Is There an Association Between Extreme Levels of Boron Exposure and Decrease in Y:X Sperm Ratio in Men? Results of an Epidemiological Study/Erkeklerde Y:X Sperm Oranindaki Azalma ile Asiri Duzeyde Bor Maruziyeti Arasinda Bir iliski Var midir? Epidemiyolojik Calisma Sonuclari.
Boric acid and borates are classified as toxic to reproduction under "Category 1B" with the hazard statement of "H369 FD" in the European Regulation on Classification, Labelling and Packaging of Substances and Mixtures. (1) The scientific background of this classification is based on the results of animal experiments (hazard assessment). In fact, the boron concentrations tested in animal experiments were too high and from this point of view these dose levels were not relevant to humans. Nevertheless, this classification triggered epidemiological studies in areas of high boron exposure in order to assess the risk of the daily boron exposure levels in those areas. The first study was conducted in mining areas located in Kuandian City, China. (2) The blood boron concentration reported in that study was 499.2[+ or -]790.6 (20.4-3568.9) ppb. In spite of this extreme level of boron exposure the reproductive toxicity biomarkers reported in that study did not indicate adverse effects on male reproduction. The second comprehensive epidemiological study was conducted by our study group in workers employed in Bandirma boric acid production plant. The mean blood boron concentration of the high exposure group in our study was 223.89[+ or -]69.49 (152.82-454.02) ng/g and no boron-mediated unfavorable effects on reproductive toxicity parameters of male workers were observed. (3,4) The results of our study were in agreement with those of the study conducted in China. Accordingly, both studies clearly indicate that boron-mediated adverse effects on male reproduction do not seem possible even under extreme occupational exposure conditions. However, in 2008 Robbins et al. (5) reported a boron-mediated decrease in Y:X sperm ratio in workers residing in mining areas of Kuandian City. Essentially, a boron-mediated decrease in Y:X sperm ratio was not observed in earlier epidemiological studies or even in animal experiments. Therefore, the results of the study published by Robbins et al. (5) had to be verified by an epidemiological study. The major aim of the present epidemiological study was to corroborate or refute the negative association between the high level of boron exposure and the decrease in the Y:X sperm ratio in men suggested by Robbins et al. (5)
MATERIALS AND METHODS
The blood and semen samples were sampled in accordance with the study protocols approved by the Ethics Committee of Hacettepe University School of Medicine (HEK 08/167, date: 22/10/2008). All participants gave their informed consent prior to participation in the project.
The present study was performed using the blood and semen samples obtained within the scope of our "Boron Project--I", which was completed in 2010. (3,4) The "Boron Project--I" was conducted in Bandirma boric acid production zone and 204 workers were enrolled in that study. After the project was completed, the remaining semen samples were stored under appropriate conditions (cryopreserved in liquid nitrogen). The total number of remaining semen samples was 163 and 86 of them were from workers employed in the boric acid production facilities and were assigned as the samples of the exposed group of workers. The rest of the semen samples, from workers employed in the steam power plant, energy supply unit, demineralized water plant, mechanical workshop etc., were assigned as the control samples (n=77). The demographic information, blood boron concentrations, semen boron concentrations, and sperm concentrations of these 163 workers had been gathered within the scope of the "Boron Project--I". More detailed information about the samples and the sampling area were provided in our previously published studies. (3,4)
Blood and semen samples were analyzed by inductively coupled plasma mass spectrometry with a flow injection system. A special sample introduction system that included a perfluoroalkoxy spray chamber and a nebulizer with an alumina injector tube in a quartz torch was used for this study. The details of the above-mentioned analyses were published in our previous study. (3)
The semen samples were sampled and analyzed in accordance with the recommendations of the World Health Organization. (6) Sperm concentrations were determined in fresh semen samples using an SQA-V Gold Sperm Quality Analyzer. The results were expressed as [10.sup.6] sperm cells/mL.
Determination of Y:X sperm ratios
Y- or X-bearing sperm cells in semen samples were detected using fluorescence in situ hybridization (FISH). The Cytocell FAST FISH prenatal X, Y, and 18 Enumeration Probe Kit (LPF 002) was used in the detection and quantification of chromosomes X, Y, and 18 by FISH. The probes are specific for the alpha satellite DNA sequences in the DXZ1, DYZ3, and D18Z1 regions of chromosomes X (green), Y (orange), and 18 (blue), respectively.
The semen samples were removed from the liquid nitrogen, thawed at room temperature in PBS solution, and centrifuged in an appropriate centrifuge tube at 500xg for 5 min as the initial step of the procedure. The supernatant was gently discarded and the precipitated sperm cells were used in sperm FISH analysis. The cells were resuspended in 10 mL of 0.075 M KCl and left at 37[degrees]C for 1 h. After centrifugation for 5 min at 1000 rpm the supernatant was discarded and the precipitated sperm cells were resuspended in 5 mL of (4[degrees]C) Carnoy's solution. The cell suspension was centrifuged again for 5 min at 1000 rpm. This process was repeated 3 times. Afterwards, a sufficient amount of these sperm cells was transferred directly onto the slide and allowed to dry at room temperature. The slides then were washed in 2 x saline-sodium citrate (SSC) (3 min), 70% ethanol (3 min), 85% ethanol (3 min), and finally in 100% ethanol (3 min). The slides and the probe were prewarmed on a 37[degrees]C hotplate for 10 min. A sufficient amount of probe mixture (10 [micro]L) was pipetted onto the sperm cells and coverslipped. The slides were placed on a hotplate at 75[degrees]C for 5 min for denaturation. Afterwards the slides were transferred into a humid and dark (lightproof) incubator at 37[degrees]C for ~18 h (overnight) for hybridization. After the waiting period, the coverslips were removed and the slides were immersed into 0.4 x SSC at 67[degrees]C for 30 s. The washing process continued with 2 x SSC + Tween-20 (room temperature) again for 30 s. The slides were left to drain. Next, 15 [micro]L of DAPI antifade was applied onto each hybridization area, which was then covered with a coverslip. The slides were left in the dark for 15 min to allow color development. Afterwards the slides were viewed under a fluorescence microscope. The sperm cells were analyzed via fluorescence microscopy using a Leica DM1000 microscope with DAPI, AQUA, G/R filters. Leica provided a suitable set-up for simultaneous visualization of DAPI and the triple fluorochromes (spectrum green, orange, and aqua). A total of 5000 morphologically preserved sperm cells were counted per sample by one experienced scorer. The microscopic images of some sperm cells are shown in Figure 1.
The Kruskal-Wallis and Wilcoxon Mann-Whitney U tests were used to analyze some of the variables in Table 1. The statistical significance between the number of girls and boys (at birth) was analyzed using the [chi square] test. Box plots (Figure 2), Pearson's correlation coefficient, and linear regression (Figure 3) show the empirical distribution and possible linear dependencies.
All statistical tests were performed with IBM SPSS Statistics Version 23. The significance levels of the tests were set at 0.05.
The study population was composed of control (n=77) and exposed (n=86) groups of workers employed in Bandirma boric acid production zone. The "ages" and "years of employment" of the workers did not differ significantly between the control and exposed groups as shown in Table 1. The participating workers were healthy and were not taking any medication during the sampling period.
The mean blood boron concentration of the exposed group (141.55 ng B/g blood) was significantly higher (p<0.05) than that of the control group (63.56 ng B/g blood) (Table 1, Figure 2). This significant difference between the control and exposed groups supports the high level of daily boron exposure for the workers assigned to the exposed group. This finding was also supported by the semen boron concentrations of workers. The mean semen boron concentrations of the control and exposed groups were significantly different (p<0.05) from each other and thereby provided additional support for the extraordinary daily boron exposure conditions in our study population (Table 1).
The high level of boron exposure in the exposed group of workers did not adversely affect the sperm concentrations or Y:X sperm ratios of workers as shown in Table 1 and Figure 2. This finding is also supported by the lack of statistically significant correlation (Pearson, P>0.05) between blood/semen boron concentrations and Y:X sperm ratios in workers (Figure 3).
Boron-mediated effects on the sex ratio at birth were also investigated within the scope of this study. However, no shift in the sex ratio at birth toward females was observed. As shown in Table 1, the percentage of girls at birth in the exposed group was not significantly higher than that in the control group (P>0.05).
Some earlier studies performed on the relation between boron exposure and sex ratio at birth reported an excess of female offspring in highly boron exposed populations. In spite of an increase in female offspring, the increase was not statistically significant in these previously published studies. (7-9) In 2008, however, Robbins et al. (5) reported a statistically significant relation between boron exposure and Y:X sperm ratio in male workers employed in a boron mining company in Kuandian City, China. The authors reported a significant association between a high level of boron exposure and a decrease in Y-bearing versus X-bearing sperm cells. Moreover, the excess of female offspring around the boron mining area in Kuandian City (China) was attributed to the decrease in the Y:X sperm ratios in highly boron exposed men. (5) Although the results of that study were criticized by some authors due to weaknesses in the study design, a study confirming or rejecting the results of this study has not been performed thus far. (10) Therefore, the major aim of the present study was to corroborate or refute the results reported by Robbins et al. (5)
The Bandirma boric acid production zone is a suitable place for investigating boron-mediated unfavorable effects on the Y:X sperm ratios in men. This study area provided a wide range of daily boron exposure, which made it possible to study dose-dependent responses of the studied parameters.
The high level of daily boron exposure in our study area was supported by using boron exposure biomarkers. Blood boron and to a lesser extent semen boron concentrations were suggested as biomarkers of boron exposure in some earlier studies. (3,4,11-16) Accordingly, blood boron and semen boron concentrations were used as the biomarkers of boron exposure in order to prove the high level of daily boron exposure of workers classified in the exposed group. The significantly high mean blood boron and semen boron concentrations in the exposed group support the extraordinary daily boron exposure level in our study population as shown in Table 1.
In spite of this high level of daily boron exposure, the mean Y:X sperm ratio of the exposed group was not significantly different from that of the control group (P>0.05) as shown in Table 1. The sex ratio (boys/girls) at birth in the control and exposed groups was 0.94 and 1.17, respectively. In this regard, the numbers of boys and girls in the control and exposed groups were not significantly different (P>0.05) as presented in Table 1.
Consequently, boron-mediated decrease in Y:X sperm ratios in men or excess of female births were not observed in our highly boron-exposed study population. Under these circumstances, our results refute the association between a high level of boron exposure and decreased Y:X sperm ratios in men that was reported by Robbins et al. (5)
The study was funded by Eti Mine Works General Management and BOREN (2008-G0207).
Conflicts of Interest: No conflict of interest was declared by the authors.
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[iD] Can Ozgur YALCIN, [iD] Aylin USTUNDAG, [iD] Yalcin DUYDU (*)
Ankara University, Faculty of Pharmacy, Department of Toxicology, Ankara, Turkey
(*) Correspondence: E-mail: email@example.com, Phone: +90 312 203 31 18 ORCID-ID: orcid.org/0000-0001-7482-086X
Received: 02.06.2018, Accepted: 26.06.2018
Table 1. Characteristics of male workers assigned to the control and exposed groups Parameters Control group, Exposed group, p value n=77 n=86 Age 42.86[+ or -]5.06 42.45[+ or -]4.61 P>0.05 (33-48) (33-48) Years of 18.02[+ or -]6.58 15.76[+ or -]7.16 P>0.05 employment (2-23) (1.62-22) Sperm 68.08[+ or -]41.37 64.96[+ or -]56.09 P>0.05 concentration (9.69-139.21) (11.06-161.98) (x[10.sup.6] /mL) Blood boron 63.56[+ or -]43.89 141.55[+ or -]80.43 p<0.05 conc., ng B/g blood (21.45-134.57) (41.28-286.30) Semen boron 1127.78[+ or -]1713.96 1703.42[+ or -]1895.09 p<0.05 conc., (66.4-5115.6) (452.60-7067.85) ng B/g semen Y:X sperm 0.99[+ or -]0.03 0.99[+ or -]0.02 P>0.05 ratio (0.94-1.01) (0.96-1.01) Fathered 136 (1.77) (*) 150 (1.74) (*) P>0.05 children Girls 70 (0.91) (*) 69 (0.80) (*) P>0.05 (at birth) Boys 66 (0.86) (*) 81 (0.94) (*) P>0.05 (at birth) Girls % 51.47 46.00 P>0.05 (at birth) The results are given as mean [+ or -] standard deviation (5th-95th), (*) mean values
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|Title Annotation:||ORIGINAL ARTICLE|
|Author:||Yalcin, Can Ozgur; Ustundag, Aylin; Duydu, Yalcin|
|Publication:||Turkish Journal of Pharmaceutical Sciences|
|Date:||Mar 1, 2019|
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