Genotoxicity assessment of perfluoroalkyl substances on human sperm

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are synthetic chemicals that have been used in industry and consumer products. Because the presence of PFAS has been identified in humans and the environment in the last decade, human exposure to PFAS is a current public health concern. It has been shown that some PFAS lead to adverse health effects in the male reproductive system. However, there is no information about probable genotoxic effects of these chemicals on sperm cells. This study aimed to investigate the possible genotoxic damage on human sperm cells exposed to certain major PFAS compounds that were selected considering their extensive usage, high persistence in the environment, and high bioaccumulation in humans. These PFAS are perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorohexanoic acid (PFHxA). The alkaline comet assay was used to detect the DNA damage to sperm. Sperm cells were treated with 0.1–1 mM of each PFAS at 32°C for 1 h to obtain optimal survival. As a result of the experiments, it was discovered that the exposure to PFOS, PFOA, PFNA, and PFHxA did not cause significant levels of cytotoxicity and did not cause damage to sperm DNA under these conditions. The results suggest that the exposure to these PFAS did not interfere with sperm DNA. Indirect toxicity mechanisms should be taken into account to assess the association between the PFAS exposure and male reproductive toxicity.

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P–033 In vitro protective effect of α -tocopherol and anthocyanin against TiO2-NPs induced genotoxicity on human spermatozoa

Human Reproduction ◽

10.1093/humrep/deab130.032 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

M Santonastaso ◽

F Mottola ◽

C Iovine ◽

N Colacurci ◽

L Rocco

Keyword(s):

Genomic Stability ◽

Human Sperm ◽

Negative Control ◽

Sperm Cells ◽

Tio2 Nps ◽

Sperm Dna Damage ◽

Sperm Dna ◽

Rapd Pcr ◽

Intracellular Ros

Abstract Study question Do α -tocopherol and anthocyanin counteract human sperm DNA damage provoked by titanium dioxide nanoparticles (TiO2-NPs)? Summary answer: ↑-tocopherol and anthocyanin are able to counteract TiO2-NPs genotoxicity on human sperm cells reducing oxidative stress. What is known already The environmental release and the extensive use of TiO2-NPs have been implicated in poor human sperm functionality.TiO2-NPs is genotoxic on human sperm cells causing a loss of sperm DNA integrity, an increase of apoptotic process and a reduction of genomic stability related to an over production of intracellular ROS.Antioxidants are the substances that can scavenge free radicals. α -tocopherol, present in vegetables, is the most important lipophilic antioxidant involved in restore sperm parameters in several experimental models. Anthocyanin, present in Aronia melanocarpaand belonging to the flavonoid family, is able to prevent damage caused by varicocele-induced ROS in rats. Study design, size, duration Semen samples from 132 men were obtained by masturbation following 3–5 days sexual abstinence and were examined for sperm concentration, viability, motility and morphology according to WHO 2010. The sperm cells, after purification with 45–90% double density gradient, were exposed in vitro to 1 µg/L of TiO2-NPs, 1 µg/L of TiO2-NPs whit 1 mg/L of anthocyanin and 1 µg/L of TiO2-NPs plus 1 mg/L of α -tocopherolfor 15,30,45 and 90 minutes at 37 °C. Participants/materials, setting, methods Sperm motility and concentration were analyzed with Makler camber while sperm viability and morphology were evaluated by Eosin-Nigrosin Test and by Testsimplets® prestained slides respectively. Antigenotoxicity was evaluated by Comet assay, TUNEL test and RAPD-PCR technique and Genomic Template Stability (GTS,%) calculation. The intracellular ROS level was assessed by DFC Assay. The data were analyzed using ANOVA test by GraphPad Prism 6 and considered significant if p-value ≤ 0.05. Main results and the role of chance Sperm analyses showed none statistically significant changes in sperm viability and motility (progressive and non-progressive) for each treatment. Anthocyanin and α -tocopherol counteracted sperm DNA damage induced in vitro by TiO2-NPs neutralizing ROS in a time-dependent way. Comet assay displayed that both antioxidants reduced sperm DNA strand breaks produced by TiO2-NPs, in particular the damage was no longer statistically significant starting from 30 and 90 minutes of anthocyanin-TiO2-NPs and α-tocopherol-TiO2-NPs co-exposure respectively. The antioxidant supplementation induced a statistically decrease of sperm DNA fragmentation provoked by TiO2-NPs after 45 co-treatment minutes.The RAPD-PCR technique evidenced variations of bands number in the TiO2-NPs treated sperm compared to the negative control and anthocyanin and α -tocopherol-TiO2-NPs co-treated samples. Human sperm genomic stability increased after anthocyanin and α -tocopherol TiO2-NPs co-exposure respect to the TiO2-NPs single treatment, until it almost reaches the negative control at 90 minutes. Intracellular ROS percentage was significantly lower both in anthocyanin and α -tocopherol TiO2-NPs co-treated compared to TiO2-NPs alone starting from 45 minutes. Limitations, reasons for caution In vitro study. Wider implications of the findings: Our results showed a protective effect of anthocyanin and α -tocopherol on human DNA by neutralizing intracellular ROS induced by TiO2NPs. We suggest anthocyanin and α -tocopherol as suitable molecules to defend human sperm DNA from oxidative stress, with a potentially role in treatmentof male infertility due to environmental factors. Trial registration number None

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Research Article. Perfluoroalkylated substances in human urine: results of a biomonitoring pilot study

Biomonitoring ◽

10.1515/bimo-2017-0001 ◽

2017 ◽

Vol 4 (1) ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Christina Hartmann ◽

Wolfgang Raffesberg ◽

Sigrid Scharf ◽

Maria Uhl

Keyword(s):

Human Urine ◽

Consumer Products ◽

Laboratory Animals ◽

Adverse Health Effects ◽

Research Article ◽

Synthetic Chemicals ◽

Wide Range ◽

Study Population ◽

Long Chain ◽

Perfluoroalkylated Substances

AbstractPerfluoroalkylated substances (PFASs) are a class of synthetic chemicals used in a wide range of processes and products due to their unique physicalchemical properties. Through intake of PFASs via food or several consumer products, humans can be exposed. Long-chain PFASs have been associated with adverse effects in laboratory animals, and there is also evidence for adverse health effects in humans. Although investigations of human exposure are mainly conducted in blood samples, some studies have shown that especially short-chain PFASs can be detected in human urine. In the present study, a sensitive analytical method was adapted for the measurement of 12 PFASs in human urine samples by HPLC-MS/MS. For verifying this method, concentrations in 11 male and female participants aged 25-46 years were analysed. In the study population, ranges of urinary PFASs concentrations were n.d.- 8.5 ng/l for perfluoropentanoic acid, <LOQ-3.0 ng/l for perfluorohexanoic acid, n.d.-1.8 ng/l for perfluorohexane sulphonate, n.d.-0.99 ng/l for perfluoroheptanoic acid, 0.79-5.1 ng/l for perfluorooctanoic acid, <LOQ-4.9 ng/l for perfluorooctane suphonate, and <LOQ-0.57 ng/l for perfluorononanoic acid. For the other investigated PFASs, no urinary exposure could be identified in any of the samples. The present study shows that several shortchain PFASs are detectable in human urine.

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In Vitro Effects of Titanium Dioxide Nanoparticles (TiO2NPs) on Cadmium Chloride (CdCl2) Genotoxicity in Human Sperm Cells

Nanomaterials ◽

10.3390/nano10061118 ◽

2020 ◽

Vol 10 (6) ◽

pp. 1118 ◽

Cited By ~ 3

Author(s):

Marianna Santonastaso ◽

Filomena Mottola ◽

Concetta Iovine ◽

Fulvio Cesaroni ◽

Nicola Colacurci ◽

...

Keyword(s):

Oxidative Stress ◽

Titanium Dioxide ◽

Dna Fragmentation ◽

Titanium Dioxide Nanoparticles ◽

Human Sperm ◽

Sperm Cells ◽

Sperm Dna Fragmentation ◽

Sperm Dna ◽

And Oxidative Stress

The environmental release of titanium dioxide nanoparticles (TiO2NPs) associated with their intensive use has been reported to have a genotoxic effect on male fertility. TiO2NP is able to bind and transport environmental pollutants, such as cadmium (Cd), modifying their availability and/or toxicity. The aim of this work is to assess the in vitro effect of TiO2NPs and cadmium interaction in human sperm cells. Semen parameters, apoptotic cells, sperm DNA fragmentation, genomic stability and oxidative stress were investigated after sperm incubation in cadmium alone and in combination with TiO2NPs at different times (15, 30, 45 and 90 min). Our results showed that cadmium reduced sperm DNA integrity, and increased sperm DNA fragmentation and oxidative stress. The genotoxicity induced by TiO2NPs-cadmium co-exposure was lower compared to single cadmium exposure, suggesting an interaction of the substances to modulate their reactivity. The Quantitative Structure-Activity Relationship (QSAR) computational method showed that the interaction between TiO2NPs and cadmium leads to the formation of a sandwich-like structure, with cadmium in the middle, which results in the inhibition of its genotoxicity by TiO2NPs in human sperm cells.

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P–121 Selection and separation of X- and Y- chromosome-bearing mammalian sperm using SPERMAN technology

Human Reproduction ◽

10.1093/humrep/deab130.120 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

B Özkösem ◽

A Fiori ◽

O Sami

Keyword(s):

Y Chromosome ◽

Correlation Coefficient ◽

Fluorescent Dyes ◽

Human Sperm ◽

Quality Parameters ◽

Cellular Damage ◽

Sperm Cells ◽

Dna Quality ◽

Sperm Dna ◽

Frozen Semen

Abstract Study question How can we select and separate X and Y-chromosome bearing sperm cells without causing cellular damage and reduced fertility? Summary answer AI powered SPERMAN technology can sort X and Y-chromosome bearing sperm populations without using harmful fluorescent dyes and lasers. What is known already Most common and reliable method to sex sorting (separation of X and Y- chromosome bearing sperm cells) is by using fluorescence activated cell sorting ( FACS) which takes advantage of the difference of amount of DNA in X and Y-chromosomes. Unfortunately this method causes reduced fertility and cellular damages due to the lasers and fluorescent dyes that are used. There are new and experimental developments in sex-sorting such as using immunological approaches to separate sperm cells. Current sorting method damages sperm cells. However, there is no automated and easy to use sperm sorting technology available. Study design, size, duration In this study, we have compared the quality parameters (motility, viability, AR status, DNA packaging) of sorted sperm cells to unsorted sperm cells. We have used epididymal sperm samples from 10 C57BL/6 mice (Charles River) and frozen semen samples from 6 Holstein bulls (Sexing Technologies) and frozen semen samples from 8 human sperm donors (Fairfax Cryobank). Samples were divided into two groups: sex-sorted and unsorted sperm. Participants/materials, setting, methods Sperm samples from 8-week-old C57BL/6 mice were collected from epididymal region, frozen bull semen samples and human semen samples were thawed at 37ºC then separated into two groups as sorted and unsorted sperm. Sorted samples in modified SP-TALP medium were loaded on the microfluidic chip of Sperman device, after the 30min long sorting process ended, all samples were centrifuged gently at 100g for 5 min, quality parameters were measured by using CASA and FACS. Main results and the role of chance Sperman device sorted X-bearing sperm cells at 81% purity and Y-bearing cells at 73% in mouse samples. Sperman device was able to sort X-bearing sperm cells at 78% purity and Y-bearing cells at 70% in bull samples.S perman device sorted X-bearing sperm cells at 85% purity and Y-bearing cells at 76% in human samples. Our study shows that in mouse, bull and human sperm samples, sperm DNA quality, sperm concentration, progressive motility and AR status results from the sorting with SPERMAN device are comparable with the unsorted samples. For sperm DNA quality, both the Spearman rank correlation coefficient and concordance correlation coefficient are above 0.97, indicating a high agreement between the unsorted samples and SPERMAN sorted samples. Limitations, reasons for caution In this study we only compared sperm quality parameters between unsorted sperm cells and Sperman-sorted sperm cells. Ideally a follow up study would be to look in detail at fertilization success rates and embryo growth. Also, genomic and proteomic profiles of unsorted and sorted sperm cells could be different. Wider implications of the findings: In livestock, sperm sex sorting has high value in terms of economic impact on the livestock management. Unfortunately, there is one expensive single technology dominates the sex-sorting market causing high fees poor outcomes. We believe that Sperman technology provides farmers more cheaper and better sex-sorting technology. Trial registration number Not applicable

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P–051 Differential resilience of sperm from different mammals to DNA decondensation

Human Reproduction ◽

10.1093/humrep/deab130.050 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

J Ribas-Maynou ◽

E Garcia-Bonavila ◽

M Llavanera ◽

J Miró ◽

S Bonet ◽

...

Keyword(s):

Human Sperm ◽

Dna Condensation ◽

Proteinase K ◽

Dna Integrity ◽

Sperm Cells ◽

Core Diameter ◽

Bovine Sperm ◽

Sperm Dna ◽

Protamine 1 ◽

Protamine 2

Abstract Study question Does sperm from different species with different protamine 1/protamine 2 ratios have different resilience to sperm decondensation? Summary answer Sperm cells from species whose DNA is condensed with both protamine 1 and protamine 2 require less time in deprotamination steps. What is known already Sperm cells present a highly particular DNA condensation that is acquired during sperm differentiation, where most part of histones are replaced by protamines. Protamines are key elements for DNA condensation and, while protamine 1 is more conserved among species, protamine 2 has evolved differentially, existing only a few species that retain the mature protein in their sperm DNA. Changes in protamine expression rates have been described to be associated to head sperm size and shape. In addition, reduced amounts of protamine 2 are related to male infertility in species in which this protein is present. Study design, size, duration Cryopreserved sperm samples were treated with lysis solutions to induce DNA decondensation and formation of sperm haloes. In these treatments, the effect of different incubation times with proteinase K added to the lysis solution upon DNA decondensation was tested by analyzing core diameter, halo diameter and the Halo/core ratio in at least 50 sperm per sample. Participants/materials, setting, methods Species included in the study were Human, Equine, Donkey, Porcine and Bovine. Sperm samples from five different individuals for each species were included in the study. DNA decondensation included three lysis steps: first, a SDS + DTT incubation for 30 minutes; second, a DTT + NaCl treatment for 30 minutes; and third, a DTT + NaCl + Proteinase K treatment with a variable time of 0, 30 or 180 minutes. Main results and the role of chance The halo/core diameter, used as a representation of the degree of DNA decondensation, for 0 minutes, 30 minutes and 180 minutes of proteinase K incubation were: 4.68±0.51, 4.32±0.51 and 4.77±0.64, respectively for human sperm; 4.15±0.41, 4.57±0.53 and 4.68±0.63, respectively for Equine sperm; 4.40±0.64, 4.00±0.37 and 4.17±0.19, respectively for donkey sperm; 1.77±0.2, 3.05±0.14 and 4.13±0.39, respectively for porcine sperm; and 2.40±0.40, 3.36±0.22 and 4.19±0.38, respectively for bovine sperm. Differences of halo/core ratio in different times were only observed in porcine and bovine sperm, where increasing degrees of DNA decondensation were found (p < 0.05). Therefore, these results show that while longer incubations in lysis solutions with proteinase K lead to higher DNA decondensation in porcine and bovine, they do not induce higher decondensation in human, equine and donkey. This evidence, coupled to the fact that porcine and bovine sperm present null or very low protamine 2 content, suggests that its presence might confer higher DNA decondensation susceptibility. Limitations, reasons for caution Only sperm cells with normal sperm haloes were analyzed in the present study. As multiple studies show, haloes exhibited by sperm cells with DNA damage display higher diameter, that is why they were strictly excluded in this study with the aim to elucidate the average DNA decondensation. Wider implications of the findings: Sperm DNA might have different degrees of DNA condensation, which can be associated to a higher difficulty of DNA decondensation, thus having implications in the sensitivity tests that assess sperm DNA integrity. Trial registration number Not applicable.

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Lead Exposure: A Summary of Global Studies and the Need for New Studies from Saudi Arabia

Disease Markers ◽

10.1155/2014/415160 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 16

Author(s):

A. P. Shaik ◽

S. A. Sultana ◽

A. H. Alsaeed

Keyword(s):

Saudi Arabia ◽

Genetic Susceptibility ◽

Lead Poisoning ◽

Lead Exposure ◽

Reproductive Toxicity ◽

Lead Toxicity ◽

Cardiovascular Effects ◽

Developed Countries ◽

Health Concern ◽

Adverse Health Effects

Lead poisoning (plumbism) can cause irreversible genetic and reproductive toxicity, hematological effects, neurological damage, and cardiovascular effects. Despite many efforts to minimize lead poisoning, it continues to be a major health concern in many developing and developed countries. Despite efforts to control lead exposure and toxicity, serious cases of lead poisoning increasingly occur as a result of higher vehicular traffic and industrialization. The biomarkers for identification of genetic susceptibility to a particular disease are useful to identify individuals who are at risk for lead poisoning. Although many such studies have been taken up elsewhere, very few studies were performed in Saudi Arabia to assess susceptibility to lead poisoning. This indicates an urgent need for testing of susceptible individuals. The present paper was planned to understand the genetic susceptibility to lead toxicity in the various population studies conducted worldwide and also to correlate it with the current scenario in Saudi Arabia. Such studies are necessary for appropriate precautions in terms of diet and avoiding exposure to be used in order to prevent adverse health effects.

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