Grantee Research Project Results
Final Report: Correlation of the CALUX Screening Assay with the HRGC/MS Measurement of Serum TCDD
EPA Grant Number: R831604Title: Correlation of the CALUX Screening Assay with the HRGC/MS Measurement of Serum TCDD
Investigators: Sweeney, Anne , del Junco, Deborah J. , Päpke, O.
Institution: Texas A & M University
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2004 through September 30, 2005
Project Amount: $31,275
RFA: Issues in Human Health Risk Assessment (2001) RFA Text | Recipients Lists
Research Category: Human Health , Pesticides
Objective:
Concerns have been raised recently regarding the validity and feasibility of using the chemically activated luciferase expression (CALUX) bioassay as a screening tool to measure dioxin exposure in large-scale epidemiologic studies. The objective of this research project was to validate the CALUX assay by comparison with the gold standard method, high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS), in 105 blood samples obtained from male Vietnam veterans during the period from 2001-2002. This grant was a follow-on to STAR Grant No. R825817.
Summary/Accomplishments (Outputs/Outcomes):
This effort was an outgrowth of a study conducted to examine the relationship between paternal serum dioxin levels, a candidate genetic susceptibility gene (CYP1A1), and neural tube defects (NTDs) among the offspring of Vietnam veterans. A total of 52 cases (Vietnam veterans who fathered a child with an NTD after return from service in the Vietnam Conflict) and 53 controls (Vietnam veterans who fathered only unaffected children after their return from service) contributed blood samples for determination of serum dioxin levels and CYP1A1 genotype. The CALUX assay and HRGC/HRMS analyses were conducted on all 105 samples. For each analytic method, distributions for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) were determined. Nonparametric and parametric correlations using log-transformed data were calculated comparing the two methods for PCDD/PCDF measurement.
Results
A total of 105 blood samples from 105 Vietnam veterans (52 cases and 53 controls) were analyzed successfully. The sample consisted of male veterans with an age range from 46-67 years (mean and median age = 53 years) at time of the blood draw. Using the CALUX screening assay for dioxins, toxic equivalency (TEQ) levels above the method’s limits of quantification were detected for only 29 samples (28%), with a range of 21.2-920.3 ppt, lipid-adjusted. Using the HRGC/HRMS method, the corresponding TEQs for PCDD/PCDFs (all 105 samples) ranged from 6.95-131.0 ppt, lipid-adjusted (geometric mean 19.38 ppt). The Pearson correlation coefficient comparing the 29 CALUX TEQs with their PCDD/PCDF TEQ counterparts was r = .27 (p = 0.15). A paradoxical higher mean PCDD/PCDF TEQ by HRGC/HRMS for the 76 samples lacking detectable CALUX TEQs (23.05 vs. 17.75 for the 29 samples with detectable CALUX TEQs, p = 0.01) further emphasizes the lack of correlation between the two methods.
Because of the poor correlation between the gold standard (HRGC/HRMS) and the CALUX, the remaining analyses present the results obtained by HRGC/HRMS. PCDD/PCDF levels were highest in the 32 Hispanic veterans (21 ppt) compared with the 71 White non-Hispanic veterans (19 ppt) and 2 African American veterans (11.6 ppt), although these differences did not reach statistical significance (p = 0.09). In contrast, differences across the three ethnic groups were highly significant for TCDD levels (p = 0.01): 2.97 ppt for Hispanic veterans, 2.33 ppt for White non-Hispanic veterans, and 1.54 ppt for the African American veterans. The overall geometric mean TCDD by the HRGC/MS method was 2.58 ppt, with a range of 0.6-9.2 ppt (n = 102, 97 percent detected).
The mean and median interval between the end of service in Southeast Asia and time of blood collection was 31 years, indicating the occurrence of approximately four half-lives for the blood dioxin levels. The crude odds ratio (OR) for the association between paternal TCDD and NTDs in offspring (expressed per unit increase in logTCDD values) was 1.78 (95% CI = 0.82-3.87). Oversampling of Hispanic controls (a result of our records linkage methodology) and the observed higher TCDD levels in Hispanic veterans necessitated appropriate adjustment for ethnicity, which increased the OR to 2.86 (95% CI = 1.18-6.93). Further adjustment for veterans’ age, year in which the veterans’ tour in Southeast Asia began, months served there, and military rank during the tour, did not change materially the OR for paternal TCDD level (2.80, 95 percent CI = 1.11-7.05). Although an inverse relationship with military rank came close, none of the other covariates were associated independently with NTDs in offspring.
Associations between NTDs in offspring and paternal blood levels were examined separately for the 6 other dioxin congeners and the 10 dibenzofuran congeners. All the ORs (also adjusted for ethnicity) were close to 1.0 and nonsignificant except for log1,2,3,7,8PeCDD (adjusted OR = 2.57, 95% CI = 1.01-6.52). Interestingly, only TCDD and PeCDD have World Health Organization TEQ weights of 1.0, whereas the remaining dioxins and dibenzofurans have TEQ weights ranging from 0.0001 to 0.5. Including both logPeCDD and logTCDD simultaneously in a logistic model reduced the ORs for logPeCDD and logTCDD to 1.49 and 2.25 respectively (a 42% reduction for logPeCDD vs. a 21% reduction for logTCDD), and only logTCDD remained borderline significant (p = 0.06 vs. p = 0.39).
As with paternal TCDD levels, important ethnic differences were observed for CYP1A1 exon7 genotype. Both African American subjects were homozygous wild type (1 case and 1 control). Of 72 Whites, 89 percent were homozygous wild type, 11 percent heterozygous, and 0 percent homozygous rare variant. Of 34 Hispanics, 47 percent were homozygous wild type, 44 percent heterozygous, and 9 percent homozygous rare variant. By itself, paternal CYP1A1 exon7 genotype was not associated with NTDs in offspring (after adjusting for ethnicity); OR was equal to 1.18 (95% CI = 0.40-3.41) when the risk-gene was defined as the homozygous wild type and the referent gene as either the heterozygous or homozygous rare variant. The association between paternal TCDD and NTD in offspring, however, was concentrated in the subgroups of fathers homozygous for the common wild-type variant, regardless of ethnicity. For example, in Whites homozygous for the common variant, the OR for TCDD-NTDs was 4.01 versus 0.75 in Whites with one or more rare variants. In Hispanics homozygous for the common variant, the OR for TCDD-NTDs was 3.60 versus 1.87 for Hispanics with one or more rare variants. The interaction term for the joint effect of logTCDD and CYP1A1 genotype, however, did not attain significance (p = 0.29). This suggestion of interaction was more pronounced when a cutpoint was used for logTCDD (i.e., > median logTCDD) than when the actual logTCDD value (on a continuous scale) was used in the logistic regression models. Although the CYP1A1 genotype X TCDD interaction term was not significant in these data, future studies with larger sample sizes and perhaps a broader array of candidate susceptibility genes are needed to assess potential interaction.
Conclusions:
Further assessments of and improvements in the validity and reliability of the CALUX assay to estimate dioxin exposures seem indicated prior to large-scale use in epidemiologic research. Paternal blood TCDD level was associated strongly and significantly with NTDs in veterans’ offspring despite the passage of about four half-lives since the veterans’ return from Vietnam service. Because paternal blood PeCDD level also appeared to be associated with NTDs in veterans’ offspring and PeCDD is not a known contaminant of Agent Orange, the observed association between NTDs in offspring and paternal dioxin exposure may not be confined to Agent Orange. Interactions with paternal CYP1A1 exon7 genotype, though not statistically significant, warrant further research to elucidate the mechanisms underlying this possible example of male-mediated developmental toxicity.
Supplemental Keywords:
risk assessment, exposure, dioxin, PCDD, PCDF, TCDD, epidemiology,, RFA, Health, Scientific Discipline, Toxics, PESTICIDES, Genetics, Epidemiology, Chemistry, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, genetic susceptability, Pesticide Types, Biology, dioxin, sensitive populations, Vietnam veterans, P450 genes, exposure, genetic polymorphisms, human exposure, environmentally caused disease, human susceptibility, bioassay, reproductive health, herbicides, dioxin exposure, genetic susceptibility, environmental hazard exposuresThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.