 226.5 660.2 ± 149.4 756.1 ± 144.1 781.5 ± 207.9

Fmax (N)*** 25.4 ± 4.6 29.6 ± 4.3 28.9 ± 6.7 21.9 ± 3.6 22.4 ± 5.2

Emax (mJ) 11.5 ± 3.1 13.6 ± 5.7 12.3 ± 5.0 10.7 ± 3.3 10.7 ± 3.6

Dy (µm) 257.0 ± 89.6 226.0 ± 61.2 257.2 ± 90.6 283.3 ± 95.0 287.6 ± 78.4

Fy (N)* 13.9 ± 4.6 16.3 ± 3.0 16.2 ± 4.6 13.1 ± 4.1 13.3 ± 3.7

Ey (mJ) 1.9 ± 1.1 1.9 ± 0.7 2.2 ± 1.4 2.0 ± 1.1 1.9 ± 0.8

77 days

N 18 18 19 20 20

k (N/mm) 348.8 ± 53.7 332.9 ± 73.9 372.2 ± 40.0 348.0 ± 62.2 351.2 ± 42.4

Dmax (µm) 642 ± 98.5 567.3 ± 236.9 656.3 ± 112.3 600.4 ± 169.2 674.4 ± 114.0

Fmax (N) 114.5 ± 14.3 123.3 ± 16.8 126.1 ± 15.0 114.2 ± 14.9 119.3 ± 17.2

Emax (mJ) 48.3 ± 9.3 54.4 ± 12.4 54.0 ± 14.2 49.5 ± 7.5 55.6 ± 15.6

Dy (µm)** 185.0 ± 35.6 314.0 ± 213.9ǁ 176.1 ± 31.7 232.5 ± 144.5 181.6 ± 31.2

Fy (N) 62.8 ± 12.7 78.5 ± 24.0 65.2 ± 10.5 67.1 ± 16.6 63.5 ± 7.6

Ey (mJ)* 6.0 ± 1.1 14.4 ± 14.3aǁ 5.9 ± 1.9 8.7 ± 8.3 5.8 ± 1.5

350 days

N 18 19 19 17 20

k (N/mm) 301.0 ± 47.8 374.0 ± 101.7 283.8 ± 54.2 345.4 ± 97.2 344.9 ± 82.9

Dmax (µm) 500.0 ± 148.7 556.7 ± 154.5 542.6 ± 161.9 550.0 ± 135.4 502.3 ± 144.6

Fmax (N) 113.7 ± 25.2 145.3 ± 48.2 111.7 ± 30.5 136.0 ± 44.8 132.3 ± 29.4†

Emax (mJ) 39.1 ± 24.8 55.1 ± 36.4 37.7 ± 20.8 49.4 ± 26.8 38.8 ± 18.5

Dy (µm) 265.2 ± 89.9 244.8 ± 41.1 263.3 ± 44.5 252.7 ± 46.8 271.4 ± 41.0

Fy (N) 76.5 ± 24.5 89.2 ± 23.1 73.6 ± 15.5 89.1 ± 30.5 89.3 ± 19.3

Ey (mJ) 11.7 ± 9.8 11.4 ± 3.4 10.2 ± 3.4 12.0 ± 5.5 12.5 ± 3.5

Treatment effects on stiffness (k) as well as yield (y) and maximal (max) force (Fy, Fmax), deformation (Dy,

Dmax), and energy (Ey, Emax). NCM treatment effect as indicated by MANOVA *p < 0.05, **p < 0.005,

*** p < 0.001 and with Tukey’s HSD post hoc test ǁ p < 0.05. A1254 treatment effect as indicated by

independent Student’s t-test †p < 0.05.

In maternally TCDD exposed rat offspring the tibial yield force was decreased by

28% and maximal bending force decreased by 16 % at PND 35. At PND 70

stiffness and maximal bending force were decreased by 14% and 20%,

respectively.

58

In NMC and A1254 exposed offspring both females and males responded

similarly as indicated by non-significant treatment-by-gender interaction, thus

biomechanical data from males and females were combined. At PND 35

MANOVA indicated significant treatment effect for stiffness and both maximal

and yield force (Table 2). However, group-wise comparison indicated that there

was no significant change between any of the dose groups compared to controls.

Later at PND 77 MANOVA indicated significant treatment effect for yield energy

and force. Post-hoc group-wise comparison showed that these parameters are

increased at the dose of 0.05 mg/kg (Table 2). At PND 350 none of the

biomechanical parameters showed treatment response for NCM. In the NCM

exposed offspring the maximum force and energy absorption of the femoral neck

were affected at every time point but group-wise comparison revealed significant

reduction only for maximum force at PND 35 with highest dose (data not shown).

Also for A1254 the MANOVA test did not show gender difference for

biomechanical properties and thus data was combined. Similarly to NCM,

stiffness and both maximal force and energy absorption of the femoral diaphysis

showed decreasing trend at PND35, but this reduction was not statistically

significant (Table 2). These parameters were not affected at PND 77 or PND 350

but rather overcompensated by age. For the femoral neck stiffness and both

maximum force and energy absorption were significantly decreased by 22%, 15%

and 24% respectively at PND 35 offspring. Again, no significant effects were

observed at PND 77 or PND 350 (data not shown).

5.4 Intrinsic matrix properties

As described previously, intrinsic matrix properties partly define overall

mechanical behavior of bone as well as provide cues to cell activation or

differentiation. Here we probed these properties using nanoindentation in

maternally TCDD exposed rats (study I) as well as in control and TCDD exposed

AHR knockout mice with corresponding wild type counterparts (study IV).

In study I, the bone material properties were not different between exposed

and control animals at PND 35 or PND 70. However, when we compared the

bone properties at PND 35 to those at PND 70 within groups significant

differences were seen. The normal maturation pattern in the control group showed

significant changes in the intrinsic properties measured from dynamic loading

protocol between PND 35 and PND 70, where the plasticity index Ip, decreased

by 5.3% (p < 0.05), while the complex modulus E*, storage modulus Estor and the

59

universal hardness HIT increased respectively by 12%, 13%, and 26%. In the

TCDD-exposed animals, there were no significant changes in these parameters of

the bone maturation process between PND 35 and PND 70 and especially

plasticity remained almost the same at PND 35 and PND 70.

As it is known that mineral fraction is related to intrinsic properties (Oyen et

al. 2008, Zioupos 2005), we investigated the relationship between

nanomechanical data and the mineral content in controls and TCDD exposed

animals separately. There was significant increase between both indentation

hardness and complex modulus and mineral content, while plasticity decreased,

and this was the case in both controls and TCDD bones. Analysis of covariance

(ANCOVA) indicated that the slopes for control and TCDD exposed animals were

not different between EStor, HIT, and Ip vs. BMC (F1,26(EStor) = 0.014;

F1,26(HIT) = 1.071; F1,26(Ip) = 1.371).

As these parameters are novel, and how they should be measured and

analyzed is still under debate, we examined the interrelationships between the

various nanomechanical parameters. There were significant correlations between

the plasticity index IP and overall creep strain amplitude A (R = 0.58, p < 0.001),

early rate creep strain B (R = −0.53, p < 0.01), and hardness value HIT (R = 0.74,

p < 0.001).

In study IV, TCDD exposure in male Ahr+/+ mice increased the dynamic

hardness of cortical bone (by 9%) and the static hardness, as well as both static

and dynamic elastic modulus, of trabecular bone (by 12%, 13% and 28%,

respectively), while the creep amplitude of trabecular bone was decreased (by

10%). Furthermore increased static hardness (by 9%), as well as both static and

dynamic elastic modulus (8% and 7%) of cortical bone were observed in female

Ahr+/+ mice after TCDD exposure.

AHR ablation lead to increased (5%) static hardness of cortical bone in

unexposed (controls) males, while the plasticity index was 2% decreased

(p < 0.05). The trabecular bone matrix properties did not differ between the

genotypes in males. In female Ahr-/- mice, the trabecular bone had decreased static

and dynamic elastic modulus (by 14% and 10%, respectively) as compared to

wild type counterparts, while females did not show any differences in cortical

bone matrix properties. TCDD exposure did not affect cortical or trabecular bone

matrix properties of Ahr-/- mice.

60

5.5 Serum markers

Serum markers were evaluated in study IV. TCDD exposure decreased the

PINP/CTX ratio by 43% (p < 0.001) in Ahr+/+ female mice, while in males this

was present only as a trend. AHR ablation increased bone resorption as indicated

by increased levels of CTX (by 60% (p < 0.05) in female Ahr-/- mice compared to

corresponding Ahr+/+ mice, while the PINP/CTX ratio did not differ between the

genotypes. TCDD treatment neither affected the PINP nor CTX levels, nor the

ratio, in either gender Ahr-/- mice.

Fig. 11. Bone remodeling balance as PINP/CTX ratio for controls and TCDD exposed

for wild type (WT) and knockout (KO) females (white) and males (grey). *** p < 0.05.

5.6 Gene expression

Of the 84 osteogenesis-related genes for which the expression was analyzed, 20

genes were altered (fold change ≥2) by TCDD-exposure of Ahr+/+ mice. Only

three of these were up-regulated and they were associated with skeletal

development (TnF) and extracellular matrix proteins (Itgam, Mmp8). Remaining

17 genes were down-regulated and associated with bone mineral metabolism

(Bmp5, Cdh11, Col4a2), skeletal development (Bmp2, Bmp5, Dmp1, Fgfr2, Phex,

Sost1, Vdr, Ahsg), extracellular matrix proteins (Cstk, Dmp1, Fgfr2, Mmp2, Sost1,

Tgfb2), cell growth and differentiation (Fgf1), transcription factors and regulators

(Msx1) and cell adhesion molecules (Col12a1).

WT:C

TRL

WT:T

CDD

KO:CTRL

KO:TCDD

WT:C

TRL

WT:T

CDD

KO:CTRL

KO:TCDD

0.0

0.5

1.0

1.5

2.0

2.5

***

PIN

P/C

TX

61

Control AHR ablated mice differed for two genes as compared with wild type

controls. One up-regulated was associated with extracellular matrix proteins and

down-regulated with skeletal development (Ahsg). The down-regulated gene was

lower also both in TCDD-exposed Ahr+/+ and Ahr-/- mice.

For Ahr-/- mice, TCDD-exposure altered the expression of only five genes.

Three of these were up-regulated and associated with skeletal development

(Tfip11, Col2a1), bone mineral metabolism (Col2a1) and extracellular matrix

proteins (Col10a1), while the two down-regulated genes were associated with

skeletal development (Ahsg) and with cell growth and differentiation (Vegfb).

5.7 Dose modelling

For NCM the pQCT and biomechanical variable with highest mean square values

got the lowest BDs indicating highest sensitivity. At PND 35 the 5% decrease was

estimated to 0.8 and 1.2 mg/kg bw/d for maximal breaking force and stiffness of

femoral diaphysis, while for the femoral the neck maximal breaking force

corresponding dose was 1.4 mg/kg bw/d. Also the strength estimates from pQCT

got low BD values being 0.9 and 1.1 mg/kg bw/d for polar moment of inertia and

polar strength strain index. Interestingly, the tibial length and morphological

values had higher BD estimates being 3.2, 1.6, 2.2 and 4.8 mg/kg bw/d for tibial

length, cortical area, cortical thickness and periosteal circumference, respectively.

All BD estimates had BD/BDL within the well tolerable range of 1.2–1.5. It is

possible to extract also maximal response from BD analyses. Generally maximal

response decrease with age (publication II, table 2B), while BD values first

decreased and then increased at PND 77 and PND 350 respectively, while the

BD/BDL were highest at PND 77 compared to other age points.

The TEQ for the used A1254 was 0.123 µg/kg bw/d with major contributions

from dioxin-like PCB congeners 126, 118 and 105 by 46%, 29%, and 12%,

respectively. Thus in this experiment the total dose of A1254 was estimated to

5.289 µg/kg bw. When the measured responses of the various bone parameters to

this dose of A1243 were compared against dose models of classical TCDD

exposure, the equivalents settled between 0.83–1.22µg/kg bw giving a range of

16–23% against the theoretical TEQ (publication III, table 4).

62

63

6 Discussion

The main goal of this study was to better understand the effects of POPs in bone

strength and morphology and the role of AHR in bone toxicity and skeletal

development at multiple hierarchical levels, thus bringing new mechanistic data

for environmental health risk assessment. This is crucial since according to the

WHO (WHO 2000) developmental defects are the most sensitive toxic endpoint

of dioxin-like compounds, and at current exposure levels the safety margin for

these effects is very narrow and simultaneous exposure to a wide variety of

different chemicals emphasize the potential significance of mixture effects (e.g.

additivity or synergism) that are dismissed when studying single chemicals

(WHO 2013).

6.1 Novel methods in bone toxicology

Part of the research novelty arises in applying recent biomedical engineering

methodologies in such an exciting field as environmental toxicology. The added

scientific value is visually clear when comparing pQCT and µCT images to each

other of the same sample Fig. 12.

Although pQCT provides a better picture of the skeletal changes than DXA

and captures some key features as with µCT (total area, cortical area) (Schmidt et

al. 2003) the method has problems separating the endosteal border of bone thus

leading to potential underestimation of BMD with increased trabecular bone

volume fraction and medullary area. Also the cortical porosity is mixed in cortical

BMD due to partial volume effects, while µCT can provide separate estimates of

TMD and cortical porosity.

Where traditional mechanical testing can be used to quantify mechanical

properties of whole bone, nanoindentation characterizes the bone material

properties, describing the bone matrix quality. These qualitative properties

contribute to overall bone strength and can make bones brittle, harder and stiffer.

It was noticed that these properties are inter-related and appear to follow BMC.

Unfortunately by the time of study I, we did not have access to µCT equipment,

and we cannot compare TMD in that study.

64

Fig. 12. Measurement positions indicated in scout view for both cortical and

trabecular bone for pQCT (two yellow dotted lines) and µCT (between two dotted black

lines). The µCT imaging provides a more complete picture of changes based on

morphological information from longer length that can be used for 3D analyses and

visualization.

Even if we utilized latest technologies to show effects of TCDD on intrinsic

properties of extracellular bone matrix and true 3D volumetric analysis of

trabecular bone and cortical porosity, there are still some improvements that

should be implemented in future studies. The technical limitation in our

nanoindentation studies was the use of dried samples, which has been often the

case (Lewis & Nyman 2008), partly due to equipment requirements, partly due to

the practical difficulty in preparing moist bone specimens without embedding,

especially from animals as small as mice. However, it has been shown, that

strontium ranelate induced improvements in material properties were more

pronounced in samples stored in a physiological solution as compared to dried

samples, where the effect disappeared (Ammann et al. 2007). Thus, it is tempting

to speculate that the effects observed in this study are more likely an

underestimation of real effects that should be probed in physiological conditions.

65

Further, the porosity analyses here were performed from images with

resolution of 5 µm, although it has recently been suggested that a resolution of as

low as 2 µm should be used for murine vascular structures (Palacio-Mancheno et

al. 2014). The same study demonstrated that osteocyte lacunas could be

quantified and visualized at resolutions of 1µm or better.

6.2 Model compound and genetic model studies

Study I showed delayed maturation after maternal TCDD exposure. More

specifically, bone matrix became more ductile, softer and less able to store energy

than control bones. Based on the correlation between intrinsic material properties

and mineralization parameters we suspect these changes are mainly due to

decreased mineralization. However, the role of the organic part cannot be

excluded since these changes are also supported by notification of previously

reported decrease in water and collagen content (Lind et al. 2000b). In this case

the increased mature cross-links could not provide improved strength and the role

of hydroxyproline in bone strength is still unknown. On the other hand, these

results are also supported by previously reported increased amount of osteioid

(Lind et al. 1999), while the time course is surprising. In humans ERα

inactivation has been shown to delay bone maturation (Quaynor et al. 2013,

Smith et al. 1994).

The toxic effects of TCDD and other dioxin-like compounds are mostly

mediated through AHR signaling. Thus, the role of AHR was studied in normal

skeletal development and dioxin toxicity at various hierarchical levels. Bone

remodeling was increased in Ahr-/- mice and they presented a skeletal phenotype

with softer and more elastic bones. Interestingly, also the bones of CA-AHR mice

were less stiff and more bendable, which most likely reflects to material level

alterations as they had also increased periosteal circumference (Wejheden et al.

2010). Ahr-/- also showed increased trabecular bone volume fraction and the

number of trabeculae were higher in these mice, similar to another recent report

(Iqbal et al. 2013). The previous studies have indicated reduced osteogenesis by

RANK-L, BaP (Iqbal et al. 2013) and less active osteoblastic cells (Korkalainen

et al. 2009, Ryan et al. 2007) in absence of AHR in vitro. However, in our Ahr-/-

mice high serum CTX indicated increased osteoclastic resorption, which might

increase osteoblastic activity through coupling. Furthermore, dioxin

administration had hardly any effects on the bone properties of the Ahr-/- mice.

66

In Ahr+/+ mice, dioxin exposure caused increased cortical porosity and a

strong anabolic effect on trabecular bone formation. Here porosity analysis

consisted mainly of blood vessels (Haversian and Volkman channels), but might

include some collagen fibers from tendon attachment at lateral side. However,

still it would be logical that osteoclastic resorption would take place in close

proximity to blood vessels and with reduced bone formation the filling of cavity

would not be complete. These effects appeared only in females. Additionally,

females showed reduced maximal and yield strengths. Reduced yield strength

indicates increased microfracture susceptibility and is probably due the increased

hardness and reduced elasticity of tissue. Generally these results indicate early

onset of skeletal senescence further supported by an observed reduced remodeling

ratio.

Similarly to TCDD exposed Ahr+/+ mice, increases in trabecular bone volume

fraction and decreased cortical thickness have been reported in ERα-/- mice

(Lindberg et al. 2001, Lindberg et al. 2002, Sims et al. 2002). Furthermore there

is evidence that AHR activation could cause ERα (Beischlag & Perdew 2005) and

ERβ (Ruegg et al. 2008) transrepression supporting the view that phenotype

could be due ER inactivation (but not antiestrogenic). However, it should be

pointed out that there are other animal models with similar skeletal phenotype e.g.

IL11α1-/- and IL6-/- (Sims et al. 2005). In PCB126 exposed adult female rats an

increase in expression of ER mRNAs has been reported with no effect on the ER

content (Lind et al. 2004).

6.3 Towards studies with naturally occurring POP mixtures

As most toxicological studies have been previously based on exposure to model

compounds, such as TCDD and individual PCB congeners, here we studied two

mixtures that resemble more of the existing POP body burden of human

populations (Van Oostdam et al. 2005). The used mixtures NCM and A1254

included 28 and 21 separate chemicals, respectively, while they did not include

the most potent PCDD/Fs. Still used mixtures appeared to have skeletal toxicity

compatible with the profile of bone changes elicited by perinatal TCDD and / or

PCB exposure described in chapter Table 1. Also the both mixtures increased

Trab.BMD later at PND 77.

Effects of A1254 were somewhat lower than what could be estimated via

TEQ estimation based on the assumption of additivity of effects of individual

chemicals. Recently it has been suggested that various types of POPs might have

67

even synergistic effects (Koskela et al. 2012). This will set a huge challenge for

chemical risk assessment, since there are over 800 chemicals that could

potentially disturb the endocrine system (WHO 2013) and thus the skeletal

system.

In the population of the Canadian Arctic, 2- to 10-fold higher levels of

environmental contaminants have been shown than in general populations living

in regions further south (Van Oostdam et al. 1999). Recently it has been shown

that POP intake in these communities exceeds the Canadian provisional tolerable

daily intake, and in one of these communities, 7% of young women exceed the

Health Canada tolerable serum level of 5 μg/L total PCB as A1254 (AMAP 2009).

However, in population based studies the correlation between skeletal properties

and POP burden has been mild (Paunescu et al. 2013b) or absent (Paunescu et al.

2013a) in both Canada and Greenland (Cote et al. 2006).

6.4 Translation of findings to human health

There have been a few known major chemical accidents or incidents that have

heavily increased POP exposure of affected populations. In Turkey the use of

HCB for treating grain in 1955–1959 resulted in HCB-exposure via contaminated

food and caused a porphyria cutanea tarda epidemic in the affected population

(Peters et al. 1987). Various skeletal manifestations have been observed within

this population including arthritis and osteomyelitis soon after exposure (Dean

1961), small hands, peripheral arthritis with osteoporosis and joint space

narrowing even decades after exposure (Gocmen et al. 1989, Peters et al. 1987).

Later in 1968 in northern Japan, ingestion of PCB and PCDF contaminated

rice oil (Yusho) lead to fetal PCB syndrome including abnormal calcification of

skull and feet malformation with dark pigmentation, gingival hyperplasia and

exophthalmic edematous eyes (Yamashita & Hayashi 1985). A recent study

showed a lack of association between serum PCB, PCQ, PCDF and BMD and

CTX (Yoshimura et al. 2009). However, there has been about 120-fold decrease

in serum TEQ between 1969 and 2007 (Masuda 2009).

Finally, a major incident took place in Seveso, Italy on 10th of July 1976,

where an explosion in a chemical factory resulted in a release of up to 30 kg of

TCDD, spread over the surrounding 18 km2 area. It has been recently reported

that DXA bone scans did not reveal adverse effects on bone health 32 years later

in women who were < 20 years old during the explosion (Eskenazi et al. 2014).

68

There are reports showing relationships between exposure to various POPs

and altered skeletal parameters. DDE (Beard et al. 2000) and PCB28 have been

associated with decreased skeletal mass, while PCB28 and γ-HCH with increased

mass (Glynn et al. 2000). Often, when adjusting with confounding factors the

effect dilutes and is no longer statistically significant. Thus the skeletal toxicity of

POPs has been mild or difficult to detect.

However, the socioeconomic burden of reduced bone strength arises from the

fractures rather than osteoporosis per se. Increased fracture incidence was found

in fishermen and their wives exposed to POP through dietary intake of fatty fish

from the Baltic sea (Alveblom et al. 2003). This causality was not present in a

questionnaire based study on the following year (Wallin et al. 2004). Thus the

attempts to correlate clinical findings with POP exposure levels for predicting

fracture risk are necessary. DXA based BMD has been the gold standard for

fracture risk assessment, but evidence shows that BMD is not a sufficient tool to

indicate patients at risk of fracture (Schuit et al. 2004, Stone et al. 2003). Also the

currently used ultrasound parameters mainly reflect the overall bulk properties

(Gluer 2007). Furthermore, the animal models in these studies present often with

weaker and smaller bones, however with increased trabecular bone fraction. This

indicates that POPs decrease bone strength, but in such a way that they are

difficult to diagnose due to increased amount of trabecular bone inside cortical

bone with poorer quality and smaller dimensions. For these reasons it is not

surprising that in some studies no statistically significant associations between

BMD/ultrasound and POP were found (Beard et al. 2000, Cote et al. 2006,

Eskenazi et al. 2014, Glynn et al. 2000, Hodgson et al. 2008, Paunescu et al.

2013a, Paunescu et al. 2013b, Rignell-Hydbom et al. 2009, Wallin et al. 2005).

Therefore in future more advanced diagnostic tools such as high resolution pQCT

should be used to study skeletal properties in POP exposed populations to

elucidate fracture risk in adult population. Some studies showed weak association

with POP burden and skeletal properties but they disappeared after various

adjustments for all possible confounding factors (Cote et al. 2006, Glynn et al.

2000, Rignell-Hydbom et al. 2009, Wallin et al. 2005). Age alone is such a strong

predictor of skeletal properties. In our NCM study the age explained about 200

times higher correlation to radiological and biomechanical parameters compared

to treatment. However, it should be realized that the treatment was relatively short

as NMC exposure was stopped at PND 23, whereas the whole study period

extended over 360 days.

69

However, it should be pointed out that the skeletal manifestations were

dependent on exposure timing. In adults TCDD exposure leads to decreased

diaphyseal bone size, increased porosity and brittleness of tissue. The maternal

exposure, on the other hand, delayed maturation of bone tissue increasing a risk

of green stick fractures. This implies that early POP exposure might increase bone

ductility and later brittleness. Interestingly, in all studies increased trabecular bone

quantity was observed. Nevertheless, in all cases POP exposure decreased bone

strength and it would be surprising if this would not be the case in humans as well.

70

71

7 Conclusions

The main goal of the thesis was to understand the effects of POPs on bone

strength and morphology, and the role of AHR in bone toxicity and skeletal

development. Based on the results of this study, it can be concluded that;

1. Developmental TCDD does not alter bone matrix properties at the level that

they would contribute to decreased bone strength. However, TCDD exposure

was shown to delay bone matrix maturation.

2. Exposure to the NCM decreases bone size and strength at relevant but

relatively high dose levels. NCM was observed to increase Trab.BMD.

3. PCB mixture A1254 causes similar changes as TCDD exposure leading to

shorter, thinner and weaker bones in juvenile rats. Again as in the NCM study

Trab.BMD was observed to be increased in adult animals after discontinued

exposure.

4. The AHR receptor has a major role in TCDD induced bone toxicity and bone

homeostasis. A new insight was gained for cortical porosity and tissue

senescence as well as increased trabecular bone quantity.

5. In general the opposite effects on trabecular and cortical bone compartments

demonstrate a need for a new view when analyzing radiological data from

humans.

72

73

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Original publications

I Finnilä MA, Zioupos P, Herlin M, Miettinen HM, Simanainen U, Håkansson H, Tuukkanen J, Viluksela M & Jämsä T (2010) Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on bone material properties. J Biomech 43: 1097–1103.

II Elabbas LE, Finnilä MA, Herlin M, Stern N, Trossvik C, Bowers WJ, Nakai J, Tuukkanen J, Heimeier RA, Åkesson A & Håkansson H (2011) Perinatal exposure to environmental contaminants detected in Canadian Arctic human populations changes bone geometry and biomechanical properties in rat offspring. J Toxicol Environ Health A 74: 1304–1318.

III Elabbas LE, Herlin M, Finnilä MA, Rendel F, Stern N, Trossvik C, Bowers WJ, Nakai J, Tuukkanen J, Viluksela M, Heimeier RA, Akesson A & Håkansson H (2011) In utero and lactational exposure to Aroclor 1254 affects bone geometry, mineral density and biomechanical properties of rat offspring. Toxicol Lett 207: 82–88.

IV Herlin M, Finnilä MAJ, Zioupos P, Aula A, Risteli J, Miettinen HM, Jämsä T, Korkalainen M, Tuukkanen J, Håkansson H & Viluksela M (2013) New insights to the role of aryl hydrocarbon receptor in bone phenotype and in dioxin-induced modulation of bone microarchitecture and material properties. Toxicol Appl Pharmacol 273(1): 219–226.

Reprinted with permission from Elsevier (I, III, IV) and Taylor & Francis (II).

Original publications are not included in the electronic version of the dissertation.

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1252. Jämsä, Ulla (2014) Kuntoutuksen muutosagentit : Tutkimus työelämälähtöisestäoppimisesta ylemmässä ammattikorkeakoulutuksessa

1253. Kaikkonen, Leena (2014) p38 mitogen-activated protein kinase and transcriptionfactor GATA-4 in the regulation of cardiomyocyte function

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UNIVERSITY OF OULU P .O. B 00 F I -90014 UNIVERSITY OF OULU FINLAND

A C T A U N I V E R S I T A T I S O U L U E N S I S

S E R I E S E D I T O R S

SCIENTIAE RERUM NATURALIUM

HUMANIORA

TECHNICA

MEDICA

SCIENTIAE RERUM SOCIALIUM

SCRIPTA ACADEMICA

OECONOMICA

EDITOR IN CHIEF

PUBLICATIONS EDITOR

Professor Esa Hohtola

University Lecturer Santeri Palviainen

Postdoctoral research fellow Sanna Taskila

Professor Olli Vuolteenaho

University Lecturer Veli-Matti Ulvinen

Director Sinikka Eskelinen

Professor Jari Juga

Professor Olli Vuolteenaho

Publications Editor Kirsti Nurkkala

ISBN 978-952-62-0508-3 (Paperback)ISBN 978-952-62-0509-0 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)

U N I V E R S I TAT I S O U L U E N S I S

MEDICA

ACTAD

D 1254

ACTA

Mikko A

. J. Finnilä

OULU 2014

D 1254

Mikko A. J. Finnilä

BONE TOXICITY OF PERSISTENT ORGANIC POLLUTANTS

UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU, FACULTY OF MEDICINE,INSTITUTE OF BIOMEDICINE,DEPARTMENT OF ANATOMY AND CELL BIOLOGY;DEPARTMENT OF MEDICAL TECHNOLOGY;NATIONAL INSTITUTE FOR HEALTH AND WELFARE,DEPARTMENT OF ENVIRONMENTAL HEALTH;UNIVERSITY OF EASTERN FINLAND, FACULTY OF SCIENCE AND FORESTRY,DEPARTMENT OF ENVIRONMENTAL SCIENCE;CRANFIELD UNIVERSITY, CRANFIELD DEFENCE AND SECURITY,CRANFIELD FORENSIC INSTITUTE