The results from this study show strong and significant associations between urinary phthalate metabolites and blood biomarkers for oxidative stress and inflammation that have previously not been examined in this context. They indicate both that phthalate exposure is associated with significant physiological changes in the general US population, and that some of the markers examined may be useful in examining the oxidative stress and inflammatory effects of phthalate and potentially other low-dose environmental exposures in large human studies. Investigation of these relationships has the potential to improve our understanding of environmental contaminant contribution to human health and disease because of the critical roles oxidative stress and inflammation can play in disease etiology and progression.
Inflammation is a multi-stage multi-factorial response to infectious microorganisms, injury and toxicants that involves rapid-onset innate immune responses (characterized by cytokine release, acute phase proteins and leukocyte recruitment) and acquired immune responses (culminated by antibody response). Oxidative stress is commonly defined as a condition in which cellular protective antioxidant systems are overwhelmed by reactive oxidant chemicals, leading to oxidative damage of macromolecules such as DNA, proteins and lipids. Responses to inflammatory and pro-oxidant stimuli may overlap and interact, as well. Serum levels of CRP, an acute phase protein, and GGT, an enzyme involved in antioxidant defense response, have been widely applied as biomarkers of inflammation and oxidative stress, respectively, and were associated with various urinary phthalate metabolites in our previous report.8
In the present study, we report new results from an expanded analysis of relationships between urinary phthalate metabolites and additional potential markers of oxidative stress (bilirubin) and inflammation (ALP, ANC, ferritin and fibrinogen).
In limited human studies of phthalate exposure, there is considerable variation in results by phthalate or phthalate metabolite and oxidative stress exposure markers. Evidence for oxidative stress associations in humans have been observed with MEP and MEHP as indicated by a potential downstream effect (increased sperm DNA damage),6,34
with MEHP and MEHHP by increased MDA and 8-OHdG,35
and with MEHP and MEHP% by increased GGT as we reported recently.8
These associations with oxidative stress in humans are consistent with results from animal studies showing that exposure to the parent diesters DEHP and DBP stimulated reactive oxidant species generation in liver36
and increased MDA generation in liver.39
Likewise, DEHP stimulated 8-OHdG formation in rat liver.39–40
Here we used serum bilirubin level as a biomarker of oxidative stress. Bilirubin is a heme breakdown product with potent antioxidant properties that acts as a scavenger of reactive oxygen species (ROS).9
Upon reaction with ROS, bilirubin can be converted to biliverdin, which is then metabolized back to bilirubin by the enzyme biliverdin reductase. Alternatively, bilirubin can form oxidative metabolites that are excreted as biopyrrins into the urine.41
As a result of its antioxidant activity, decreased bilirubin, or increased biopyrrins, may serve as a useful biomarkers of increases in oxidative stress. Furthermore, its value as a marker has been evidenced by high correlations with other known markers of oxidative stress such as MDA (for total serum bilirubin < 16 mg/dL r = −0.74, p < 0.01).10
In the present study we observed a correlation in the opposite direction expected with the oxidative stress marker GGT, however it was very weak in magnitude (r = 0.05). Previously, bilirubin has been used as a marker of oxidative stress in prediction of cardiovascular disease.12–13,42
Here we observe a decrease in serum bilirubin, in association with all of the phthalate metabolites previously shown to be associated with oxidative stress, except for MEP. In regards to our earlier study where oxidative stress was indicated by an increase in GGT, we confirmed the relationship between MEHP and increased oxidative stress. However, we did not observe the inverse relationship with MEHP% and MEOHP, and we also observed many additional significant associations.8
The first inconsistency indicates a need for closer examination of the toxicological effects of the different DEHP metabolites, as their roles are unclear. The second suggests that bilirubin may be a more sensitive marker for detection of oxidative stress, or may be measuring different aspects of and/or effects beyond oxidative stress.
Adjusted ferritin has been used recently as a marker of inflammation in the study of childhood obesity.15
In that study, Skinner et al. introduced the ferritin/transferrin saturation ratio as a way to adjust ferritin values for whole-body iron status. Although serum ferritin has been used historically as an assessment of iron status and, therefore, anemia, ferritin is also an acute phase protein of the inflammatory response to infection and injury, similar to CRP.43
Despite the markedly smaller sample sizes for adjusted ferritin compared to other markers and measurement availability restricted to women of ages 12–59 years, we still observed significant positive associations with MEHP, MEHHP and MEOHP. Notably, adjusted ferritin was the only inflammatory marker to show a positive association with urinary levels of MEHP. The associations between MEHP and ferritin are supported by laboratory studies showing that MEHP stimulated release of inflammatory cytokines from human epithelial cells,44
human umbilical vein endothelial cells45
and rat alveolar macrophages.46
In addition to being an acute phase protein, ferritin synthesis is closely linked to heme oxygenase-1 expression, an enzyme in the antioxidant response pathway that initiates heme conversion to bilirubin.47
As such, the positive associations observed with adjusted ferritin are consistent with the inverse relationships observed with bilirubin as potential indicators of oxidative stress. Also, with this measure we observed an unexpected inverse relationship with MEP. As a recently introduced marker for inflammation, further studies are needed to interpret relationships between adjusted ferritin and environmental contaminants more completely.
Although elevated ALP levels are traditionally used as markers of liver damage and disease, recent reports that serum ALP was positively correlated with the inflammatory marker CRP in epidemiologic studies, particularly in women, suggest that ALP may serve as a marker of inflammation.50–53
In the present analysis, we observed strong positive relationships between ALP and the phthalate metabolites MEHHP, MEOHP, MnBP, MiBP, the combination of the DBP metabolites, MBzP, MCPP and MCNP. Our current findings include associations with more phthalate metabolites than what we previously reported with CRP, where we only noted significant relationships with MBzP and MiBP.8
We unexpectedly observed inverse associations of ALP with MEHP, the MEHP% variable and MEP. The relationships with MEHP and MEHP% were particularly surprising; because MEHP is the putative major toxic metabolite of DEHP, we anticipated that individuals with higher urinary concentrations of MEHP or higher MEHP% would exhibit higher indication of inflammation as reflected by the marker ALP due to either higher DEHP exposure or decreased ability to transform MEHP to its oxidized metabolites. The inverse relationships we observed could be a result of interferences due to the fact that ALP is also a marker for liver function and cytotoxicity. Furthermore, the lack of a clear biological explanation linking elevated serum ALP to inflammation leaves open the possibility that ALP may not serve as a reliable marker of inflammation in all circumstances. Low, null or inverse correlations observed in this study between ALP and other potential markers of inflammation, including CRP, ANC, adjusted ferritin and fibrinogen, may also add doubt to the validity of this measure.
ANC is a clinical measure of the immune status of an individual, commonly used to assess risk of infection after chemotherapy. Increased ANC has been used as an indicator of the acute phase response in studies of disease and, more recently, as a marker of inflammation in the study of environmental exposures.14,16,19
In the present study we observed significant positive relationships between ANC and MEHHP, MEOHP, MnBP, MiBP, DBPCOM, MBzP and MCPP, and no significant inverse relationships were found. However, inconsistent with previous studies, we did not observe a significant relationship with MEHP or MEP.
Lastly, despite a limited sample size, we investigated relationships between phthalate metabolites and fibrinogen, which has been used as a marker of inflammation in association with cardiovascular disease as well as more recently in studies of exposure to environmental tobacco smoke and other air pollutants.14,17–19,54
Though no significant relationships were observed here, the potential for use of fibrinogen as a biomarker of inflammation in response to phthalate exposure should be further explored with larger sample sizes, particularly as we observed this marker to be most highly correlated with the marker CRP and the other markers used in the present study.
The results from our analysis of phthalate metabolites with the inflammatory markers ALP and ANC were consistent with previous findings of significant positive associations between MnBP and MBzP with CRP.8
Furthermore, this analysis additionally indicated that the metabolites MiBP and MCPP may be associated with inflammation. However, whereas in the previous study we observed significant inverse relationships between more oxidized DEHP metabolites MEHHP and MEOHP and CRP, we observed positive relationships with all of the markers measured here. Again, this indicates a need for a better understanding of the toxicological effects of the DEHP metabolites, and more details on which facets of inflammation or oxidative stress are reflected in the various blood markers we examined.
There were several limitations to our study. First, as is always the case with cross-sectional data like NHANES, we are unable to establish a causative relationship between exposure to phthalates and adverse health outcomes. Second, since we made a large number of comparisons in our study, it is possible that some of our observations were due to chance. The strength and consistency of our results argue against chance findings. Lastly, because phthalate exposure is thought to be primarily dietary, and because compounds are metabolized relatively quickly, measurements taken from a single urine sample may not be representative of average exposure. However, several studies have indicated that a single measurement may represent long-term averages, though the temporal reliability varies by phthalate metabolite.30,55–56
Despite these limitations, this study also has many strengths. It offers a novel exploration of phthalate associations with oxidative stress and inflammation involving potentially under-utilized and powerful biomarkers of these pathways in a large representative population. Also, our findings were robust to secondary stratified and quintile analyses. Future exploration to more thoroughly investigate these associations should involve toxicological studies and also epidemiologic analyses using more robust study designs with longitudinal information and repeat urine samples for exposure assessment, as well as the examination of relationships in sensitive subgroups.
In conclusion, we observed that various urinary phthalate metabolites, particularly MBzP, MCPP and the metabolites of DEHP and DBP, are associated with increases in biomarkers of inflammation and oxidative stress. Not only does this have implications for the potential human health effects of phthalates, but our findings out also suggest that ALP, ANC and adjusted ferritin may be useful markers of inflammation, and that bilirubin may similarly be a useful marker of oxidative stress, in the study of health impacts of low-dose environmental chemical exposures.