10. in 11. november 2016

2. BALcanOSH

MEDNARODNA KONFERENCA ZA REGIONALNO

SODELOVANJE, BLED, SLOVENIJA

Vita Dolžan1, Metoda Dodič-Fikfak2, Alenka Franko2

1Pharmacogenetics Lab., Inst. of Biochemistry, Faculty of Medicine, University of Ljubljana, Slovenia 2Clinical Institute of Occupational Medicine,University Medical Centre Ljubljana, Slovenija

ASBESTOS RELATED DISEASES

asbestosis

pleural diseases: pleural plaques, diffuse pleural

thickening, pleural effusion

malignant mesothelioma of pleura or peritoneum

lung cancer

other cancers: buccal mucosa, pharynx, larynx, ovary,

kidney, gastointestinal tract

ASBESTOSIS

asbestos

exposure

genetic

factors ? ? • most common among diseases caused by asbestos

• interstitial pulmonary process develops slowly after a long latency period

• develops into diffuse pulmonary fibrosis

• associated with increased risk of lung cancer.

reactive oxygen

species (ROS) reactive nitric

species (RNS)

NO

iNOS

asbestos

redox active Fe in

asbestos fibers

alveolar macrophages

activated by

phagocytosis

asbestos fibers

ROS and NO and their reactive products damage

biomolecules: lipids, proteins and DNA

MOLECULAR MECHANISMS ASSOCIATED WITH

PATHOGENESIS OF ASBESTOS RELATED DISEASES

- still poorly understood

DEFENCE MECHANISMS AGAINST ROS

antioxidant enzymes

proteins binding Fe and Cu

antioxidants (endogenous, exogenous)

Superoxide dismutases

MnSOD, EcSOD, Cu/ZnSOD

Catalase

CAT

ANTIOXIDANT ENZYMES

- primary line of enzymatic defence against ROS

O2• - + O2

• - + 2H+ H2O2 + O2

2H2O2 2H2O + O2

SOD

CAT

GLUTATHIONE S-TRANSFERASES: GSTM1, GST T1, GSTP1

- secondary line of enzymatic defence against ROS

asbestos

electrophiles + glutathione

ROS and RNS GST

Inactivate the electrophiles

produced by ROS and RNS

The genes coding for

MnSOD, ECSOD, CAT, GSTM1, GST T1, GSTP1 and iNOS

are polymorphic

GENETIC VARIABILITY IN

ANTIOXIDANT DEFENCE

GENETIC POLYMORPHISM

• presence of 2 or more alleles in the population

• the freuency of the rare allele > 1 %

• Single nucleotide polymorphisms - SNPs

• Copy number variations – CNVs; including gene deletions and duplications

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• Besides environmental and/or occupational exposure to

different hazards and lifestyle factors,

• genetic factors as well as the interactions between

different genotypes, between genotypes and lifestyle

factors, and between genotypes and

environmental/occupational exposure to hazards

• may also have an important influence on the development

of diseases and should be further investigated

GENE-ENVIRONMENT INTERACTIONS

THE AIM OF THE STUDY

to investigate influence of

gene-gene and gene-environment

interactions on the risk of

developing asbestosis

COHORT OF 2,080 WORKERS

>> nested case control study

All 356 cases with asbestosis

356 controls

76 female

186 male

82 female

183 male

40 died, 2 cancer, 52 refused to participate 262

29 died, 9 cancer, 63 refused to participate 265

• Data on smoking

• Duration of exposure

• Data on cumulative exposure

• Diagnosis of asbestosis

SALONIT ANHOVO

Anhovo Cumulative

exposure

DIAGNOSIS OF ASBESTOSIS

State Board for Recognition of Occupational Asbestos Diseases

The Helsinki Criteria

American Thoracic Society recommendations

MOLECULAR GENETIC METHODS

ANALYSES OF POLYMORPHISMS Real-time PCR:

MnSOD Ala-9Val

ECSOD Arg213Gly

CAT -262C>T

GSTP1 Ile105Val and Ala114Val

Multiplex PCR:

GST T1-null

GSTM1-null

Short tandem repeat

(CCT T T)n in the iNOS gene

STATISTICAL METHODS

• Standard descriptive statistics

• T-test: differences in the means of variables between cases and controls

• Chi-square test: differences in proportions between groups

• Univariate logistic regression analysis: associations between asbestosis and individual variables

• Multivariate logistic modeling : simple categorical models followed by logistic regression models using dummy variables

• OR and 95 % CI

OR 95%CI

Cumulative exposure 3.21 2.4 - 4.23

Smoking 0.98 0.69 - 1.39

Environmental factors and asbestosis risk

RESULTS

Genotype OR (95 % CI) OR (95 % CI)

Adj for smoking

OR (95 % CI)

Adj for cumulative

asbestos exposure

MnSOD –9Ala/Ala vs.

Ala/Val+Val/Val

1,50

(1,01–2,24)

1,49

(1,00–2,23)

1,48

(0,96–2,28)

ECSOD Arg/Gly vs.

Arg/Arg

1,63

(0,62–4,27)

1,65

(0,63–4,32)

2,07

(0,72–5,94)

CAT −262 TT vs.

CT+CC

1,36

(0,70–2,62)

1,37

(0,71–2,66)

1,91

(0,93–3,91)

GSTM1-null vs. present 1,01

(0,71–1,43)

0,99

(0,70–1,41)

0,97

(0,67–1,42)

GSTT1- null vs. present 0,61

(0,40–0,94)

0,63

(0,41–0,97)

0,60

(0,38–0,96)

GSTP1 high vs.

inter/slow conjugation

1,49

(1,06–2,10)

1,50

(1,06–2,13)

1,36

(0,94–1,98)

iNOS LL vs. SL+SS 1,20

(0,85–1,69)

1,17

(0,83–1,66)

1,19

(0,82–1,73)

Genetic factors and asbestosis risk

MODEL OF CAUSATION

?

?

?

??

iNOS

MnSOD

ECSOD

GSTM1

GSTT1

CAT

cumulative

exposure

smoking

Interaction between MnSOD and CAT and asbestosis risk

MnSOD Cases Controls

Ala/Val and

Val/Val 183 205

Ala/Ala 75 56

OR = 0.67 (95% CI = 0.44–1.01)

CAT TT

MnSOD Cases Controls

Ala/Val and

Val/Val 16 8

Ala/Ala 6 8

OR = 2.67 (95% CI = 0.57–13.07)

CAT CT in CC

MnSOD Cases Controls

Ala/Val and

Val/Val 164 195

Ala/Ala 68 48

OR = 0.59 (95% CI = 0.38–0.93)

Interaction: OR = 4.49 (95% CI = 1.08–18.61); p = 0.038

Biologically plausible: MnSOD and CAT catalyse sequential reactions

iNOS Cases Controls

LL 132 121

SL +SS 126 138

OR = 1.20 (95% CI = 0.85–1.69)

CAT TT

iNOS Cases Controls

LL 15 5

SL +SS 7 12

OR = 5.14 (95% CI = 1.30–20.36)

CAT CT in CC

iNOS Cases Controls

LL 115 116

SL +SS 116 126

OR = 1.08 (95% CI = 0.75–1.55)

Interaction: OR = 4.78 (95% CI = 1.15–19.81); p = 0.031

Interaction between iNOS and CAT and asbestosis risk

?

?

?

??

smoking

cumulative

exposure

CATGSTT1

GSTM1

ECSODMnSOD

iNOS

MODEL OF CAUSATION

Smoking Cases Controls

ever 117 120

never 145 145

OR = 0.98 (95% CI = 0.69–1.39)

GSTM -null

Smoking Cases Controls

ever 76 63

never 75 92

OR = 2.67 (95% CI = 0.57–13.07)

GSTM -null

Smoking Cases Controls

ever 41 57

never 61 47

OR = 0.55 (95% CI = 0.31–1.00)

Interaction: OR = 2.67 (95% CI = 1.31–5.46); p = 0.007

Interaction between GSTM1 and smoking and asbestosis risk

• both asbestos and smoking increase ROS production • role of GSTM1

?

?

?

??

iNOS MnSOD ECSOD

GSTM1

GSTT1CAT

cumulative

exposure

smoking

MODEL OF CAUSATION

Cum. exposure Cases Controls

> 11.23 146 59

≤ 11.23 116 206

OR = 4.40 (95% CI = 3.01-6.46)

iNOS LL

Cum. exposure Cases Controls

> 11.23 68 31

≤ 11.23 64 90

OR = 3.09 (95% CI = 1.81–5.25)

iNOS SL + SS

Cum. exposure Cases Controls

> 11.23 72 26

≤ 11.23 54 112

OR = 5.74 (95% CI = 3.30–9.99)

OR = 0.55 (95% CI = 0.31–0.97); p = 0.037

Interaction between iNOS and cumulative exposure

GENETIC FACTORS

GENE-ENVIRONMENT

INTERACTIONS

ASBESTOSIS

CONCLUSIONS

• molecular markers as basis for the development of new

methods for an earlier diagnosis of diseases

• understanding of pathogenesis of diseases and enable their

prevention

• to identify new targets for a more effective treatment

GENETIC FACTORS

DISCRIMINATION

THANK YOU

FOR YOUR

AT TENTION

Supported by:

ARRS projects: L3-9129, L3-3648

ARRS programme: P1-0170]

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Antioxidant enzyme polymorphisms

MnSOD

EcSOD

CAT

GCT

CGG

-9 Ala

213 Arg

GTT >

> >

> -9 Val

DNA protein

GGG 213 Gly

-262 C > -262 T ↓ expression

GST polymorphisms

GSTP1 exon 5

GSTP1 exon 6

GSTM1

GSTT1

c.313 A >

G

Gene present

p.105 Ile>Val

>

>

DNA protein

p.114 Ala>Val

Gene deletion

c.341 C>T

>

Gene present Gene deletion >

iNOS polymorphisms