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«FACULTY OF HEALTH SCIENCES PH.D. THESIS EFFECTS OF WOOD DUST: Inflammation, Genotoxicity and Cancer. JETTE BORNHOLDT LANGE SUBMITTED APRIL 29th 2008 ...»

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NATIONAL RESEARCH CENTRE FOR

THE WORKING ENVIRONMENT

FACULTY OF HEALTH SCIENCES

PH.D. THESIS

EFFECTS OF WOOD DUST:

Inflammation, Genotoxicity and Cancer.

JETTE BORNHOLDT LANGE

SUBMITTED APRIL 29th 2008

PREFACE

This thesis is submitted to the Faculty of Health Science at the University of Copenhagen, Denmark in order to meet the requirements for obtaining the Ph.D.-degree. The work was carried out at the National Institute of Occupational Health, Lersø Park Allé 105, Copenhagen, Denmark. During my Ph.D. studies I received supervision from Professor Håkan Wallin, Associated Professor Ulla Vogel and Professor Steffen Loft. The project was funded by the National Institute of Occupational Health and the EU 5th Framework Programme, Key Action 4, Environment and Health, Quality of Life and Management of Living Resources, project no.

QLRT-2000-00573.

First at all, I would like to express my gratitude to Håkan Wallin and Ulla Vogel for their excellent supervision during my studies and for their enthusiasm and daily guidance. I would also like to thank my supervisor, Steffen Loft at University of Copenhagen for his support and interest. Also very special thanks to the rest of the WOOD RISK collaboration who made this project possible by their enthusiasm and endless patience with me. Thanks to the pathology departments at hospitals all over Denmark for taking time out of their busy schedule to help me with the collection of tumour blocks and for lending me this priceless material. Especially thanks to Specialechef. Dr.Med. Torben Steiniche and Dr. Med. Annemarie Antonsen for your priceless help in selecting the tumour blocks and for making sure that I was not lost forever in the pathology archives.

During my project I have had the opportunity of working in a dynamic laboratory, surrounded by wondeful persons. Whom I would like to thanks for a lot of things; from their enlightening spirit on grey cold mornings to always being helpful even on a late afternoon. Especially Lourdes Petersen for technical assistance in the sequencing of the mutations. Most of all I would like to thank my dear office-mate Anne Saber for enlightening conversations, priceless teamwork during the cell exposures and her always pleasant spirit. I really enjoyed sharing our 8 m2.

Last, but not least, I would like to thank my family for being there for me or not being there in the final stage of this thesis and a very special thanks to my mother for taking care of my children.

Copenhagen, April 2008 Jette Bornholdt Lange SUMMARY The present thesis focuses on the inflammatory, genotoxic and carcinogenic effects of exposure to different species of wood dust. The experimental work performed in this thesis consists of two parts. The first part of the study was carried out in an in vitro model with the human lung epithelial cell line A549 measuring inflammatory and DNAdamaging effects. The second part consists of a molecular analysis of the K-ras gene for mutations in the hotspots codons in human sinonasal cancers. Design, calibration and validation of the assays were performed.

Cancer at the sinonasal cavities is rare with incidence rates between of 0.3 to 1.4 per 100,000 for men and 0.1 to 0.8 per 100,000 for women in Europe, depending on country. However, cancer at this site is associated with occupational exposures including wood dust. Especially the adenocarcinoma subtype is strongly associated with exposure to wood dust primarily from hard woods. Non-malignant symptoms like allergy, asthma, rhinitis and chronic bronchitis have also been associated with occupational exposure to wood dust in epidemiological studies. In most epidemiological studies hardwoods (e.g. oak and beech wood dust) seems to have greater association to both the malignant and non-malignant symptoms compared to softwoods (e.g. pine and spruce wood dust). Since, well standardized experiments of wood dust exposure are limited and difficult to execute in humans, knowledge of the cellular mechanisms underlying wood dust induced carcinogenicity and non-malignant symptoms are still poorly understood. Particulate induced inflammation as well as extractives are suggested to be involved in the carcinogenesis.

In this thesis wood dust potential to induce DNA damage and inflammation was investigated exposing the human lung epithelial cell line A549 to various species of wood dust and endpoints for inflammation and genotoxicity was evaluated. The experiments showed that the different species of wood dust vary in their ability to cause DNA strand breaks and inflammation. There was no apparent correlation between the species potential to initiate inflammation and their potential to cause DNA damage. Contrary to our hypothesis, we showed that pure wood dust is able to cause primary DNA damage, independent of inflammation as well as hardwoods had no higher inflammatory or genotoxic potential than softwoods.

To investigate the molecular mechanisms behind the wood dust induced carcinogenesis, we examined human sinonasal tumours for mutations in the ras genes. The mutational spectrum, which is the type, site and frequency of mutations, provides useful clues to etiological factors and identification of exposure related tumours.We examined all incident cases of sinonasal adenocarcinoma and squamous cell carcinoma reported to the Danish Cancer Registry between 1991 and 2001. After a very careful inclusion process in order only to include sinonasal cancer of the nasal cavities and sinuses with correct histology, we included 174 cases. Wood dust exposure was assessed by interview and job/trade codes from the National Pension Fund and self reported job titles in the Central Personal Register. Among the cases wood dust exposure occurred in 21 percent of our patients and was 7 times more frequent in patients with adenocarcinomas than in squamous cell carcinoma (p0.0001). In the mutational analysis, the K-ras gene was mutated in adenocarcinoma (13%) which is in the range reported in earlier studies, whereas the frequency was very low in the squamous cell carcinoma (1%). By analysis of all published K-ras mutations, the GGTGLY →GAT ASP transition was the most common K-ras codon 12 mutation. Despite that 65 percent of these patients had been exposed to wood dust, it was not significantly different from the patients without a G→A mutation.





The main conclusions based on the results obtained in this thesis are:

• All wood dust species cause inflammation. However, the species vary in their potential to induce the inflammation.

• Wood dust species vary in their potential to cause DNA damage. The DNA damage observed seemed to be caused by a direct genotoxic effect of the dust it selves.

• The current study provides evidence that the distinction between hard and softwoods dust may not be that clear cut in relation to health effects.

• The previously reported association between adenocarcinomas and exposure to wood dust could be confirmed by our study.

• Mutations activation of K-ras was restricted to a small subpopulation of the adenocarcinomas. The predominant mutations among these tumours were G•C→A•T transitions.

• Overall, the study suggests a limited role for K-ras mutations in development of sinonasal cancer.

DANSK RESUMÉ Denne ph.d. afhandling omhandler inflammatoriske, genotoksiske og karcinogene effekter efter eksponering for træstøv fra forskellige træsorter. Eksperimenterne udført i forbindelse med dette projekt, var delt i to. Den første del omhandlede en in vitro model, hvor den humane lunge epitel celle linje A549 blev brugt, til at bestemme inflammatoriske og DNA skadende effekter. Den anden del bestod i en molekylær analyse af humane sinonasale tumourer for mutationer i det kræft relaterede gen K-ras. Mutationer i kodon 12, 13, og 61, såkaldte ’hotspot’ kodons blev bestemt. I forbindelse med disse forsøg blev kalibrering og validering af alle de brugt metoder udført.

Kræft i næse og bihuler er sjældent. I Europa er forekomsten af nydiagnosticerede tilfælde på mellem 0,3 til 1,4 per 100 000 indbyggere for mænd og mellem 0,1 og 0,8 per 100 000 for kvinder, afhængigt af hvilket land man ser på. Til trods for den lave hyppighed ses en stærk sammenhæng mellem kræft i næse og bihuler og erhvervsmæssig udsættelser for især træstøv.

Professionel udsættelse for træstøv, især fra hårde træsorter, er blevet stærkt associeret med histologi typen adenokarcinom. Ikke maligne symptomer så som allergi, astma, rhinitis og kronisk bronkitis kan ligeledes i epidemiologiske undersøgelser relateres til en professionel udsættelse for træstøv. I de fleste studier er træstøv fra hårde træsorter (f.eks. eg og bøg) mere karcinogene og har en stærkere association til de ikke maligne symptomer sammenlignet med støv fra bløde træsorter (f.eks. fyr og gran). Da standardiserede og kontrollerede forsøg med hensyn til eksponering er svært at udføre på mennesker, er de underliggende mekanismer bag træstøvs effekter mangelfuldt kortlagt. Det er blevet foreslået at partikel induceret inflammation, så vel som de indholdsstoffer der er i selve træet kan virke karcinogene.

I dette projekt eksponerede jeg den humane lunge epitel celle linje A549 for træstøv fra forskellige træsorter og målte markører for inflammation og genotoksicitet. Resultaterne viste, at de forskellige træsorter varierer i deres potentiale til at give DNA streng brud og til at kunne inducere en inflammations proces. Der var umiddelbart ingen sammenhæng mellem en træsorts evne til at forårsage inflammation og dets genotoksiske potentiale. I modsætning til vores hypotese, var inflammation ikke den direkte årsag til DNA streng brudene, ligesom træstøv fra hårdt træ ikke var mere inflammatoriske eller genotoksiske i forholdt til støv fra blødt træ.

For at undersøge de molekylære mekanismer bag træstøvs induceret kræftudvikling, undersøgte vi tumorer fra næse og bihuler for mutationer i ras generne. Et mutations spektrum, indeholdende oplysninger omkring typen, placeringen og hyppigheden af mutationer, giver et godt værktøj til, at vurdere hvorvidt en tumor type er eksponerings betinget eller ej. Ved hjælp af cancer registeret identificerede vi samtlige adeno- og planocellulære karcinomer fra næse og bihuler, diagnosticeret mellem 1991 og 2001. Væv fra 174 patienter, gennemgik en grundig udvælgelses proces, der sikrede os at de inkluderede tumorer var lokaliseret i næse og bihuler med en korrekt diagnose. Eksponeringen for træstøv blev vurderet ud fra interviews, branche- og job koder fra ATP registeret og selvrapporterede job titler til det Centrale Person Register.

Blandt patienterne var 21 pct. udsat for træstøv i forbindelse med deres arbejdsliv. Træstøvseksponering var syv gange mere hyppigt forekommende blandt patienter med adenokarcinomer end blandt patienter med planocellulære karcinomer (p0,0001). Mutationsanalyserne viste at Kras genet var muteret i 13 procent af adenokarcinomerne, hvilket svarer til hyppigheden fundet i tidligere studier. Blandt de planocellulære karcinomer var kun en procent muteret. Ved en analyse af alle publicerede mutationer i K-ras genet, var GGTGLY →GAT ASP mutationen var den mest almindelige. Til trods for at 65 procent af disse patienter havde været udsat for træstøv, adskilte dette sig ikke signifikant fra patienterne uden en G → A mutation.

Hovedkonklusioner, der kan blive draget ud fra resultaterne i denne ph.d. afhandling:

• Alle testede træ sorter kan forårsage inflammation, men variere i deres potentiale til at inducere inflammationen.

• Træsorter varierer i deres potentiale til at forårsage DNA skade. Den observerede DNA skadende effekt, var forårsaget af en direkte genotoksisk effekt af selve træstøvet.

• Resultaterne fra dette studie tyder på, at brugen af kategorierne hårdt og blødt træ ikke er de mest optimale, når det gælder helbreds effekter.

• Sammenhængen mellem adenokarcinomer og erhvervsmæssig udsættelse for træstøv blev bekræftet i dette studie.

________________________________________________________________________

• Mutationer i K-ras genet er begrænset til en lille population af adenokarcinomer. Den hyppigst forekomne mutation blandt disse tumorer var G•C→A•T mutationer.

• Alt i alt, viser studiet kun en begrænset rolle for K-ras mutationer i udviklingen af kræft i næse og bihuler.

________________________________________________________________________

TABLE OF CONTENTS

Table of Contents

Introduction

Background

The wood industry

Exposure Levels to Wood Dust by Legalisative and Measurements

Classification and composition of wood

Secondary metabolites and their biological effects

Wood dust particles deposition in the respiratory tract

Clearance of wood particles from the respiratory tract

Effects of the deposition of wood dust particles in the nose and lung

Particle induced inflammation

Interleukin 6

Interleukin 8

Inflammation and cancer.

Cancer development

Genotoxicity of wood particles

Carcinogenesis

The Ras genes

Function of Ras proteins

RAS as human oncogenes

Ras mutations as a result of exposure to chemical agents?

Epidemiological studies of the biological effects of wood dust exposure- non malignant.............34 Epidemiological studies of the biological effects of wood dust exposure- malignant

Sinonasal cancer

Sinonasal cancer and wood dust exposure

Sinonasal cancer and other occupational exposures than wood dust.

Wood dust and cancer at other localisations than the nose and paranasal sinuses

Hypotheses and Aims

________________________________________________________________________

Methods and Materials

Ethical considerations

The In Vitro Exposure

Choice of model

The doses

Choice of time perspectives

Cell culture

Wood dust exposure

Assessment of cytotoxicity by lactatedehydrogenase (LDH) activity

Assessment of inflammation



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