Molecular epidemiologic evidence for diabetogenic effects of dioxin exposure in U.S. Air force veterans of the Vietnam war.

Many veterans of the Vietnam War were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin), which was present as a contaminant in the herbicide formulation Agent Orange [Booker 2001; Institute of Medicine (IOM) 2000, 2003]. Indeed, significant residues of dioxin have been found in their serum, even after many years, attesting to the fact of their initial exposure and to the long half-life of dioxin, which is estimated to be 7.6 years (95% confidence interval, 7.0–8.2 years) (Michalek and Tripathi 1999). Those dioxin residue levels are relatively high among veterans of Operation Ranch Hand (ORH) (Michalek and Tripathi 1999), the operation responsible for the handling and the actual aerial spraying of Agent Orange and other dioxin-contaminated herbicides in Vietnam from 1962 to 1971. Because of the health implications, many epidemiologic studies have been conducted to identify the possible harmful effects of dioxin on exposed veterans (Frumkin 2003; IOM 2000). Among the recent findings, one of the most consistently identified diseases statistically associated with dioxin exposure is type 2 diabetes mellitus (Henriksen et al.1997; Michalek et al. 1999). This conclusion is also supported by the results of similar epidemiologic studies such as those of the population exposed to high levels of dioxin in Seveso, Italy (Bertazzi et al. 1998; Pesatori et al. 2003), industrial workers (Vena et al. 1998), and Korean Vietnam veterans who were also exposed to Agent Orange (Kim et al. 2003). Despite the epidemiologic evidence for the positive correlation, it has been difficult to explain the mechanistic basis of action of dioxin in causing type 2 diabetes in humans. Because the actual levels of dioxin residues among U.S. veterans are not as high as those of the Seveso population (Bertazzi et al. 1998) or the industrially exposed cohort (Vena et al. 1998) and because small amounts of dioxin and dioxin-type chemicals are also found among general populations in industrialized nations, some scientists question the cause–effect relationship between dioxin residues in humans and an increased incidence of diabetes (Remillard and Bunce 2002). To address this important environmental health question, we conducted a molecular epidemiologic study on the state of expression of selected biomarkers in adipose tissue samples from participants in the Air Force Health Study. The Air Force Health Study was designed to determine whether veterans of ORH experienced adverse health effects and whether those health effects, if they exist, can be attributed to exposure to herbicides or their dioxin contaminant (Wolfe et al. 1990). The ORH veterans were exposed to herbicides during flight operations, herbicide preparation, and maintenance of the aircraft and herbicide spray equipment. The examination content of the Air Force Health Study emphasized detection of medical end points suspected of being associated with exposure to dioxin, so extensive data on health status and health behaviors were collected on ORH veterans and a matched comparison group of background-exposed veterans. Adipose tissue was selected for study because of its significant role in the etiology of type 2 diabetes (Scheen 2003). Type 1 diabetes, considered genetic with onset at an early age, was not included as an end point in the Air Force Health Study protocol and so was not considered in this study. We hypothesized two possible pathways for TCDD-induced glucose intolerance. In one pathway involving oncogenes, Src [GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"M16243","term_id":"338449","term_text":"M16243"}}M16243 (GenBank 2006)], would activate MYC (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"V00568","term_id":"34815","term_text":"V00568"}}V00568; Jahner and Hunter 1991), which is a direct controller of CCAAT/enhancer binding protein-α (C/EBPα; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_004364","term_id":"551894987","term_text":"NM_004364"}}NM_004364; Freytag and Geddes 1992). Src is known to be essential for TCDD-induced wasting in mice (Dunlap et al. 2002), and C/EBPα is a master switch for adipocytes that causes the up-regulation of lipid accumulation processes (MacDougald and Lane 1995). In the other pathway, which involves inflammation, there would be increased production of the cytokine tumor necrosis factor-α (TNF-α; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_000594","term_id":"395132451","term_text":"NM_000594"}}NM_000594), one of the major mediators of dioxin-induced cell inflammatory reactions (Alsharif et al. 1994; Kern et al. 2002; Matsumura 2003; Taylor et al. 1992). TNF-α is produced by adipocytes also in response to obesity, causing increased expression of the transcription factor NFκB, and leading eventually to down-regulation of the insulin receptor and the major insulin-responsive glucose transporter GLUT4 (Halle et al. 1998). To test the contributions of each pathway we measured mRNA expression of the genes c-Src, C/EBPα, NFκB, and GLUT4 in adipose tissue. In addition we measured GAPDH, a housekeeping gene (Gorzelniak et al. 2001), for use as a normalization standard to correct for differences in cDNA synthesis efficiency.