evaluation of biochemical changes associated with DDT exposure

1. evaluation of biochemical changes associated with DDT exposure Prof. R Delport1,4, Prof. C de Jager2,3,4, Dr. N AneckHahn2,4, Prof. PJ Becker3, Prof. R Bornman2,4. 1 Department of Chemical Pathology, University of Pretoria, Pretoria, South Africa 2 Department of Urology, University of Pretoria, Pretoria, South Africa 3 School of Health Systems and Public Health, University of Pretoria 4 University of Pretoria Centre for Sustainable Malaria Control.

2. Introduction • Malaria estimated 627,000 deaths in 2012, mostly African children <5 yrs (WHO, 2013) • Indoor residual spraying (IRS) with technical dichlorodiphenyl-trichloroethane (DDT) • Estrogen-like properties, largely due to the o,p’–DDT isomer • Primary metabolite p,p ′-DDE marker of chronic DDT exposure • Several reproductive and developmental effects associated with DDT and DDE • p,p’-DDE inhibits androgen binding to the androgen receptor and androgen-induced transcriptional activity • Anti-androgen properties affect testosterone levels – impact on male reproductive function

3. Introduction On going UP study • Previous observations • Impaired semen quality • Lower RBP, deleterious effect on thyroid function Delport R,Bornman R, MacIntyre UE, Oosthuizen NM, Becker PJ, Aneck-Hahn NH, de Jager C. Changes in retinol-binding protein concentrations and thyroid homeostasis with non-occupational exposure to DDT. Environ Health Perspect. 2011;19(5):647-51.

4. Introduction • Previous reports • Increase in circulating t-Test and Estradiol • Association between vitamin A and spermiogenesis (RA initiates meiosis and may generate the cycle of the seminiferous epithelium and the spermatogenicwave) • Association between thyroid hormone levels and spermiogenesis

5. Introduction • Research questions • How severe is the exposure? What are the biochemical effects of exposure to DDT? What is the temporal relationship between IRS, DDT-isomer concentrations and biochemical changes? • Does Vitamin A (RBP)mediate changes in sperm morphology and kinetics? • Is there a relationship between thyroid- and sex hormone changes and sperm morphology and kinetics?

6. Methods • Cross-sectional observational study • Samples from 5 visits, three in IRS villages and 2 in non-IRS villages • Exposure assessment • Biochemical analyses • Semen analyses • NB Cytoplasmic droplets greater than one-third of the area of a normal sperm head

7. Cytoplasmic droplets

8. Results • Study population • The mean (SD) age of the participants was 22.00 (±4.34) years and the mean (minimum maximum time of residence in the area was 19 (1-37) years • IRS: • IRS is performed between October and April usually at two occasions per year in each village that was targeted for IRS.

9. Results • All subjects residing in IRS villages (n=175) had evidence of DDT uptake with at least one of the isomers above the LOD. • Of the subjects residing in non-IRS villages 92 (42%) had evidence of DDT uptake as reported in Table 1. • How severe is the exposure? • What are the biochemical effects of exposure to DDT? • What is the temporal relationship between IRS, DDT-isomer concentrations and biochemical changes?

10. Results Table 1 Comparison of level of exposure between sprayed and unsprayed villages in subjects with evidence of DDT uptake

11. Results Table 2 Comparison of biochemical insult between sprayed and unsprayed villages in subjects with evidence of DDT uptake Sub-group: Sprayed villages n=90, unsprayed villages n=74 subjects

12. Results • ▲ALP and GGT: disorders that affect the drainage of bile: • Gallstone or tumours • Alcoholic liver disease • Drug-induced hepatitis (AST 2xALT) • Malaria (+ ALT + AST + Tot Bili) • Mild or moderate ▲GGT in the presence of a normal ALP: • Alcohol or medications • ▲GGT: All forms of liver disease • ▼ALP: Malnutrition, magnesium deficiency, hypothyroidism

13. Results

14. Results

15. Results Temporal relationship between IRS, DDT-isomer concentrations and biochemical changes Figure 1 Duration of residence in villages Polynomial contrasts were determined on ranked data to assess significance of linear or quadratic trend across duration of residence quartiles in IRS villages. No significant trend was observed for ppDDE or for any biochemical variable. Time_res percentiles: 25th 14.000 50th : 19.000 75th:21.000

16. Results Figure 2 Temporal associations between variables with visits: DDT IRS is usually performed between October and April at two occasions per year in all village targeted for IRS. Visits for non-IRS villages sample collections Visit 5: July 2006 Visit 6: October 2006 Visits for IRS villages sample collections Visit 1: November 2003 Visit 2: March 2004 Visit 4: July 2005

17. Results Figure 3 Temporal associations between variables with visits: GGT Visit Mean Homogeneous Groups 4 31.080 A 2 27.429 AB 1 23.865 BC 6 20.600 C 5 20.123 C

18. Results Figure 4 Temporal associations between variables with visits: Percentage cytoplasmic droplets Visit Mean Homogeneous Groups 4 12.816 A 2 10.761 AB 1 10.116 B 5 6.7123 C 6 5.8167 C

19. Results Figure 5 Temporal associations between variables with visits: Relatively low RBP

20. Results Figure 1 Frequency distribution of % cytoplasmic droplets for normal or relatively low RBP concentrations

21. Results • The number of subjects categorized as having normal RBP-concentrations (RBP ≥ 34 mg/L) was 156 while the remainder of subjects (n=150) had relatively low (RBP < 34 mg/L) concentrations. • A significant association was observed between RBP status and evidence of DDT uptake and (chi-square 28.03; p<0.0001) in that 10% of subjects with no evidence of DDT uptake had relatively low RBP concentrations, while 55% of subjects with evidence of DDT uptake had relatively low RBP concentrations. • Does Vitamin A (RBP)mediate changes in sperm morphology and kinetics?

22. Results 77% (n=120) with DDT uptake 97% (n=146) with DDT uptake Table 2Comparison of Semen parameters associated with RBP status

23. Results Table 2Comparison of Semen parameters associated with RBP status (Cont.) All velocity patterns were recorded on the rapid progressive cells (type ‘a’ according to WHO)

24. Results Logistic regression analyses • Is there a relationship between thyroid- and sex hormone changes and sperm morphology and kinetics? • Risk for %Cytoplasmic droplets>4 (Crude) • IRS 2.63 (95% CI 1.31-5.25) • T-Test (higher) 2.38 (95% CI 1.18-4.81) • E2 (higher) 1.95 (95% CI 1-3.79) • RBP (lower) status 2.27 (1.31-3.96) • Independent risk for t-Test (Adjusted) • IRS 2.38 (95% CI 1.18-4.79) • T-Test 2.22 (95% CI 1.09-4.54) • T-Test 1.12 (0.63-1.98) • E2 2.10 (0.97-4.18) • Independent risk for RBP status • IRS 2.03 (95% CI 0.98-4.21) • RBP status 1.91 (1.07-3.43)

25. Results Logistic regression analyses • Risk for %Cytoplasmic droplets>4 (Crude) • T3 (lower) status 2.47 (95% CI 1.10-5.53) using 5th percentile as cut-off • T4 (lower) status 1.43 (95% CI 0.72-2.85) using 5th percentile as cut-off • Independent effect for IRS only when adjusted for T3 and RBP • IRS 3.29 (95% CI 1.44-7.47) • RBP status 1.61 (95% CI 0.88-2.93) • T3 status 1.29 (95% CI 0.52-3.23)

26. Summary of findings • DDT was detected in plasma of approximately 40% of subjects from non-IRS villages • A considerable variation in ppDDT and ppDDEacross visits which may be explained by time of sampling following IRS (IRS data incompletely recorded) • Peak ppDDT and ppDDE concentrations during a specific visit associated with a similar trend in GGT concentrations and % sperm with Cytoplasmic droplets • RBP levels did not mirror these findings, suggesting a time lapse for detectable effect of exposure • A cumulative trend across residence years divided into quartiles could not be established for ppDDE nor for any of the biochemical variables.

27. Summary of findings • IRS associated with significantly higher GGT, lower ALP, lower Free-T3, Free-T4 and TSH, and higher Estradiol. • Relatively low RBP levels associated with • higher semen volume and total sperm count, • significantly higher % spermatozoa with cytoplasmic droplets, • significantly lower % sperm with progressive and total motility and higher % of immotile sperm, • significantly lower number of round cells and leucocytes • Significant changes observed with CASA in Straightness index (%). Mean motility (%) was significantly higher in contrast to standard sperm analysis findings. • A shift to the right was observed in the distribution of % cytoplasmic droplets of sperm when subjects with relatively low RBP were compared to other subjects with normal RBP

28. Summary of findings • Total testosterone, Estradiol, RBP status and Free T3 were risk factors for increased% sperm with cytoplasmic droplets (>4%). Adjustment of IRS risk for RBP status resulted in establishment of RBP as independent risk factor and attenuation of risk associated with IRS. • Free-T3 below 5th percentile of the group with no DDT uptake (4.05 pmol/L) was associated with increased risk for % sperm with cytoplasmic droplets (>4%). • IRS was independently related to risk for increase % sperm with cytoplasmic droplets while risk relating to Free-T3 and RBP status was attenuated following adjustment for these risk factors.