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Laser printers emit high levels of nanoparticles (PM0.1) during operation. Although it is well established that toners contain multiple engineered nanomaterials (ENMs), little is known about inhalation exposures to these nanoparticles and work practices in printing centers. In this report, we present a comprehensive inhalation exposure assessment of indoor microenvironments at six commercial printing centers in Singapore, the first such assessment outside of the United States, using real-time personal and stationary monitors, time-integrated instrumentation, and multiple analytical methods. Extensive presence of ENMs, including titanium dioxide, iron oxide, and silica, was detected in toners and in airborne particles collected from all six centers studied. We document high transient exposures to emitted nanoparticles (peaks of ∼500 000 particles/cm3, lung-deposited surface area of up to 220 μm2/cm3, and PM0.1up to 16 μg/m3) with complex PM0.1chemistry that included 40–60 wt % organic carbon, 10–15 wt % elemental carbon, and 14 wt % trace elements. We also record 271.6–474.9 pmol/mg of Environmental Protection Agency-priority polycyclic aromatic hydrocarbons. These findings highlight the potentially high occupational inhalation exposures to nanoparticles with complex compositions resulting from widespread usage of nano-enabled toners in the printing industry, as well as inadequate ENM-specific exposure control measures in these settings.

We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menadione and DMNQ induced damage to sperm membranes after 1 h (P < 0.001; P < 0.05) and after 3 h of incubation (P < 0.001, P < 0.05). Pre-incubation with cysteine ameliorated both the menadione- and DMNQ-induced increase in 4-hydroxynonenal (P < 0.001). As cysteine is a precursor of GSH, we hypothesized that stallion spermatozoa are able to synthesize this tripeptide using exogenous cysteine. To test this hypothesis, we investigated the presence of two enzymes required to synthesize GSH (GSH and GCLC) and using western blotting and immunocytochemistry we detected both enzymes in stallion spermatozoa. The inhibition of GCLC reduced the recovery of GSH by addition of cysteine after depletion, suggesting that stallion spermatozoa may use exogenous cysteine to regulate GSH. Other findings supporting this hypothesis were changes in sperm functionality after BSO treatment and changes in GSH and GSSG validated using HPLC-MS, showing that BSO prevented the increase in GSH in the presence of cysteine, although important stallion to stallion variability occurred and suggested differences in expression of glutamate cysteine ligase. Mean concentration of GSH in stallion spermatozoa was 8.2 ± 2.1 μM/109 spermatozoa, well above the nanomolar ranges per billion spermatozoa reported for other mammals.

Although stallion spermatozoa produce significant quantities of reactive oxygen species, a lag between 4-hydroxynonenal (4HNE) adduction and the loss of motility in stallion spermatozoa suggests the presence of a robust aldehyde detoxification mechanism. Because there is a paucity of studies characterizing the role of aldehyde dehydrogenase (ALDH) in sperm functionality, the aim of this study was to ascertain the relationship between 4HNE production and motility and ALDH expression by stallion spermatozoa. PCR analysis revealed the presence of the ALDH1A3, ALDH1B1, and ALDH2 isoforms in these cells. Strong correlations (P< 0.001) were found between ALDH expression and various motility parameters of stallion spermatozoa including the percentage of progressive (r= 0.79) and rapidly motile (r= 0.79) spermatozoa, whereas repeated measurements over 24 h revealed highly significant correlations among progressive motility loss, 4HNE accumulation, and ALDH expression (P≤ 0.001). ALDH inhibition resulted in a spontaneous increase in 4HNE levels in viable cells (21.1 ± 5.8% vs. 42.6 ± 5.2%; P≤ 0.05) and a corresponding decrease in total motility (41.7 ± 6.2% vs. 6.4 ± 2.6%; P≤ 0.001) and progressive motility (17.0 ± 4.1% vs. 0.7 ± 0.4%; P≤ 0.001) of stallion spermatozoa over 24 h. Similarly, inhibition of ALDH in 4HNE-challenged spermatozoa significantly reduced total motility over 4 h (35.4 ± 9.7% vs. 15.3 ± 5.1%, respectively; P≤ 0.05). This study contributes valuable information about the role of the ALDH enzymes in the maintenance of stallion sperm functionality, with potential diagnostic and in vitro applications for assisted reproductive technologies.

The spermatozoa of many stallions do not tolerate being cooled, restricting the commercial viability of these animals and necessitating the development of a chemically defined room temperature (RT) storage medium. This study examined the impact of two major modulators of oxidative phosphorylation, pyruvate (Pyr) and L-carnitine (L-C), on the storage of stallion spermatozoa at RT. Optimal concentrations of Pyr (10 mM) and L-C (50 mM) were first identified and these concentrations were then used to investigate the effects of these compounds on sperm functionality and oxidative stress at RT. Mitochondrial and cytosolic reactive oxygen species, along with lipid peroxidation, were all significantly suppressed by the addition of L-C (48 h MitoSOX Red negative: 46.2% vs. 26.1%; 48 and 72 h dihydroethidium negative: 61.6% vs. 43.1% and 64.4% vs. 46.9%, respectively; 48 and 72 h 4-hydroxynonenal negative: 37.1% vs. 23.8% and 41.6% vs. 25.7%, respectively), while the Pyr + L-C combination resulted in significantly higher motility compared to the control at 72 h (total motility: 64.2% vs. 39.4%; progressive motility: 34.2% vs. 15.2%). In addition, supplementation with L-C significantly reduced oxidative DNA damage at 72 h (9.0% vs. 15.6%). To investigate the effects of L-C as an osmolyte, comparisons were made between media that were osmotically balanced with NaCl, choline chloride, or L-C. This analysis demonstrated that spermatozoa stored in the L-C balanced medium had significantly higher total motility (55.0% vs. 39.0%), rapid motility (44.0% vs. 25.7%), and ATP levels (70.9 vs. 12.8 ng/ml) following storage compared with the NaCl treatment, while choline chloride did not significantly improve these parameters compared to the control. Finally, mass spectrometry was used to demonstrate that a combination of Pyr and L-C produced significantly higher acetyl-L-carnitine production than any other treatment (6.7 pg/106spermatozoa vs. control at 4.0 pg/106spermatozoa). These findings suggest that Pyr and L-C could form the basis of a novel, effective RT storage medium for equine spermatozoa.

The relationship between stallion fertility and oxidative stress remains poorly understood. The purpose of this study was to identify criteria for thoroughbred fertility assessment by performing a logistical regression analysis using “dismount” sperm parameters as predictors and weekly per-cycle conception rate as the dependent variable. Paradoxically, positive relationships between fertility and oxidative stress were revealed, such that samples that produced pregnancies exhibited higher rates of 8-hydroxy-2′-deoxyguanosine release (1490.2% vs. 705.5 pg/ml/24 h) and lower vitality (60.5% vs. 69.6%) and acrosome integrity (40.2% vs. 50.1%) than those that did not. We hypothesized that the most fertile spermatozoa exhibited the highest levels of oxidative phosphorylation (OXPHOS), with oxidative stress simply being a by-product of intense mitochondrial activity. Accordingly, an experiment to investigate the relationship between oxidative stress and motility was conducted and revealed positive correlations between mitochondrial ROS and total motility (R2= 0.90), rapid motility (R2= 0.89), average path velocity (VAP; R2= 0.59), and curvilinear velocity (VCL; R2= 0.66). Similarly, lipid peroxidation was positively correlated with total motility (R2= 0.46), rapid motility (R2= 0.51), average path velocity (R2= 0.62), and VCL (R2= 0.56), supporting the aforementioned hypothesis. The relative importance of OXPHOS in supporting the motility of equine spermatozoa was contrasted with human spermatozoa, which primarily utilize glycolysis. In this study, mitochondrial inhibition significantly reduced the velocity (P< 0.01) and ATP (P< 0.05) content of equine, but not human, spermatozoa, emphasizing the former's relative dependence on OXPHOS. The equine is the first mammal in which such a positive relationship between oxidative stress and functionality has been observed, with implications for the management of stallion fertility in vitro and in vivo.

DNA damage in the male germ line is associated with failed fertilization, impaired preimplantation development and poor pregnancy outcomes. Large-scale epidemiological studies have also suggested that DNA damage in spermatozoa is associated with adverse impacts on the health and wellbeing of children, including dominant genetic disease and childhood cancer. In light of these considerations, the iatrogenic introduction of DNA damaged spermatozoa into the oocyte during the course of assisted conception therapy, particularly when intracytoplasmic sperm injection (ICSI) is involved, may be a source of some concern. Although the oocyte actively repairs the DNA damage brought into the zygote by the fertilizing spermatozoon, we have hypothesized that errors in this repair process may generate mutations that impair the progress of pregnancy and the health of the offspring. Evidence from the study of dominant genetic diseases suggests that the incidence of mutations capable of generating phenotypic change in the offspring is so low that it might be several generations before the full consequences of using DNA damaged spermatozoa in assisted conception cycles are understood. Given the potential significance of DNA damage in human spermatozoa, it is of interest to consider the mechanisms by which this damage is induced. In this context we have advanced a two-step hypothesis for the origins of DNA damage in the male germ line. The first step involves poor chromatin remodeling as a consequence of which sperm DNA is inadequately protaminated and the chromatin remains relatively histone rich. Such defective remodeling creates a state of vulnerability that becomes manifest during the second stage of the process, during which the chromatin is subjected to attack. The latter may involve either endogenous endonucleases and/or free radicals, generating oxidative base damage, abasic sites and DNA strand fragmentation. The free radicals associated with oxidative DNA damage may arise from several sources including: (i) sperm mitochondria, (ii) the redox cycling of endogenously generated metabolites (eg. catechol estrogens) or xenobiotics (eg. quinones, retinoids), (iii) the presence of transition metals such as iron and copper, (iv) the presence of free radicalgenerating leukocytes, (v) the aberrant activation of oxidases (DUOX has recently been identified in the human sperm proteome) as a consequence of excess cytoplasmic retention (vi) exposure to electromagnetic radiation and (vii) a reduction in the free radical scavenging activity of the cells and fluids associated with spermatozoa during their descent of the male reproductive tract. These sources of oxidative stress make a particular impact on the post-meiotic stages of germ cell development when these cells are losing their capacity for apoptotic deletion as well as their competence for DNA repair. The factors that trigger oxidative stress include age- and diet- related declines in free radical scavenging activity, exposure to xenobiotics, exposure to redox cycling environmental pollutants and pharmaceuticals, exposure to sources of electromagnetic radiation and male genital tract infection. A systematic and careful examination of subfertile males in order to identify the incidence and sources of oxidative stress in the male tract would be a worthwhile undertaking with implications for the diagnosis, management and treatment of DNA damage in the male germ line.