The Healthy Men Study: An Evaluation of Exposure to Disinfection By-Products in Tap Water and Sperm Quality

BackgroundChlorination of drinking water generates disinfection by-products (DBPs), which have been shown to disrupt spermatogenesis in rodents at high doses, suggesting that DBPs could pose a reproductive risk to men. In this study we assessed DBP exposure and testicular toxicity, as evidenced by altered semen quality.MethodsWe conducted a cohort study to evaluate semen quality in men with well-characterized exposures to DBPs. Participants were 228 presumed fertile men with different DBP profiles. They completed a telephone interview about demographics, health history, water consumption, and other exposures and provided a semen sample. Semen outcomes included sperm concentration and morphology, as well as DNA integrity and chromatin maturity. Exposures to DBPs were evaluated by incorporating data on water consumption and bathing and showering with concentrations measured in tap water. We used multivariable linear regression to assess the relationship between exposure to DBPs and adverse sperm outcomes.ResultsThe mean (median) sperm concentration and sperm count were 114.2 (90.5) million/mL and 362 (265) million, respectively. The mean (median) of the four trihalomethane species (THM4) exposure was 45.7 (65.3) μg/L, and the mean (median) of the nine haloacetic acid species (HAA9) exposure was 30.7 (44.2) μg/L. These sperm parameters were not associated with exposure to these classes of DBPs. For other sperm outcomes, we found no consistent pattern of increased abnormal semen quality with elevated exposure to trihalomethanes (THMs) or haloacetic acids (HAAs). The use of alternate methods for assessing exposure to DBPs and site-specific analyses did not change these results.ConclusionsThe results of this study do not support an association between exposure to levels of DBPs near or below regulatory limits and adverse sperm outcomes in humans

Days to onset of hypersensitivity symptoms in patch test confirmed cases of abacavir HSR (range <2–19 days, median 8 days; n = 23).

<p>Days to onset of hypersensitivity symptoms in patch test confirmed cases of abacavir HSR (range <2–19 days, median 8 days; n = 23).</p

Abacavir ELISpot responses.

<p><b>A</b>. Mean abacavir ELISpot responses for HLA B*57:01 positive or negative individuals, that were either abacavir naïve or treatment experienced. <b>B</b> Abacavir ELISpot responses in patch test confirmed abacavir HSR patients plotted relative to time from first exposure to abacavir (25-562 SFU/10⁶ PBMC, n = 12). Positivity of abacavir ELISpot responses was defined using a cut-off response of ≥ 10 SFU/10⁶ PBMC above background, and differences in proportions of positive responses were assessed using a Fishers exact test.</p

Assessment of abacavir responsive memory or naïve T cells.

<p><b>A</b>. Sorting gates used to collect total CD4+ and CD8+ memory phenotype (green polygon) and naïve phenotype T cells (blue rectangle) or <b>B</b>. CD8+ memory or naïve phenotype T cells only. <b>C</b>. Protocol used for the selection, priming and re-stimulation of sorted naïve and memory T cells from cryopreserved HLA-B*57:01 positive donor PBMC. <b>D</b>. Representative plots of in-vitro cultures of HLA-B*57:01 positive memory (left most pairs of plots) or naïve (right most pairs of plots) phenotype T cells derived from sorting strategy A. Cultures were re-stimulated with APCs treated with abacavir (C1R.B57.ABC) or untreated (C1R.B57), respectively. <b>E</b>. Representative plots of in-vitro cultures of HLA-B*57:01 positive memory (left most pairs of plots) or naïve (right most pairs of plots) phenotype CD8+ T cells derived from sort strategy B. Cultures were re-stimulated with APCs as described above.</p

Abacavir responsive CD8+ T cells can be expanded from unsorted, memory and naïve phenotype T cells from abacavir-unexposed HLA-B*57:01 positive donors.

<p>CD8+/IFN-γ frequencies are compared across T-cell populations which had been cultured ± abacavir, and then re-stimulated with APCs treated with abacavir (yes) or untreated (no), respectively. <b>A</b>: CD8+/IFN-γ frequencies in unsorted PBMC from HLA-B*57:01 negative (n = 3) and positive donors (n = 8). CD8+/IFN-γ frequencies in HLA-B*57:01 positive donors according to memory and naïve phenotype in <b>B</b>: sorted CD4+ and CD8+ T cells (n = 8) and <b>C</b>: sorted CD8+ T cells only (4 samples, n = 3 donors). Each donor is represented by the same symbol in the different figures; group median CD8+ /IFN-γ+ T-cell frequencies are indicated by a horizontal line. Pairwise differences in observed frequencies according to abacavir stimulation are assessed by a donor-stratified Wilcoxon test.</p

Immunization of an HLA-B*57:01 donor with the yellow fever vaccine results in the expansion of populations of CD8+ T cells that detect both the wild type epitope and abacavir dependent variant epitopes.

<p>T cells expanded from a yellow fever vaccinated B*57:01 donor were labelled with anti-CD3 and CD8 and a mixture of K9<u>F</u>-B*57:01-PE, K9<u>A</u>-ABC-B*57:01-APC, and K9<u>V</u>-ABC-B*57:01-BV421 tetramers. CD8+ T cells were gated and the combination of K9<u>F</u>-B*57:01-PE and K9<u>A</u>-ABC-B*57:01-APC tetramer stained CD8+ T cells are shown in A. Subpopulations of tetramer stained cells are gated: P4 (pink); P5 (yellow); P6 (green); P7 (blue); P8 (orange); P9 (purple). The combinations of K9<u>F</u>-B*57:01-PE and K9<u>V</u>-ABC-B*57:01-BV421 tetramer stained CD8+ T cells are shown in B. The colors identify the position of the gated tetramer stained populations in panel A.</p