# Presentation: 106475

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

- By
- Phillip Fujiyoshi, Joel Michalek and Fumio Matsumura

## 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.

## 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.

## 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.

## 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 were:GLUT4 (glucose transporter), NFҝB (inflammation),C/EBPα (adiposity index), c-Src (a signal messenger for toxic action of dioxin) and GAPDH (a housekeeping gene, used here as a normalization standard).

## 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.

## Figure 1

## Interim conclusions

- The ratio of GLUT4:NFҝB (= G:N 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.

## 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?

## Strategies for Phase 2 analysis

- Make efforts to answer the above questions raised.
- Concentrate in analyzing the relationships between GLUT4:NFҝB (G:N ratio) and Dioxin.
- Try to find the most affected subgroups by dioxin.

## Figure 2

**(C)**

**(RH)**

**C = Control**
**RH = Ranch Hand**

**p = 0.01**

**p = 0.02**

**Dioxin**

**Dioxin**

## 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.

## Figure 3

**O, ND, (C)**

**L, D, (RH)**

**O, ND, F (C+RH)**

**L, ND, F (C+RH)**

**C = Control**
**RH = Ranch Hand**

**O = Obese**
**L = Lean**
**F = Family History**

**Dioxin**

**Dioxin**

**D = Diabetic**
**ND = Non-diabetic**

**p = 0.09**

**p = 0.001**

**p = 0.04**

## Conclusion from Fig. 3  analysis

- 1.Subgroups differ their G:N responses to dioxin
- 2.Obese subgroups respond positively and Lean subgroups negatively to dioxin

## Possible hypothesis for the difference in slopes

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

## 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 G:N ratio and recent changes in body fat.

## Figure 4

**ND (C+RH)**

**D (C+RH)**

**C = Control**
**RH = Ranch Hand**
**D = Diabetic**
**ND = Non-Diabetic**

**p = 0.03**

**Change in Body Fat**

**Change in Body Fat**

**p < 0.001**

## Conclusion from Fig.4 analysis 

- The G:N ratio among nondiabetic subjects is negatively correlated to obesity, one of the risk factors for diabetes.
- However, among diabetic subjects the tendency is opposite.

## Figure 5

**Change in Body Fat**

**Change in Body Fat**

**ND, NF (C)**

**ND, NF (RH)**

**ND, F (C)**

**ND, F (RH)**

**C = Control**
**RH = Ranch Hand**

**ND = Non-Diabetic**

**F = Family History**
**NF = No Family History**

**p = 0.02**

**p = 0.003**

**p = 0.03**

## Brief Conclusions from Phase 3 analysis

- 1. The G:N 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.

## 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) G:N ratio, (b) % body fat  and (c) dioxin residue levels.

## Figure 6

**C = Control**
**RH = Ranch Hand**
**D = Diabetic**
**ND = Non-Diabetic**

**p = 0.009**

**ND (C+RH)**

**D (C+RH)**

**p = 0.001**

## Conclusion from Fig 6 analysis

-  The G:N ratio is a reliable marker of diabetes, since it is highly correlated to the fasting glucose levels.

## Figure 7

**C = Control**
**RH = Ranch Hand**
**D = Diabetic**
**ND = Non-Diabetic**

**p = 0.09**

**ND (C+RH)**

**D (C+RH)**

**p < 0.001**

## Conclusion from Fig 7 analysis

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

## Figure 8

**(C)**

**(RH)**

**C = Control**
**RH = Ranch Hand**

**p = 0.02**

## Conclusion from Fig. 8 analysis

- The fasting glucose levels are also directly affected by dioxin

## Brief mechanistic explanation/ hypothesis

- Dioxin causes TNFα-type inflammation (NFҝB 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.

## 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

## 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.

## 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).