In this study, we found that long-term treatment with low concentrations of the herbicide, ATZ, induced insulin resistance and weight gain in Sprague Dawley rats. We acknowledge that, unlike the current results, previous studies have reported that the body weights of ATZ-treated animals were generally decreased or unchanged 
. However, the doses of ATZ used in these other studies were 10–100 fold higher (2.7 to 50 mg kg−1
) than those used in our study. Thus, our interpretation is that acute exposure to high concentrations of ATZ is toxic, and thus prevents weight gain and possibly causes weight loss. In contrast, chronic low-dose ATZ exposure might lead to mild mitochondrial damage that mimics the characteristic of the insulin-resistance state, and hence, leads to weight gain.
Because ATZ treatment in our study induced obesity without changing food intake or physical activity, ATZ presumably lowered energy metabolism. Indeed, indirect calorimetry measurements revealed that the ATZ-induced weight gain was associated with decreased energy metabolism. Furthermore, in vitro experiments provided evidence that ATZ interferes with electron transfer through OXPHOS complex at Q sites in mitochondria, resulting in reduced oxygen consumption. We have found that treatment with rotenone, a complex I inhibitor, or dideoxydytidine, an mtDNA-replication inhibitor, directly inhibits insulin-mediated phosphorylation of Akt without affecting upstream signaling molecules (data not shown). In addition, overexpression of dominant-positive Akt (myr-Akt) completely reverses downstream Akt function. For example, Akt-mediated phosphorylation of Foxo1, which is blocked by mitochondrial dysfunction, is abrogated by myr-Akt overexpression, implying that Akt is the focal point of the mitochondrial dysfunction (manuscript in preparation). As part of this study, we tested whether ATZ also blocked Akt function, and found, as expected, that ATZ blocked insulin-mediated Akt phosphorylation in skeletal muscle cells.
Treatment with ATZ changed the ultrastructure of mitochondria in liver and muscle of rats, producing morphological alterations such as ring- and cup-shaped mitochondria without significantly altering the expression of mitochondrial OXPHOS complex proteins. This observation is in agreement with the findings of others that ATZ induced mitochondrial damage in fresh water mussels 
and rainbow trout 
. In our study, we also found that ATZ decreased the membrane potential of mitochondria and reduced intracellular ATP content in various cells (data not shown). Collectively, these data led us to conclude that ATZ treatment damaged both mitochondrial respiratory function and morphology.
Intramuscular lipid accumulation is known to be closely related to insulin resistance 
, and is considered as an early phenomenon in the development of obesity. Increased lipid content in muscle is also associated with decreased ATP synthesis and diminished mitochondrial function 
. The observed increase in intracellular lipid content in ATZ-treated rats also supports the interpretation that ATZ-induced mitochondrial damage affects the insulin-signaling pathway, and consequently induces insulin resistance and fat accumulation in metabolically active tissue, such as muscle.
There is epidemiological evidence that human exposure to POPs such as TCDD may also disturb glucose metabolism and induce insulin resistance 
, and it has been reported that exposure to herbicides or pesticides, including ATZ, is associated with an increased risk of gestational diabetes 
. However, there is scant information in the literature to indicate the level of human exposure to ATZ or similar herbicides. Obviously, there is no direct evidence of ATZ accumulation in diabetic or obese human subjects. However, a toxicological report has shown that acute occupational or dietary exposure of humans to ATZ was in the range of 0.2–90 µg kg−1
, and the annual average exposure (chronic) was between 0.046 and 0.286 µg kg−1
We believe that ATZ or its metabolites may be introduced to humans through air, water and and/or corn products as contaminants, and accumulate in tissues. One such pathway by which ATZ or its metabolites might be introduced into humans is through corn-derived foods (e.g., high fructose corn syrup or corn oil). Since corn syrup 
and fast foods served in the USA 
are suspected of causing an obesity epidemic, this seems a reasonable supposition. Recently, it was reported that of 160 food products purchased at a fast food restaurant throughout the USA, not a single item could be traced back to a non-corn source 
. This work also identified corn as the overwhelmingly predominant animal feed for the beef and chicken served at fast food restaurants.
Considering that the process of corn wet milling requires a huge amount of fresh water for steeping (digesting), which in turn generates 1,300 to 1,600 L of light steepwater per ton of corn that is then evaporated or dried to make intermediate products, it is conceivable that ATZ and related herbicides are present in steepwater and may be concentrated during the process 
. Because herbicides like ATZ and their metabolites are present in streams and groundwaters throughout USA and are at highest concentrations in agricultural areas where wet corn milling plants are located 
, there is a substantial possibility of ATZ contamination of corn products. However, in 2006 the U.S. Environmental Protection Agency issued a cumulative risk assessment of triazine herbicides, concluding that they posed “no harm that would result to the general U.S. population, infants, children, or other consumers” 
Based on evidence presented in this study, we conclude that environmental ATZ might be an important contributing factor to the obesity epidemic in the United States. It damages mitochondrial function, affects insulin signaling, and induces insulin resistance and obesity, especially when exposure is associated with a high-fat diet. Sanguine official assessments notwithstanding, further studies are definitely needed to clarify issues related to human exposure to ATZ and related herbicides in the US and elsewhere around the world.