Molecular Epidemiological Evidence for Diabetogenic effects of Dioxin Among Vietnam Veterans

1
Molecular Epidemiological Evidence for
Diabetogenic effects of Dioxin Among Vietnam
Veterans

• By
• Phillip Fujiyoshi, Joel Michalek and Fumio
  Matsumura

2
Molecular Epidemiology

• A new field of epidemiological science that is
  designed to increase the sensitivity of detection
  of biological effects, and to provide the logical
  bases for empirically observed correlations.

3
Brief Background

• A Molecular Epidemiology study coordinated with
  the Air Force Health Study (AFHS) design.
• Ranch Hand group (RH) was compared to the
  carefully matched dioxin unexposed Air Force
  veterans, who served in Southeast Asia,
  designated as Comparison group (C-group).
• Altogether 313 volunteers agreed to donate
  adipose samples.

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Methodologies 1

• Dioxin residues in serum lipids analyzed by CDC.
• Diabetes determined by diagnosis and by
  abnormally high 2-hour post-prandial blood
  glucose levels (fasting glucose).
• Body mass index (BMI) by weight (kg divided by
  the square of height (m).
• Body fat defined as 2.64 x BMI 13.04.

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Methodologies 2

• Selection of molecular markers was made by
  conducting preliminary animal tests
  (mice,chronic).
• All markers (mRNA expression) were detected
  through reverse transcription, DNA amplification,
  namely quantitative polymerase chain reactions
  (RT-PCR).
• Markers studied wereGLUT4 (glucose transporter),
  NF?B (inflammation),C/EBPa (adiposity index),
  c-Src (a signal messenger for toxic action of
  dioxin) and GAPDH (a housekeeping gene, used here
  as a normalization standard).

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Initial Study (Phase 1) Strategies

• To increase the power of statistics by dividing
  each service group into four quartiles according
  to the level of dioxin residues.
• Start the study by looking for statistically
  significant correlations between molecular marker
  expressions and dioxin residues.
• Search for positive correlations between dioxin
  and not only individual markers but also all
  combinations of all marker ratios.

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Figure 1
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Interim conclusions

• The ratio of GLUT4NF?B ( GN ratio) appears to
  be the best marker among all tested ones,
  detecting the difference between C- and RH-group
  in terms of their response to dioxin.
• There appears to be some subgroups which affect
  the pattern of correlations.

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Questions raised by the initial analysis

• Could such a quartile approach introduce any
  artifacts?
• Why are the directions of the slope opposite
  between RH and C ?
• Are these trends influenced by particular
  subgroups within each service group?

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Strategies for Phase 2 analysis

• Make efforts to answer the above questions
  raised.
• Concentrate in analyzing the relationships
  between GLUT4NF?B (GN ratio) and Dioxin.
• Try to find the most affected subgroups by dioxin.

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Figure 2
(C)
(RH)
p 0.02
p 0.01
Dioxin
Dioxin
C Control RH Ranch Hand
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Conclusion from Fig 2 analysis

• The use of the quartile approach is justifiable,
  since essentially the same results can be
  obtained by using undivided populations.

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Figure 3
O, ND, (C)
L, D, (RH)
p 0.09
p 0.001
O, ND, F (CRH)
L, ND, F (CRH)
p 0.04
Dioxin
Dioxin
O Obese L Lean F Family History
C Control RH Ranch Hand
D Diabetic ND Non-diabetic
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Conclusion from Fig. 3 analysis

• 1.Subgroups differ their GN responses to dioxin
• 2.Obese subgroups respond positively and Lean
  subgroups negatively to dioxin

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Possible hypothesis for the difference in slopes

1. The effect of dioxin is biphasic at low dose
   suppressing GN ratio and at high doses
   elevating.
2. Dioxin mimics obesity-type at low doses, and
   lean-type diabetes at high doses.
3. Long-term dioxin exposure at high doses induce a
   negative counteraction (such as glucocorticoid
   up-regulation) by the adipocytes.

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Phase 3 analysis

• Objective to test our hypothesis that the
  diabetogenic effect of TCDD is similar to that of
  obesity.
• Approach study the relationship between GN
  ratio and recent changes in body fat.

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Figure 4
ND (CRH)
D (CRH)
p lt 0.001
p 0.03
Change in Body Fat
Change in Body Fat
C Control RH Ranch Hand D Diabetic ND
Non-Diabetic
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Conclusion from Fig.4 analysis

• The GN ratio among nondiabetic subjects is
  negatively correlated to obesity, one of the risk
  factors for diabetes.
• However, among diabetic subjects the tendency is
  opposite.

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Figure 5
ND, NF (C)
ND, NF (RH)
p 0.003
p 0.02
ND, F (C)
ND, F (RH)
p 0.03
Change in Body Fat
Change in Body Fat
C Control RH Ranch Hand
F Family History NF No Family History
ND Non-Diabetic
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Brief Conclusions from Phase 3 analysis

• 1. The GN ratio can detect diabetogenic effects
  of obesity as well as genetic risk factors.
• 2. Dioxin exposure makes nondiabetic RH subjects
  more susceptible to diabetogenic influence of
  obesity.

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Phase 4 analysis

• Objective to relate the above findings to
  diabetes.
• Approach Use fasting glucose as the diabetes
  marker, and find its relation ships to (a) GN
  ratio, (b) body fat and (c) dioxin residue
  levels.

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Figure 6
ND (CRH)
D (CRH)
p 0.009
p 0.001
C Control RH Ranch Hand D Diabetic ND
Non-Diabetic
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Conclusion from Fig 6 analysis

• The GN ratio is a reliable marker of diabetes,
  since it is highly correlated to the fasting
  glucose levels.

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Figure 7
ND (CRH)
D (CRH)
p lt 0.001
p 0.09
C Control RH Ranch Hand D Diabetic ND
Non-Diabetic
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Conclusion from Fig 7 analysis

• Fasting glucose levels is a sensitive marker in
  detecting the diabetogenic effect of obesity

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Figure 8
(C)
(RH)
p 0.02
C Control RH Ranch Hand
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Conclusion from Fig. 8 analysis

• The fasting glucose levels are also directly
  affected by dioxin

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Brief mechanistic explanation/ hypothesis

• Dioxin causes TNFa-type inflammation (NF?B up)
  similar to that induced by obesity.
• The resulting suppression of glucose uptake
  (GLUT4 down) by adipose tissue and release of
  lipids into blood, which causes
  insulin-resistance and suppression of insulin
  secretion from pancreas.
• Inflammation also causes glycogen breakdown in
  liver resulting in massive release of glucose
  into blood, contributing to the rise in fasting
  glucose.

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The main conclusion

• We could obtain the definite molecular
  epidemiological evidence, indicating that dioxin
  is acting as a diabetogenic risk factor among
  Vietnam veterans even after many years from the
  time of exposure at such low levels of exposure

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Major Health Implication

• The diabetogenic effect of dioxin can be found
  even in C subjects whose serum levels of dioxin
  overlap with the medium to high end of the
  general public.
• The implication is very clear that we,
  environmental health scientists, should become
  concerned about the potential diabetes-related
  health effect of dioxin to the general public,
  particularly those people with known risk
  factors, even at the current level of low dioxin
  exposure.

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Acknowledgements

• This work could not have been accomplished
  without the tremendous co-operation offered by
  volunteered veterans, all AFHS personnel, Medical
  collaborators and funding managers. We would like
  thank them all.
• Supported by the US Air Force contract
  (sub-project 01-813-32-8280-906-721900).