In the present study of up to eight years of NHANES data, we report statistically significant relationships between several urinary phthalate metabolites and serum levels of GGT or CRP. Because urinary concentrations of phthalate metabolites are considered reliable indices of exposure (Silva et al. 2004
), our results suggest that exposures to phthalates may modulate oxidative stress and inflammation pathways as reflected by GGT and CRP, respectively. While numerous statistical comparisons were made and thus we cannot rule out chance findings, previous studies have shown that several phthalates are associated with oxidative stress and inflammation which may be relevant to a range of health effects potentially linked to phthalate exposure.
Serum GGT has been used in recent studies as a biomarker of oxidative stress (Whitfield 2001
). Gamma glutamyltransferase has the critical function of breaking down extracellular glutathione into amino acids that subsequently can be taken up by the cell. Because glutathione is important in protection against oxidative stress, yet must be synthesized intracellularly from constitutive amino acids, upregulation of GGT is considered part of the cellular antioxidant response pathway. Although it is not well understood how oxidative stress could increase serum concentrations of GGT, one plausible explanation is that oxidative stress or glutathione conjugation of reactive metabolites deplete intracellular GSH, followed by compensatory increase of GGT which is then released into the serum (Whitfield 2007
In addition, serum GGT is used clinically as a liver function test, which could be explained by oxidative stress effects of alcohol on the liver (Whitfield 2007
). Interestingly, several of the phthalates, most notably DEHP, cause changes in liver function in laboratory rodents (Rusyn et al. 2006
). Our findings of an association between urinary MEHP and serum GGT are consistent with these laboratory animal studies. Serum GGT has also been used as a biomarker of excessive or harmful consumption of alcohol (Whitfield 2007
). Due to missing data we did not include alcohol consumption in our final models in order to maximize the sample size of our study. Our decision to not include alcohol consumption in the final models was further supported by the lack of an association between alcohol use and urinary phthalate metabolites among participants who contributed data on alcohol use, suggesting alcohol consumption would not act as a confounder in our analysis.
MEHP, a monoester metabolite of DEHP, was positively associated with GGT, as were elevated quintiles of MEP, a monoester metabolite of DEP. Interestingly, these same two urinary metabolites were also associated with significant elevations in sperm DNA damage in 379 men recruited through a US fertility clinic (Hauser et al. 2007
), and oxidative stress is likely to be the primary cause of sperm DNA damage (Aitken and Deluliis 2010
; Aitken et al. 2008). Our findings are also consistent with experimental studies reporting MEHP induction of oxidative stress in rat testis, measured as increased lipid peroxidation (Santhosh et al. 1998
; Rusyn et al. 2006
), increased MDA and 8-OHdG levels (Seo et al. 2004
), and increased generation of reactive oxygen species with simultaneous declines in testis concentrations of the antioxidants glutathione GSH and ascorbic acid (Kasahara et al. 2002
). Likewise, our finding with MEP, the monoester metabolite of DEP, is consistent with a study of fish that found increased oxidative stress as measured by increased hepatic lipid peroxide and compensatory changes in the oxidative stress defense enzymes (Kang et al. 2010
MEHP is metabolized further to the oxidized metabolites MEHHP, MEOHP, and MECPP. In contrast to MEHP, the latter three metabolites of DEHP were inversely associated with GGT to varying degrees. While this is not consistent with a recent study of 960 Korean adults that reported positive associations between MEHHP and MEOHP with urinary markers of oxidative stress (MDA and 8-OHdG)(Hong et al. 2009
), it is consistent with a study of men from an infertility clinic that reported inverse associations between MEHHP and MEOHP and sperm DNA damage (Hauser et al. 2007
). Because MEHP is likely the most bioactive form of DEHP (Erkekoglu et al. 2010
; Chu et al. 1978
), it has been suggested that increased urine concentrations of MEHHP, MEOHP and other oxidized DEHP metabolites may reflect an individual's increased ability to convert MEHP to less toxic metabolites that are more easily excreted in urine (Hauser 2008
; Meeker et al. 2007
). As such, the positive association of MEHP with GGT and inverse associations of oxidized MEHP metabolites with GGT would be consistent with MEHP being the more toxic DEHP metabolite.
Interindividual differences in the proportion of MEHP in relation to other less toxic metabolites may serve as a marker for individual metabolic susceptibility to DEHP exposure (Hauser 2008
; Meeker et al. 2007
). This may be further supported by our secondary analysis of the MEHP% variable. We found that increased MEHP%, indicating higher levels of MEHP in relation to the oxidized DEHP metabolites (MEHHP and MEOHP), was associated with a significant increase in GGT. In addition, we found that a MEHP*MEHP% interaction term was positive and significant for both GGT and CRP. These findings indicate that an increase in the proportion of MEHP in comparison to other DEHP metabolites may result in an increased effect in relation to the same amount of MEHP, which further supports the hypothesis that the relative concentration of MEHHP and MEOHP may represent individuals who are more likely to efficiently oxidize MEHP to less toxic metabolites and thus may be less susceptible to effects related to DEHP exposure compared to individuals with a high MEHP%. Another factor that likely contributes to the variability in MEHP% between individuals relates to sample timing if human exposure to DEHP is episodic. Since the oxidized DEHP metabolites have a longer biological half-life (10-15 hours) compared to MEHP (5 hours) (Lorber et al. 2010
), a higher MEHP% for an individual may also reflect that the person experienced a more recent DEHP exposure event compared to an individual with a low MEHP%. However, high reliability in urinary MEHP% within individuals over time was recently reported (Adibi et al. 2008
) which may provide support for underlying inter-individual differences in DEHP metabolism.
Some of our findings appear to be inconsistent with the literature, which could be due to chance findings, differences in study design or populations, outcome measures being assessed, phthalate exposure levels, methods used to measure phthalate exposure, statistical methods and covariates that were considered, or other reasons. MBzP and various DBP metabolites have been reported to be associated with oxidative stress in previous studies (Hong et al. 2009
; Seo et al. 2004
), but neither were associated with GGT in the present analysis. We also observed inverse relationships between GGT and MCNP (an oxidized metabolite of diisodecyl phthalate) and MCPP (an oxidized metabolized of both DBP and di-n
-octyl phthalate). Although these latter phthalates are less studied than the other phthalates we evaluated, our findings with these phthalates may provide further indication for an inverse relationship between oxidative metabolism of phthalates and serum GGT. Further research on the metabolism and mechanisms of action for MCNP and MCPP and their parent compounds will be needed to better interpret the inverse associations of GGT with these metabolites.
Increased serum concentration of CRP is widely used as a biomarker of inflammation associated with various diseases (Marnell et al. 2005
) and, more recently, with environmental toxicant exposure (Everett et al. 2010
). Named for its ability to precipitate the so-called “C” polysaccharide of Streptococcus pneumonia
, CRP binds to phosphocholine of microorganism cell walls and damaged host cell membranes, as well as nuclear antigens (Marnell et al. 2005
). As an acute phase serum protein, CRP concentrations in serum increase markedly in response to cell injury or infection. Although CRP is made in various tissues, increased serum levels of CRP are mainly the result of increased CRP production in the liver in response to the pro-inflammatory cytokine interleukin (IL)-6 (Bottazzi et al. 2010
Serum CRP was positively associated with MBzP, MiBP, and the sum of DBP metabolites in urine. These associations are consistent with previous experimental studies. MBzP and MiBP stimulate increased PPARα expression in rodent cell lines, indicating increased peroxisome proliferation and inflammation (Hurst and Waxman 2003
, Bility et al. 2004
). Similarly, MnBP and MBzP increased release of the inflammatory cytokines IL-6 and IL-8 from human epithelial cells in vitro
(Jepsen et al. 2004
). Although MEHP stimulated pro-inflammatory cytokine release from cultured human epithelial cells (Jepsen et al. 2004
; Rael et al. 2009
), we did not observe an association between urinary MEHP and serum CRP in the present study. However, CRP was inversely associated with the oxidized DEHP metabolite MEOHP. Furthermore, consistent with our hypothesis surrounding the importance of interindividual variability in DEHP/MEHP metabolism, CRP was positively associated with a MEHP*MEHP% interaction term.
A secondary analysis of pregnant women revealed a suggestive positive relationship between MnBP and the sum of DBP metabolites with CRP despite a much smaller sample size. Interestingly, inflammation is associated with preterm birth (Romero et al. 2007
) and MnBP was the phthalate metabolite most strongly associated with preterm birth in our recent exploratory nested case-control study that measured third trimester urinary phthalate metabolites in 60 pregnant women (Meeker et al. 2009
There were several limitations to the present analysis. First, due to the cross-sectional study design we are unable to make any conclusions regarding causation in the relationships between phthalate exposure and inflammation or oxidative stress. Also, since the data were cross-sectional only one urine sample per subject was analyzed for phthalate metabolite levels, and it has been suggested that one data point may not be representative of the subject's average body burden (Fromme et al. 2007
). However, several studies have demonstrated that single spot urine sample phthalate levels may be representative of long-term averages though temporal reliability likely varies by metabolite (Hauser et al. 2004
, Teitelbaum et al. 2008
; Suzuki et al. 2009
). The dataset was also limited to single serum measures of CRP and GGT which may also vary over time (Meier-Ewert et al. 2001
; Gu et al. 2009
; Lazo et al. 2008
). In addition, there are markers other than CRP and GGT (e.g. cytokines, MDA, 8-OHdG, and many others) that may be potentially more sensitive for examining inflammation and oxidative stress responses to environmental contaminants that were not available in the NHANES datasets. Future studies of oxidative stress and inflammatory outcomes in relation to phthalate exposure should examine multiple markers for comparison, and, where possible, measure these biomarkers in fluids or tissues most relevant to the particular downstream health outcomes of interest. Finally, our secondary analysis of pregnant women, who may be particularly susceptible to inflammation- and oxidative stress-inducing xenobiotics, suffered from a much smaller sample size and lacked information on other important pregnancy factors (e.g., preeclampsia, gestational diabetes, and other complications).
Despite these limitations our study had several strengths that warrant further research on this topic. First, the study utilized state-of-the-art methods to measure exposure biomarkers of phthalate metabolites in urine and serum markers of inflammation and oxidative stress. Second, the use of the NHANES dataset allowed for a large sample size and excellent statistical power. Our primary findings were also robust to multiple statistical modeling approaches and various sensitivity analyses that we conducted in our secondary analyses. Finally, in our primary analysis of non-pregnant participants, our results are likely representative of the US population and thus have good generalizability.
In conclusion, we found that several phthalate monoester metabolites that are detected in a high proportion of urine samples from the US general population are associated with increased serum markers of inflammation and oxidative stress. Though causation cannot be determined, these results suggest that the relationships between phthalates and inflammation, oxidative stress, and related adverse health effects deserve further exploration in more detailed molecular epidemiology studies, especially among potentially sensitive subgroups. In addition, future investigations should be aimed at explaining the potential inverse (i.e., protective) relationships between oxidized phthalate metabolite concentrations in urine and serum markers of inflammation and oxidative stress.