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