In this nested case–control study, we found that some POPs (in particular, trans
-nonachlor and highly chlorinated PCBs) among the CARDIA participants at year 2 were associated with incident type 2 diabetes for the next 18 years, especially among obese persons. However, POPs did not show a traditional dose–response relation with diabetes. Instead POPs showed strong associations at relatively low exposure, making inverted U-shapes. These nonlinear shapes were anticipated, at least insofar as we discussed these possibilities in our previous cross-sectional studies on POPs (Lee et al. 2006a
). In fact, the cohort study performed in Great Lakes Sport Fish Consumers, which included only 36 cases of incident diabetes, did not show the inverted U-shaped association in their main results (Turyk et al. 2009
). However, low-dose effects were suggested among younger and heavier persons in their study, as in our study subjects (M Turyk, personal communication).
The inverted U-shaped associations have been proposed as possible biological responses of endocrine disruptors (Daston et al. 2003
; Welshons et al. 2003
), unlike the traditional paradigm of cellular toxicity in which there is a linear dose–response relation. POPs are well-known endocrine disruptors (Vasseur and Cossu-Leguille 2006
). Hormones act indirectly through binding to specific receptors. In many biological systems, there is a linearity of dose and receptor occupancy only up to a dose that occupies about 10% of receptors. At higher doses, the effect of a higher occupancy rate does not increase linearly as the dose of the hormone increases (Daston et al. 2003
; Welshons et al. 2003
). Furthermore, a linear biological response is observed only at doses that result in the lowest range of receptor occupancy (Daston et al. 2003
; Welshons et al. 2003
). In high-dose hormone exposure, there is even downregulation of receptors as the dose further increases (Medlock et al. 1991
). Thus, high doses of hormonally active chemicals can exert inhibitory effects on processes that are stimulated at much lower doses, which result in inverted U dose–response curves. Although endocrine disruption is an example of a biological system that could lead to low-dose response, we do not have specific evidence that endocrine disruption explains these epidemiologic observations.
At present, there is little knowledge about the biological mechanisms that might link POPs and type 2 diabetes. Although aryl hydrocarbon receptor (AhR) affinity has been the main focus in relation to POPs toxicity (Nebert et al. 1993
), PCBs with some affinity to AhR (PCB105, PCB118, and PCB156) were not clearly associated with the risk of type 2 diabetes. Thus, mechanisms other than AhR affinity may be involved in the pathogenesis of type 2 diabetes. In addition, when we analyzed PCBs using the classification of Wolff et al. (1997)
, we found that groupings of both estrogenic PCBs and antiestrogenic PCBs showed low-dose effects. Thus, estrogenicity, at least in the Wolff et al. (1997)
formulation, does not explain the link between PCBs and diabetes. In the analyses, using the classification of McFarland and Clarke, (1989)
, we found that PCBs in a grouping of phenobarbital-type inducers and in another grouping of weak or noninducers of CYP 450 showed the low-dose effects, whereas groupings of PCBs that were mixed-type inducers were not associated with type 2 diabetes. Interestingly, PCBs in the grouping of mixed-type inducers have been traditionally regarded as having the greatest toxic potential compared with other PCBs. Therefore, our data suggest that neither an AhR affinity nor estrogenicity explain the association between PCBs and type 2 diabetes. Rather, chronic exposure with weak or little-known toxicity may play a role in the pathogenesis of type 2 diabetes, given that highly chlorinated PCBs tend to be more persistent in the environment than PCBs with fewer chlorines. In the absence of known biological mechanism(s) and demonstrated low-dose effects of POPs on type 2 diabetes, our grouping of individual POP analyses, coupled with the consistency of our results with previous findings for prevalent diabetes (Lee et al. 2006b
), can be justified to create summary POPs measures based on results of individual POP analyses.
For valid estimation of relative risks to detect the presence of low-dose effects, a reference group is required with extremely low concentrations of relevant POPs so that low risk is uniform across values within the reference group. However, a true reference group without any exposure to POPs does not exist in the general population (i.e., in epidemiological data). In general, serum concentrations of POPs tend to be highly correlated with each other because of the simultaneous exposure to various POPs through food consumption, but the correlations are not perfect. Given these correlations, the reference group used in the analysis of an individual POP would have the lowest concentration of that specific POP but could have higher concentrations of other POPs that had a higher risk of type 2 diabetes. Therefore, in the presence of low-dose effects of POPs, the contamination of the reference group of specific POPs with higher concentrations of other important POPs may lead to an artificial increase of risk within the reference group of the specific POP, leading to underestimated risk relative to this group. In this situation, a summary POPs measures formed by summing individual ranks of the POPs, for example, those 16 POPs with ORs of ≥ 1.5 in the second quartile in individual analyses, facilitates selecting a reference group with extremely low concentrations of those POPs that are relevant to a risk of type 2 diabetes. We acknowledge that such a summary measure may to some extent overestimate ability to predict type 2 diabetes because it is data driven; however, this tendency is countered by the fact that several of the 16 POPs selected for analysis in this study were based on a priori
selection of POPs that were found to be important in previous studies (Lee et al. 2006b
In contrast, although the summary measure of all 31 POPs is not data driven, it can compromise our effort to select subjects with very low concentrations of relevant POPs as the reference group. For example, some subjects in the lowest sextile of the summary measure of all 31 POPs were those with very low concentrations of the irrelevant POPs but relatively high concentrations of the 16 POPs with ORs of ≥ 1.5 in the second quartile. In this case, the risk of diabetes in the reference group, based on the sum of all 31 POPs, would increase and the OR estimates would be biased toward the null, compared with using the reference group based on the sum of the 16 selected POPs. The true association of selected POPs with diabetes risk likely lies between the estimates based on these two summary measures. Whichever summary measure we used, the ORs detected were large, particularly among the obese participants, so the findings as presented give a good idea of the size and shape of the association of certain POPs with incident diabetes.
In this study, another important strategy to reduce POPs level in the reference group was to categorize in sextiles. This strategy could have the drawback of leading to too many categories in a study with a relatively small sample size. In fact, our study showed that the associations became stronger when we refined the lowest category by expanding from quartiles to sextiles.
One critical question about exposure assessment is whether lipid standardization of POPs is valid in epidemiological studies. In a previous study, Lee et al. (2006a)
reported that OC pesticides and PCBs were associated with dyslipidemia; that is, increased triglycerides and decreased HDL cholesterol. Our findings in this study also suggest that some POPs may disturb lipid metabolism over the extended period of observation; these findings will be reported in a separate paper. As dyslipidemia is an early manifestation of conditions characterized by insulin resistance and is often detectable before the development of postprandial or fasting hyperglycemia (Lewis et al. 2002
), lipid concentrations may be intermediaries in the associations between POPs and type 2 diabetes. In this case, any form of adjustment for circulating lipid concentrations would substantially underestimate true risk associations. However, although it can be overadjustment to control for baseline lipid concentrations, findings not adjusted for lipids can be underadjusted, because POPs are carried by lipid in the blood. In fact, the effect of lipid adjustment was most strongly observed in the highest sextile of POPs, which consisted of subjects with a broad range of particularly high lipid concentrations. The highest category tended toward increased ORs before lipid adjustment, but this tendency mostly disappeared after lipid adjustment. The true associations between the POPs studied and incident diabetes may lie between the estimates with and without lipid adjustment.
Another important methodological issue is that a single exposure assessment provides an imprecise picture of POPs exposure. In this study, serum was sampled up to 18 years before the diabetes diagnosis. Although POPs have long half-lives, body burden of POPs can change substantially during follow-up. Because the development of diabetes requires deterioration of glucose metabolism over the long term, prolonged and persistent exposure to POPs may be an important factor in a putative causal pathway linking POPs and diabetes. Because we measured POPs only once, we could not assess whether their concentrations changed during follow-up. In fact, chlorinated POP concentrations at year 2 in these CARDIA subjects (1987–1988, recognizing that they had higher BMI than the general CARDIA sample) were much higher than those in the current U.S general population—a finding that suggests that chlorinated POPs concentrations in serum of CARDIA subjects primarily reflect the body burden accumulated during the calendar time before banning chlorinated POPs in the 1970s. Based on the comparison with NHANES participants at current CARDIA ages, current concentrations of chlorinated POPs, not brominated POPs such as PBDEs, in our participants would be lower than at year 2. However, we also believe that the slow clearances of POPs over many years differ among people, and we hypothesize that the rank of POPs concentrations of the participants may have changed during follow-up. It is thought that there are interindividual and chemical-specific variations in excretion of POPs (Thomaseth and Salvan 1998
; Wolff et al. 1997
As most chlorinated POPs have already been banned and body burdens are decreasing, research interest has tended to focus on more recently introduced POPs such as PBDEs. However, the strong low-dose effects of chlorinated POPs observed here imply that their low persistent exposures throughout the body may be a problem well into the future as the banned POPs slowly clear, passing through perhaps more dangerous lower concentrations. In addition, obesity seemed to exaggerate metabolic disturbance related to POPs, with the result that in the midst of the ongoing obesity epidemic, the currently low POPs concentrations may be a potent health risk.
This study has several limitations. First, the sample size was a compromise related to the practicalities of cost and the use of precious samples. Therefore, most analyses based on individual POPs failed to reach statistical significance in terms of p for trend or p for quadratic term, although some ORs in the second quartile showed statistical significance. Second, the data analyses involved a large number of statistical tests; therefore, some of the individual statistically significant findings may have occurred by chance. In this study, we focused our interpretation more on consistency than on statistical significance. Third, under the low-dose effect, the selection of a reference group with very close to zero POP concentrations is critical to unbiased assessment of the risk of adverse health outcomes. Combining study subjects across apparently low ranges of POPs can lead to a reference group with artificially increased risk and diluted relative risk estimates. As the CARDIA subjects in 1987–1988 had higher values of chlorinated POPs than present in the NHANES studies in 2003–2004, we cannot exclude the possibility that some risk gradient was still masked in our CARDIA subgroup, even when we used the lowest control group sextile to approximate more closely a true zero risk reference group in our nested case–control study. Finally, we could not exclude the possibility that other POPs not measured in this study could play a role in the pathogenesis of type 2 diabetes, because serum concentrations of POPs are highly correlated in the general population.
In conclusion, the results from this study suggest that environmental exposure to some POPs may increase, in a nonlinear fashion, the risk of future type 2 diabetes in the general population. Various POPs, which mainly accumulate in adipose tissue, may play a critical role in the current epidemic of type 2 diabetes.