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Effects of Nano-Graphene Oxide on Testis, Epididymis and Fertility of Wistar Rats
Abstract
Nanoscale graphene oxide (NGO) has great potential in biomedicine by the virtue of its facile functionalization and tunable characteristics. Toxicity assessments have, therefore, become essential prior to its biomedical applications. The present study examined the effects of NGO exposure on male reproductive function of adult Wistar rats. Rats were exposed intraperitoneally to three increasing doses, namely low (0.4 mg/kg BW), mid (2.0 mg/kg BW) and high (10.0 mg/kg BW) dose of NGO. Repeated exposure of NGO for 15 and 30 days resulted in decreased epididymal sperm counts and elevated sperm abnormalities. Percentage of motile sperms was also significantly reduced due to the exposure. Activities of SOD, GPx and malondialdehyde concentration in testes increased in a dose-specific manner. Results of the study demonstrated that high-dose NGO (10.0 mg/kg BW) resulted in considerable histological damage to testicular tissue which included atrophy of seminiferous tubules with reduction in germinal epithelium, germ cell loss and vacuolization. Low and mid doses of NGO were not associated with sperm dysfunction or testis damage. Withdrawal of treatment for 30 days demonstrated significant recovery potential. Histology of epididymis and male fertility potential were not affected due to the NGO exposure. These findings are important for assessment of the risk involved in manufacturing, use and processing of the graphene oxide-based materials towards male reproductive function. This article is protected by copyright. All rights reserved.
... GNPs) adversely influence the sperm quality , reproductive organs structure, the activities of certain main reproductive hormones, fertility could decrease and health offspring could be effected. In rats , the intraperitoneal injection of high nanoscale graphene oxide concentration (10.0 mg / kg BW) for 15 and 30 days resulted in decreased epidididymal sperm counts and motility, and increased sperm abnormalities, as well as histological damage to testicular tissue such as seminiferous tubules atrophy with reduced germ epithelium, loss of germ cell, and vacuole formation (39) . In fish, Dasmahatra et al. (40) found a connection between graphene oxide and gonad histpathology in fish, including granulosa and leydig cell changes. ...
... Research has shown that low testosterone levels in animals have affected spermatogenesis by reducing sperm count and motility, increasing the percentage of defective sperm cells, and modifying the histomorphology of the testis and/or epididymis (49)(50) . Nirmal et al. study demonstrated that after 15 and 30 days of exposure to nano graphene oxide, total sperm count decreased with dose effects (39) . Whereas, Yoshida et al. (51) found that intratracheal administration of carbon nanoparticles to male mice resulted in decreased sperm production and tissue changes in the testes. ...
... Morphological abnormalities in epididymis tail sperm have increased significantly in all three concentration of GNPs, indicating that oxidative stress is being produced. According to the results of Nirmal et al (39) study, nanographene oxide induce oxidative stress in testicular tissue. Oxidative stress triggers the oxidation of cell membrane that rich in polyunsaturated fatty acids. ...
Due to their exceptional physical properties, graphene nanomaterials have gained a great deal of interest in recent years for their application to state-of-the-art technology. On the other hand, nanotoxicity of graphene materials has also rapidly become a major concern, especially in the field of occupational health. There is a lack of evidence that such graphene nanomaterials influence the organs of the male reproductive system. Therefore, the goal of this study was to determine the effect of Graphene nanoparticles (GNPs) on mice body weight, reproductive organs weight and sperm quality, as well as certain reproductive organs injuries, after14 days of treatment with oral gavages of 0.1 ml of 10, 20, and 30 mg/kg of GNPs in male albino mice. The results showed a decrease in animal body weight and certain reproductive organs, as well as a decrease in the proportion of live sperm and the concentration of sperm in the tail of the epididymis and an increase in sperm abnormalities in all treatment relative to the control group. Injuries in the testes and epididymis (head and tail) were shown by histopathological analysis. This study concluded that oral GNPs gavages at various concentrations have a negative effect on the male reproductive system and can affect fertility.
... Several studies have analyzed testicular parenchymal lesions after exposure to nanoparticles. Most of them first measured the evolution of testicular weight [113,119,[123][124][125]127,143,187,[194][195][196][197][198][199][200][201][202][203]. It has been shown that, irrespective of the animal model (rats, mice), the particles considered (silver, gold, carbon nanotubes, nano-graphene oxide, carbon black, titanium dioxide, zinc oxide, iron oxide, chromium-cobalt), and the exposure pathways (intravenous, intraperitoneal, inhaled, oral, intra-articular), exposure to nanoparticles do not appear to affect the testicular weight of an adult animal. ...
... Half of the studies analyzed did not report any histological abnormalities after exposure to nanoparticles [119,[125][126][127]135,146,197,199,[203][204][205][206][207], or did so only for extremely high doses [198,208]. Some studies that have focused on the intravenous exposure to carbon nanotubes [113] or silver nanoparticles [124,195,209], on the intraperitoneal exposure of nano-graphene oxide [201], or on the intra-articular exposure to chromium-cobalt nanoparticles mimicking prosthetic abrasion [123], have observed moderate testicular histological abnormalities: disorganization of the seminiferous epithelium, increased number of apoptotic cells or minor alteration of the Leydig cells. All these abnormalities were shown to be reversible upon cessation of exposure, except for the complete degeneration of some seminiferous tubules after inhalation of carbon black nanoparticles reported by Yoshida et al. [194], a result that was not, however, confirmed in a similar study by Skovmand et al. [203]. ...
... Several studies have found no impact of nanoparticle exposure on testosterone. The particles in question were carbon nanotubes [113], welding fumes particles [219], carbon black or diesel exhaust nanoparticles [203], nanoparticles of silica, [197] titanium dioxide particles [220] and graphene nanosheets [128,201,203] inhaled or injected intravenously, intraperitoneally or intrathecally, or silver nanoparticles ingested during the pubertal period [221,222] or injected during adulthood [223]. ...
The real impact of nanoparticles on male fertility is evaluated after a careful analysis of the available literature. The first part reviews animal models to understand the testicular biodistribution and biopersistence of nanoparticles, while the second part evaluates their in vitro and in vivo biotoxicity. Our main findings suggest that nanoparticles are generally able to reach the testicle in small quantities where they persist for several months, regardless of the route of exposure. However, there is not enough evidence that they can cross the blood–testis barrier. Of note, the majority of nanoparticles have low direct toxicity to the testis, but there are indications that some might act as endocrine disruptors. Overall, the impact on spermatogenesis in adults is generally weak and reversible, but exceptions exist and merit increased attention. Finally, we comment on several methodological or analytical biases which have led some studies to exaggerate the reprotoxicity of nanoparticles. In the future, rigorous clinical studies in tandem with mechanistic studies are needed to elucidate the real risk posed by nanoparticles on male fertility.
... Sheet thickness was 0.8-2 nm and average lateral dimensions were 5-10 μm (Table 1). Characterization details shown in the table have already been published (Nirmal et al. 2017b). Additional characterization data of GO is available in the Online Resource. ...
... The supernatant was then centrifuged at 10,000g for 5 min at 4°C. Resulting pellet was washed thrice with ice cold PBS and resuspended in buffered saline (Nirmal et al. 2017b). ...
... Specifications of GO used in the study (reproduced fromNirmal et al. 2017b) ...
Graphene oxide (GO) has a multitude of applications in areas of nanomedicine, electronics, textile, water purification, and catalysis among others. GO is relatively easier to manufacture and customize as compared with other carbon-based nanomaterials. In the present work, GO was administered intraperitoneally to adult Wistar rats in four incremental doses, i.e., 0.0 mg/kg (control), 0.4 mg/kg (low dose), 2.0 mg/kg (mid-dose), and 10.0 mg/kg (high dose). After 15 repeated doses over a period of 30 days, biochemical assays for alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), catalase (CAT), and malondialdehyde (MDA) were carried out. Histopathological and morphometric analyses of liver and kidney were also performed. Results demonstrated dose-dependent toxicity of GO. General behavior and liver indices remained unaffected in the study. Serum levels of ALT, ALP, and AST were altered significantly in high-dose treated animals. Changes were found insignificant in the low- and mid-dose groups. Catalase activity in liver tissue homogenates was decreased in the high-dose group. MDA levels were found elevated in treated rats. Unlike control and low dose, mid- and high-dose treated rats exhibited varying degrees of histopathological changes like inflammation around the central vein and portal veins, vacuolations, hepatocytic injury, and near normal to abnormal hepatic sinusoids. These findings show that GO has considerable toxic potential to mammalian liver and thorough toxicity studies are needed before these nanosheets are used in biomedicine.
... C mixture did not affect hatching success, reproductive toxicity is dosedependent. Various studies have shown reproductive disorders at specific doses, such as poor spermatogenesis, higher apoptosis, and lower egg production [66][67][68]. Conversely, other studies showed no reproductive toxicity at different doses [69][70][71]. ...
... Vitamin C alone did not change egg composition, indicating its effect on Vg gene expression but not protein production or egg composition. doses, such as poor spermatogenesis, higher apoptosis, and lower egg production [66][67][68]. ...
The use of nanoparticles in the industry carries the risk of their release into the environment. Based on the presumption that the primary graphene oxide (GO) toxicity mechanism is reactive oxygen species production in the cell, the question arises as to whether well-known antioxidants can protect the cell or significantly reduce the effects of GO. This study focused on the possible remedial effect of vitamin C in Acheta domesticus intoxicated with GO for whole lives. The reproduction potential was measured at the level of Vitellogenin (Vg) gene expression, Vg protein expression, hatching success, and share of nutrition in the developing egg. There was no simple relationship between the Vg gene’s expression and the Vg protein content. Despite fewer eggs laid in the vitamin C groups, hatching success was high, and egg composition did not differ significantly. The exceptions were GO20 and GO20 + Vit. C groups, with a shift in the lipid content in the egg. Most likely, ascorbic acid impacts the level of Vg gene expression but does not affect the production of Vg protein or the quality of eggs laid. Low GO concentration in food did not cause adverse effects, but the relationship between GO toxicity and its concentration should be investigated more thoroughly.
... Male reproductive organs viz: testis, epididymis and seminiferous tubules bioaccumulate nanoparticles and thus become the targets of their toxicity (Zhao et al. 2014; Wang et al. 2018). Graphene oxide was found to induce histopathological changes in testis i.e. atrophy of seminiferous tubules, reduction in germinal epithelium, loss of germ cells and necrosis (Nirmal et al. 2017). Male rats exposed to silver nanoparticles also showed histopathological changes in seminiferous tubules (Lopes et al. 2019). ...
... SEM Observations supported the histopathological results. Exposure of rats to grapheme oxide was also reported to induce similar morphological changes in rat testes (Nirmal et al. 2017). Other reports on silver nanoparticles (Lopes et al. 2019) and cerium oxide nanoparticles (Adebayo et al. 2018) also suggested similar effects on testes morphology. ...
Exposure to nickel nanoparticles is known to cause adverse health effects in man and animal models. Present investigations were undertaken to determine the histo-morphological changes induced by nickel nanoparticles in testis of Wistar rats with special reference to duration of exposure. Suitable numbers of rats were fed with NiONPs and NiOMPs (5mg/kg b.w.each) for 15 and 30 days. Testes were removed and processed for histopathological, histochemical and SEM observations. Bioconcentration of nickel in testis and serum level of testosterone were also estimated. The results showed that NiONPs and NiOMPs could induce specific time dependent lesions in the testis of rat. Degeneration of germinal epithelium, spermatocytes, hypertrophy of seminiferous tubules and necrosis were observed. Localization of alkaline phosphatase was also altered. SEM results also indicated specific morphological changes in cellular components of tubules. In conclusion, present study suggests that testis is a target organ of NiONPs toxicity where its exposure time dependent effects are expressed. The results will be helpful in understanding the reproductive toxicity of new materials.
... However, during inflammation and reduced sulfation, shedding of the GL components could be expected, with more available cationic binding sites exposed on the cell surface, enabling NPs to be more easily taken up by scavenger receptors. Most of the GBNs and graphene safety studies mainly consider the hydrophobicity of the cell membrane, but do not take into account the effect of the negatively charged intact sulfated GL, and the phenomenon of undersulfation, or a degraded GL due to the shedding of glycosaminoglycans, especially during inflammatory conditions [44]. (8) In addition to the hydrophobicity of GBNs that plays a role in its interaction with the lipid bilayer of cells, surface energy may be modified in vivo by the formation of a protein corona on the surface, modifying the membrane response to GBNs [34]. ...
... Although the continuous wear of activated carbon masks has been established to pose a higher risk of fiber-like microplastic inhalation [74], the risk of inhaling GBNs from masks has not been researched extensively. However, inhaled GNSs can easily penetrate the tracheobronchial airways and then travel down to the lower lung airways, where it destroys the ultrastructure and biophysical properties of the pulmonary epithelial GL layer [27,43], therefore, the first line of innate host defense [44]. GO was demonstrated to disrupt the alveolar-capillary barrier, allowing inflammatory cells to infiltrate the lungs, thus stimulating the release of pro-inflammatory cytokines, resulting in epithelioid granulomas, interstitial inflammation, and lung fibrosis [75][76][77]. ...
Graphene-based materials (GBMs) possess remarkable physiochemical properties, making them promising for diverse applications in biomedicine, agriculture, food, and industrial applications. Human and environmental exposure to GBMs is increasing at an unprecedented rate, yet there is still a knowledge gap regarding the safety of GBMs. This review summarizes the physiochemical properties of GBMs and critically examines the possible effects of GBMs, both at the level of molecular mechanism and at the level of the organism. While oxidative stress-mediated cell damage has been proposed as a primary cytotoxicity mechanism for GBMs, various in vivo biodistribution and cytotoxicity mechanisms are also highlighted. This review of the literature provides an overview of the cytotoxicity of GBMs, raising concerns about their widespread application with potential hazardous consequences on the environment and in human health.
... John and coworkers [72] studied the adverse effects of GO (0.8-1 nm thickness, 101-258 nm diameters) at 0.01, 0.1, 1, 10, and 100 mg/L concentrations on the embryogenesis of zebrafish and reported that GO had entered and induced hypoxia in the chorion, generated an anoxic space close to the chorion, and also enforced mechanical pressure on its surface area. Given the substantial role of chorion in the development of zebrafish embryos, the aforementioned effects along with the envelopment of chorions by GO resulted in the decreased embryo movement and delayed hatching and development of the embryos. ...
... Decreased sperm production in the testes, spermatogonia loss, cell cycle arrest, and occurrence of cell death within the sperm production pathways are potential explanations for this matter. Of note, spermatogonia and spermatid numbers were also reported to have decreased in this study [72]. The high-dose subgroup of the AS2 group also experienced declined sperm motility values, which were restored to normal after a recovery period. ...
With the glorious discovery of graphene back in 2004, the field of nanotechnology was faced with a breakthrough that soon attracted the attention of many scientists from all over the world. Owing to its unique bidimensional structure and exquisite physicochemical properties, graphene has successfully managed to cave its way up to the list of the most investigated topics, while being extensively used in various fields of science and technology. However, serious concerns have been raised about the safety of graphene, for which numerous studies have been conducted to evaluate the toxicity of graphene derivatives in both in vitro and in vivo conditions. The reproductive toxicity of graphene is one of the most important aspects of this subject as it not only affects the individual but can also potentially put the health of one’s offsprings at risk and display long-term toxic effects. Given the crucial importance of graphene’s reproductive toxicity, more attention has been recently shifted toward this subject; however, the existing literature remains insufficient. Therefore, we have conducted this review with the aim of providing researchers with assorted information regarding the toxicity of graphene derivatives and their underlying mechanisms, while mentioning some of the major challenges and gaps in the current knowledge to further elucidate the path to exploring graphene’s true nature. We hope that our work will effectively give insight to researchers who are interested in this topic and also aid them in completing the yet unfinished puzzle of graphene toxicity.
... With respect to the effects due to non-metallic or semi-metallic particles having different shapes, different outcomes have been reported. A study conducted by Nirmal et al. (2017a)on Wistar rats, exposure to 2.0 and 10.0 mg/kg bwt doses of OH-f MWCNTs resulted in sperm dysfunction and degeneration in seminiferous tubules [49]. In another study by the same group, Wistar rats were exposed with high doses of nanoscale GO (NGO) intraperitoneally which reduced sperm motility and total sperm count and increased sperm abnormalities [50]. ...
... The exposure did not affect epididymal histology[37]. Examination of histology demonstrated normal lumen and epithelial lining of epididymis in treated (10.0 mg/kg) as well as control group with both OH-functionalized MWCNTs and GO in our previous reports[49,50]. These results are in contrast with the findings ofFarombi et al. (2014). ...
There are numerous chemical substances that man has produced, many of them, apart from other factors, are toxic to the male reproductive function in animals, including man. A new addition in this list is the nanoscale materials. Worldwide production of nanomaterials is increasing at high rates. Response of the male reproductive system towards exposure to nanomaterials is being studied across the world. Studies have demonstrated adverse effects on male reproductive function in various animals and human resulting from exposure to nanomaterials. The review focuses on properties, types, applications of engineered nanomaterials and human exposure scenarios followed by a detailed review of recent reports on male reproductive toxicity. It also highlights the challenges associated with nanotoxicity studies.
... However, the use of this synthetic methodology does not ensure a precise characterization of the final product that may differ in terms of exfoliation degree (single, SLGO, or few layered sheets, FLGO) [48], oxygen content (either in terms of C/O w/w, that may vary from 0.25 [59] to 0.39 [48] or as final oxygen weight with respect to the total weight, i.e., 20% [41] or >36% [60]), dimensions of the sheets (i.e., from 297 nm mean size [61] to 3-5 µm [41,47]), surface charge (i.e., from ζ -potential −14.13 ± 11.1 mV [60] to −56.7 ± 1.5 mV [46]). In a few studies GO sheets are named nano-GO (nanoGO or NGO) [62,63] or are distinguished in small GO (S-GO) and large GO (L-GO) [64] sheets when the protocol of preparation involves a further step of cutting into small pieces by sonication [64] or harsh oxidation conditions. ...
... Their findings demonstrated a dose-dependent reduction in the number of spermatozoa, spermatogonia and spermatids, a decreased sperm motility, and some morphological abnormalities in the groups that were treated with the highest NGO concentrations. Moreover, some oxidative stress was confirmed with the increased activity of antioxidant enzymes, without causing damage to the testis or reducing the fertility potential, data confirmed with the healthy offspring derived from the matching with female rats [63]. ...
Since its discovery, graphene and its multiple derivatives have been extensively used in many fields and with different applications, even in biomedicine. Numerous efforts have been made to elucidate the potential toxicity derived from their use, giving rise to an adequate number of publications with varied results. On this basis, the study of the reproductive function constitutes a good tool to evaluate not only the toxic effects derived from the use of these materials directly on the individual, but also the potential toxicity passed on to the offspring. By providing a detailed scientometric analysis, the present review provides an updated overview gathering all the research studies focused on the use of graphene and graphene-based materials in the reproductive field, highlighting the consequences and effects reported to date from experiments performed in vivo and in vitro and in different animal species (from Archea to mammals). Special attention is given to the oxidized form of graphene, graphene oxide, which has been recently investigated for its ability to increase the in vitro fertilization outcomes. Thus, the potential use of graphene oxide against infertility is hypothesized here, probably by engineering the spermatozoa and thus manipulating them in a safer and more efficient way.
... On the other hand, the few studies that evaluated the sexual/mating behaviors, with neurohormonal regulation, did not show any changes after exposure to CdTe QDs (1.9 ± 0.4 nm; 0.2-2.0 nmol; 1-90 d; i.v.; , GO NPs (0.8-2 nm; 0.4-10 mg/kg; 15-30; i.p.; Nirmal et al., 2017b), and GQDs; (5.25 ± 1.63 nm; 25-300 mg/kg; 7-10 d; i.v., oral; Zhang et al., 2019a) in adult rodents. Further studies are needed to confirm the lack of effects by NMs in this aspect of male reproductive function. ...
... Other alterations induced by Ag NPs (20-200 nm; 5-10 mg/kg; 1-28 d; i.v.), CdTe QDs (1.9 ± 0.4 nm; 0.2-2.0 nmol; 3 h-90 d; i.v.), CNTs (10-16 nm; 4 mg/kg; 1-35 d; i.v.), GO NPs (0.8-2 nm; 0.4-10 mg/kg; 15-30 d; i.p.), SiO 2 NPs (64 nm; 20 mg/kg; 13-60 d; i.v.), TiO 2 NPs (5.5 nm; 1.25-5 mg/kg; 6 mo; oral), ZnO NPs (5-300 mg/kg; 35 d; oral), PbSe NPs (30-70 nm; 10 mg/kg; 60 d; i.p.), such as reduction in the number of sperm in the lumen of epididymal duct observed in histopathological analyses, decrease or increase in epididymis weight, and changes in the epididymal sperm such as reduced sperm concentration in the cauda, motility, acrosome integrity, increases in DNA damage (Gromadzka-Ostrowska et al., 2012) and morphological abnormalities Xu et al., 2014b;Hong et al., 2015a;Nirmal et al., 2017b;Zhou et al., 2019;Mohammadi et al., 2020) were considered to be mostly of testicular origin or in response to imbalance in androgen levels (Kempinas and Klinefelter, 2014). ...
The nanotechnology enabled the development of nanomaterials (NMs) with a variety of industrial, biomedical, and consumer applications. However, the mechanism of action (MoA) and toxicity of NMs remain unclear, especially in the male reproductive system. Thus, this study aimed to perform a bibliometric and systematic review of the literature on the toxic effects of different types of NMs on the male reproductive system and function in mammalian models. A series of 236 articles related to the in vitro and in vivo reproductive toxicity of NMs in mammalian models were analyzed. The data concerning the bioaccumulation, experimental conditions (types of NMs, species, cell lines, exposure period, and routes of exposure), and the MoA and toxicity of NMs were summarized and discussed. Results showed that this field of research began in 2005 and has experienced an exponential increase since 2012. Revised data confirmed that the NMs have the ability to cross the blood-testis barrier and bioaccumulate in several organs of the male reproductive system, such as testis, prostate, epididymis, and seminal vesicle. A similar MoA and toxicity were observed after in vitro and in vivo exposure to NMs. The NM reproductive toxicity was mainly related to ROS production, oxidative stress, DNA damage and apoptosis. In conclusion, the NM exposure induces bioaccumulation and toxic effects on male reproductive system of mammal models, confirming its potential risk to human and environmental health. The knowledge concerning the NM reproductive toxicity contributes to safety and sustainable use of nanotechnology.
... Došlo také ke změnám v testikulární tkáni, k atrofii semenotvorných tubulů s redukcí zárodečného epitelu a k vakuolizaci. 46 Rovněž hydroxylované MWCNT snižovaly počty spermií a jejich motilitu a ovlivňovaly morfologii buněk (nálezy bezhlavých spermií a poruch ocasu). Histologická analýza testikulární tkáně odhalila poškození semenotvorných tubulů a vakuolizaci v důsledku zeslabení zárodečného epitelu. ...
... Currently, confocal Raman spectroscopy seems to be the most promising method for the detection and measurement in cells, allowing the detection of very low concentrations of GBNPs [67]. Nonetheless, graphene has intrinsic photoluminescence properties [27,59,69,70], which makes it easier to establish the presence of GBNPs by dark field optical microscopy [71]. ...
Abstract: Graphene-based nanoparticles possess remarkable physiochemical properties, making them promising for diverse applications in biomedicine, agriculture, food, and industrial applications. These nanoparticles have also been used in the fight against COVID-19. Human and environmental exposure to graphene-based nanomaterials is increasing at an unprecedented rate. However, there is still a huge knowledge gap regarding its safety in clinical applications. The topic remains controversial; although several routes of degradation exist, the cytotoxicity of graphene-based nanoparticles has been demonstrated. Various factors that can influence the cytotoxicity of graphene-based materials are discussed. This review summarizes the physiochemical properties of graphene-based materials and critically examines the possible effects of graphene-based nanoparticles on the molecular level and adverse health outcomes. While oxidative stressmediated cell damage has been proposed as a primary cytotoxicity mechanism for graphene-based materials, various in vivo biodistribution and cytotoxicity mechanisms are also highlighted. Therefore, this review of the
literature provides an overview of the cytotoxicity of GBMs and raises concerns about their widespread application with potential hazardous consequences on the environment and human health.
... Both light and electron microscopic results of withdrawal groups I and II of the present study reflected the gradual reversibility of AuNPs-induced damage to the testicular tissues. Similar reversible effects for the damage of the testicular tissues were detected by Bai et al. [2], who used carbon nanotubes, Ren et al. [30], who used silica nanoparticles,and Nirmal et al. [29], who used graphene oxide nanoparticles. ...
Nanotechnology has become a trending area in science all over the world. Although gold nanoparticles (AuNPs) have been utilized widely in biomedical fields, potential toxicities may arise from their interactions with biological systems. The current study aimed at evaluating the toxic effects of AuNPs on the reproductive system of adult male albino rats and assessing the recovery probability. In this study, AuNPs (13 ± 4 nm in diameter) were synthesized, and the experimental work was conducted on 60 adult male albino rats divided into the following groups: control group (received deionized water daily intraperitoneally (IP) for 28 days), test group, and withdrawal groups I and II (received 570 μg/kg of 13 ± 4 nm AuNPs daily IP for 28 days). Withdrawal groups I and II were left for another 30 and 60 days without sacrification, respectively. The test group showed significant decreases in final body and absolute testicular weights, testosterone hormone level, sperm count and motility, and spermatogenesis score, as well as significant increase in the percentage of sperms of abnormal morphology compared to the control group, associated with significant light and electron microscopic histopathological changes. Partial improvement of all studied reproductive parameters was detected after one month of withdrawal in withdrawal group I, and significant improvement and reversibility of all these parameters were reported after two months of withdrawal in withdrawal group II. So, AuNPs induce male reproductive toxicity, which partially improves after one month of withdrawal and significantly improves and reverses after two months of withdrawal.
Supplementary Information
The online version contains supplementary material available at 10.1007/s43188-023-00203-2.
... Studies using male rats and mice as animal models revealed that NPs of titanium dioxide (TiO 2 ) and graphene oxide (GO) caused damage to testicular tissue and sperm deterioration (Morgan et al., 2017;Nirmal et al., 2017). Fathi et al. (2019) reported reduced motility and viability, in addition to reducing the number of testicular tubules, sperm cells, Sertoli cells and Leydig cells. ...
One of the prominent peculiarities of nanoparticles (NPs) is their ability to cross biological barriers. Therefore, the development of NPs with different properties has great therapeutic potential in the area of reproduction because the association of drugs, hormones and other compounds with NPs represents an alternative for delivering substances directly at a specific site and for treatment of reproductive problems. Additionally, lipid-based NPs can be taken up by the tissues of patients with ovarian failure, deep endometriosis, testicular dysfunctions, etc., opening up new perspectives for the treatment of these diseases. The development of nanomaterials with specific size, shape, ligand density and charge certainly will contribute to the next generation of therapies to solve fertility problems in humans. Therefore, this review discusses the potential of NPs to treat reproductive disorders, as well as to regulate the levels of the associated hormones. The possible limitations of the clinical use of NPs are also highlighted.
... NGOs caused a dose-dependent reduction in sperm, spermatogonia, and spermatids. Furthermore, a decrease in sperm motility and morphological abnormalities (atrophy of seminiferous tubules with reduction in germinal epithelium, germ cells, and vacuolization) was detected in animals treated with the highest doses of NGOs [61]. OH-MWCNTs decreased sperm count and motility in a dose-dependent manner, viability was not affected; however, a significant increase in sperm abnormalities (headless sperms, absence of normal hook, amorphous head, bent tail, folded tails) was documented. ...
The presented review aims to summarize the knowledge regarding the reproductive and developmental toxicity of different types of carbon nanoparticles, such as graphene, graphene oxide, multi- and single-walled nanotubes, fullerenes, and nanodiamonds. Carbon nanoparticles have unique chemical and physical properties that make them an excellent material that can be applied in many fields of human activity, including industry, food processing, the pharmaceutical industry, or medicine. Although it has a high degree of biocompatibility, possible toxic effects on different tissue types must also be taken into account. Carbon nanoparticles are known to be toxic to the respiratory, cardiovascular, nervous, digestive system, etc., and, according to current studies, they also have a negative effect on reproduction and offspring development.
... Another study found that GO can promote sperm damage. 207 The physical interaction of the sharp edge of GBNs with cells may cause cell membrane damage. For graphene, hydrophobicity may cause variation of cell membrane lipids and induce toxicity. ...
Graphene-based nanomaterials (GBNs) have been the subject of research focus in the scientific community because of their excellent physical, chemical, electrical, mechanical, thermal, and optical properties. Several studies have been conducted on GBNs, and they have provided a detailed review and summary of various applications. However, comprehensive comments on biomedical applications and potential risks and strategies to reduce toxicity are limited. In this review, we systematically summarized the following aspects of GBNs in order to fill the gaps: (1) the history, synthesis methods, structural characteristics, and surface modification; (2) the latest advances in biomedical applications (including drug/gene delivery, biosensors, bioimaging, tissue engineering, phototherapy, and antibacterial activity); and (3) biocompatibility, potential risks (toxicity in vivo/vitro and effects on human health and the environment), and strategies to reduce toxicity. Moreover, we have analyzed the challenges to be overcome in order to enhance application of GBNs in the biomedical field.
... The literature on nanotechnology abounds with reports describing both the positive and negative effects of GO nanoparticles on various species of organisms [37][38][39][40][41]. In some respects, this study is a different view on the nanotoxicity aspect. ...
Interest in graphene oxide nature and potential applications (especially nanocarriers) has resulted in numerous studies, but the results do not lead to clear conclusions. In this paper, graphene oxide is obtained by multiple synthesis methods and generally characterized. The mechanism of GO interaction with the organism is hard to summarize due to its high chemical activity and variability during the synthesis process and in biological buffers’ environments. When assessing the biocompatibility of GO, it is necessary to take into account many factors derived from nanoparticles (structure, morphology, chemical composition) and the organism (species, defense mechanisms, adaptation). This research aims to determine and compare the in vivo toxicity potential of GO samples from various manufacturers. Each GO sample is analyzed in two concentrations and applied with food. The physiological reactions of an easy model Acheta domesticus (cell viability, apoptosis, oxidative defense, DNA damage) during ten-day lasting exposure were observed. This study emphasizes the variability of the GO nature and complements the biocompatibility aspect, especially in the context of various GO-based experimental models. Changes in the cell biomarkers are discussed in light of detailed physicochemical analysis.
... In male rats, oral administration of nanosized TiO 2 and nickel (Ni) NPs had toxic effects on the reproductive system, causing marked changes in weight of the testis and epididymis (Morgan et al. 2017). A histological study has also shown that graphene oxide exposure was associated with structural damage in the testis, which included atrophy of seminiferous tubules with a reduction in germinal epithelium, germ cell loss, and vacuolization (Nirmal et al. 2017). It was reported that male rats exposed to silver nanoparticles (AgNPs) induced alterations in the testis seminiferous tubule morphometry (Lopes et al. 2019). ...
The field of nanotechnology has allowed for increasing nanoparticle (NP) exposure to the male reproductive system. Certain NPs have been reported to have adverse consequences on male germ and somatic cells. Germ cells are the bridge between generations and are responsible for the transmission of genetic and epigenetic information to future generations. A number of NPs have negative impacts on male germ and somatic cells which could ultimately affect fertility or the ability to produce healthy offspring. These impacts are related to NP composition, modification, concentration, agglomeration, and route of administration. NPs can induce severe toxic effects on the male reproduction system after passing through the blood–testis barrier and ultimately damaging the spermatozoa. Therefore, understanding the impacts of NPs on reproduction is necessary. This review will provide a comprehensive overview on the current state of knowledge derived from the previous in vivo and in vitro research on effects of NPs on the male reproductive system at the genetic, cellular, and molecular levels.
... In contrast, accumulation of GO in the testicles has been reported to result in a significant decrease in sperm motility in the epididymis, sperm DNA damage and an increase in ROS production (Akhavan et al. 2015). Although exposure to GO resulted in structural abnormalities in the testis, but a gradual recovery was observed within 30 days and fertility of the rats was not significantly affected (Nirmal et al. 2017). Note that rGO did not alter concentrations of estrogen in the serum of non-pregnant female mice, while mice in the late stages of pregnancy exposed to rGO resulted in loss of fetus and mother . ...
Due to its unique physical structure and chemical properties, graphene family nanomaterials (GFNs) and derived commodities have been widely used in commercial products, particularly biomedical applications, which has significantly increased the risk of human exposure. There exists significant evidence that GFNs are accumulated in a number of tissues and organs through different exposure pathways, and further cause toxicity manifested as lesions or functional impairment. Moreover, GFNs can be internalized by varing cell types and induce cytoskeletal disorders, organelle dysfunction, and interact directly with biological macromolecules such as DNA, mRNA and proteins, ultimately resulting in greater rates of cell apoptosis, necrosis and autophagic cell death. The toxicological effect of GFN is closely related to its lateral size, surface structure, functionalization, and propensity to adsorb proteins. Using major data published over the past four years, this review presents and summarizes state of current understanding of GFN toxicology and identifies current deficiencies and challenges. This review aims to help improve evaluation of the biocompatibility of GFNs and provides theoretical guidance for their safe application.
... Повреждение структурных компонентов гемато-эпидидимального барьера приводит к развитию инфертильных состояний и формированию спермальных гранулем [1, 15,19,32]. Повреждения эпителия эпидидимиса могут быть вызваны повышением давления семенной жидкости (обтурационные повреждения), а также в результате действия внешних факторов, таких, как бактериальная инфекция, воспаление, химические агенты и лекарственные препараты [1, 12,15,19,27,30,37]. Клеточное обновление является важнейшим свойством всех эпителиев и в первую очередь это касается эпителиальных клеток, формирующих плотные контакты, обеспечивающие барьерные свойства ряда эпителиев. ...
... First, it is a relatively hydrophilic molecule able to interact with biological structures in aqueous phase (as blood or other biological fluids). Moreover, the possibility that GO could exert a detrimental effect on reproductive function in some animal models had been suggested [14][15][16] , in particular when the spermatozoa are exposed to different GO forms [17][18][19] . ...
Graphene Oxide (GO) is a widely used biomaterial with an amazing variety of applications in biology and medicine. Recently, we reported the ability of GO to improve the in vitro fertilization (IVF) outcomes in swine, a validated animal model with a high predictive value for human fertility. For that reason, here we characterized the mechanisms involved in this positive interaction by adopting an experimental approach combining biological methods (confocal microscopy analysis on single cell, flow cytometry on cell populations and co-incubation with epithelial oviductal cells), physical-chemical techniques (Differential Scanning Calorimetry and Thermogravimetric Analysis), and chemical methods (mass spectrometry and lipid measurement). As a result, we propose a model in which GO is able to extract cholesterol from the spermatozoa membrane without causing any detrimental effect. In this way, the cholesterol extraction promotes a change in membrane chemical-physical properties that could positively affect male gamete function, modulating sperm signalling function and increasing in this way the fertilizing potential, without losing the ability to physiologically interact with the female environment. In conclusion, these data seem to suggest new intriguing possibilities in engineering sperm membrane for improving assisted reproduction technologies outcomes, even in human medicine.
... Histological examination revealed that graphene oxide exposure was associated with structural damage in the testicles, but the alteration in structure showed recovery within 30 days. 40 Gold alloys might induce the inflammation of the epididymis, which is important because the inflammation decreases sperm motility. 41 Alterations in the testis seminiferous tubule morphometry in rats treated with 200 nm AgNPs were observed. ...
With the vigorous development of nanometer-sized materials, nanoproducts are becoming widely used in all aspects of life. In medicine, nanoparticles (NPs) can be used as nanoscopic drug carriers and for nanoimaging technologies. Thus, substantial attention has been paid to the potential risks of NPs. Previous studies have shown that numerous types of NPs are able to pass certain biological barriers and exert toxic effects on crucial organs, such as the brain, liver, and kidney. Only recently, attention has been directed toward the reproductive toxicity of nanomaterials. NPs can pass through the blood–testis barrier, placental barrier, and epithelial barrier, which protect reproductive tissues, and then accumulate in reproductive organs. NP accumulation damages organs (testis, epididymis, ovary, and uterus) by destroying Sertoli cells, Leydig cells, and germ cells, causing reproductive organ dysfunction that adversely affects sperm quality, quantity, morphology, and motility or reduces the number of mature oocytes and disrupts primary and secondary follicular development. In addition, NPs can disrupt the levels of secreted hormones, causing changes in sexual behavior. However, the current review primarily examines toxicological phenomena. The molecular mechanisms involved in NP toxicity to the reproductive system are not fully understood, but possible mechanisms include oxidative stress, apoptosis, inflammation, and genotoxicity. Previous studies have shown that NPs can increase inflammation, oxidative stress, and apoptosis and induce ROS, causing damage at the molecular and genetic levels which results in cytotoxicity. This review provides an understanding of the applications and toxicological effects of NPs on the reproductive system.
Three-dimensional (3D) printing is becoming a promising tool for drug delivery, tissue modeling, fabrication, and regeneration. This chapter discusses the most recent advances and the state-of-the-art applications of 3D printing and bioprinting, including the use of models, scaffolds, biopapers, and bioinks, in nanoneurosciences and neuroscience in general. In particular, the use of nanomaterials in bioprinting and related technical concerns is also covered. Besides, the chapter describes the widespread clinical use of 3D printing at the macro-scale, significantly improving education and surgical planning in recent years. In this case, 3D printing offers a unique opportunity to provide personalized medicine and 3D visualization in real life, helping patients and healthcare providers better understand the pathology and possible approaches for its management. Finally, the endpoint of this chapter describes the future perspectives of using 3D and four-dimension printing technologies in neuroscience.
Present investigations were undertaken to record the vulnerability of testis to nickel oxide nano and microparticles in Wistar rat with special reference to their preferred bioaccumulation, consequent generation of reactive species, reciprocal influence on testosterone synthesis, DNA damage in spermatids and histopathological changes. Suitable numbers of rats were gavaged NiONPs or NiOMPs (5 mg/kg b.w.each) for 15 and 30 days. Testes en bloc were removed and processed for the estimation of selected parameters. Results showed that rat testes could accumulate nickel in an exposure time dependent manner. Generation of malondialdehyde, a denominator of ROS, increased significantly in the testes of NiONPs treated rats. Moreover, serum testosterone values also increased in NiONPs treated rats. Higher DNA damage in sperms was also recorded. Nano and microparticles of nickel, both could induce specific dose and time dependent lesions in the testis of rat. Histopathological results revealed degeneration of germinal epithelium and spermatocytes; hypertrophy of seminiferous tubules and necrosis. SEM results also indicated specific morphological changes in cellular components of tubules. This study suggests that testis is also vulnerable to the adverse effects of NiONPs alike liver and kidney. Both micro and nanoparticles of nickel elicited differential effects in a dose and exposure time dependent manner. However, NiONPs induced greater overall toxicity than NiOMPs. The results are expected to be helpful in determining the human reproductive health risks, associated with environmental/ occupational exposure to nanoparticles of nickel.
The inheritable impact of exposure to graphene oxide nanoparticles (GO NPs) on vertebrate germline during critical windows of gamete development remain undetermined to date. Here, we analyzed the transgenerational effects of exposure to nano-graphene oxide particles (nGO) synthesized in house with lateral dimensions 300-600 nm and surface charge of -36.8 mV on different developmental stages of germ cells (GCs): (1) during GCs undergoing early development and differentiation, and (2) during GCs undergoing gametogenesis and maturation in adulthood. Biocompatibility analyses in Japanese medaka embryos showed lethality above 1 µg/ml and also an aberrant increase in germ cell count of both males and females at doses below the lethal dose. However, no lethality or anomalies were evident in adults up to 45 µg/ml. Long term exposure of embryos and adults for 21 days resulted in reduced fecundity. This effect was transmitted to subsequent generations, F1 and F2. Importantly, the inheritable effects of nGO in adults were pronounced at a high dose of 10 µg/ml, while 1 µg/ml showed no impact on the germline indicating lower doses used in this study to be safe. Further, expressions of selected genes that adversely affected oocyte maturation were enhanced in F1 and F2 individuals. Interestingly, the inheritance patterns differed corresponding to the stage at which the fish received the exposure.
Two-dimensional (2D) materials have attracted tremendous interest ever since the isolation of atomically thin sheets of graphene in 2004 due to the specific and versatile properties of these materials. However, the increasing production and use of 2D materials necessitate a thorough evaluation of the potential impact on human health and the environment. Furthermore, harmonized test protocols are needed with which to assess the safety of 2D materials. The Graphene Flagship project (2013–2023), funded by the European Commission, addressed the identification of the possible hazard of graphene-based materials as well as emerging 2D materials including transition metal dichalcogenides, hexagonal boron nitride, and others. Additionally, so-called green chemistry approaches were explored to achieve the goal of a safe and sustainable production and use of this fascinating family of nanomaterials. The present review provides a compact survey of the findings and the lessons learned in the Graphene Flagship.
The unique characteristics of nanoparticles (NPs) have captivated scientists in various fields of research. However, their safety profile has not been fully scrutinized. In this regard, the effects of NPs on the reproductive system of animals and humankind have been a matter of concern. In this article, we will review the potential reproductive toxicity of various types of NPs, including carbon nanomaterials, dendrimers, quantum dots, silica, gold, and magnetic nanoparticles, reported in the literature. We also mention some notable cases where NPs have elicited beneficial effects on the reproductive system. This review provides extensive insight into the effects of various NPs on sperm and ovum and the outcomes of their passage through blood-testis and placental barriers and accumulation in the reproductive organs.
Graphene oxide (GO) is a new type of graphene material, but its effects on the male reproductive system are unclear. Here, we investigated the effects of GO on human sperm in vitro. Sperms were incubated with various doses of GO (0, 10, 20, or 40 μg/mL) for different times (1, 3, or 6 h) at 37 °C, followed by analyses of the sperm motility, viability, abnormalities, and DNA fragmentations. GO exposure significantly decreased sperm motility and viability, increased sperm abnormalities, and DNA fragmentation. Moreover, GO exposure resulted in a significant reduction of sperm mitochondrial membrane potential (MMP), which was confirmed by the ultrastructural changes of chromatin and mitochondria caused by GO. These data revealed the adverse effects of GO on sperm. Further research showed that GO exposure led to a significant increase in malondialdehyde (MDA) and reactive oxygen species (ROS) in sperm cells and a significant decrease in total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px). In addition, western blot analysis showed that the levels of Nrf-2 and HO-1 protein expression in GO-treated sperm cells were significantly increased compared to the control. These results indicated that GO had adverse effects on human sperm through oxidative stress, which was associated with Nrf-2/HO-1 signaling pathway.
Carbon nanotropes, fullerene, carbon nanotubes, and graphene have gained extensive attention due to their robust nature, ultrathin structure, high specific surface area, typical synthesis procedure and become a new vehicle in cancer therapy as a drug carrier as well as in photothermal therapy, photodynamic therapy and bioimaging. Although major challenges like hydrophobicity, toxicity, and poor biocompatibility are addressed, due to their excellent surface modification, tuning, and functionalization, all these problems have been overcome and occupied an attractive arena in biomedical applications. Their interactions with electromagnetic radiation generate heat to resulting in hyperthermia in cancer cells and interact with molecular oxygen to generate reactive oxygen species (ROS) to induce apoptosis of cancer cells through various signaling pathways. Their high optoelectronic, photoluminescence, and fluorescence property, light stability, and low cellular toxicity make them promising materials. Here, we have summarized carbon nanotropes, their unique properties, fabrication methodologies, drug delivery carrier, and other biomedical applications.KeywordsNanoparticlesCarbonNanotropesSynthesisToxicityDrug delivery and bioimagingPhototherapy
In the past few years, the development in the construction and architecture of graphene based nanocomplexes has dramatically accelerated the use of nano-graphene for therapeutic and diagnostic purposes, fostering a new area of nano-cancer therapy. To be specific, nano-graphene is increasingly used in cancer therapy, where diagnosis and treatment are coupled to deal with the clinical difficulties and challenges of this lethal disease. As a distinct family of nanomaterials, graphene derivatives exhibit outstanding structural, mechanical, electrical, optical, and thermal capabilities. Concurrently, they can transport a wide variety of synthetic agents, including medicines and biomolecules, such as nucleic acid sequences (DNA and RNA). Herewith, we first provide an overview of the most effective functionalizing agents for graphene derivatives and afterward discuss the significant improvements in the gene and drug delivery composites based on graphene.
Over the last decades, photomedicine has made a significant impact and progress in treating superficial cancer. With tremendous efforts many of the technologies have entered clinical trials. Photothermal agents (PTAs) have been considered as emerging candidates for accelerating the outcome from photomedicine based cancer treatment. Besides various inorganic and organic candidates, 2D materials such as graphene, boron nitride, and molybdenum disulfide have shown significant potential for photothermal therapy (PTT). The properties such as high surface area to volume, biocompatibility, stability in physiological media, ease of synthesis and functionalization, and high photothermal conversion efficiency have made 2D nanomaterials wonderful candidates for PTT to treat cancer. The targeting or localized activation could be achieved when PTT is combined with chemotherapies, immunotherapies, or photodynamic therapy (PDT) to provide better outcomes with fewer side effects. Though significant development has been made in the field of phototherapeutic drugs, several challenges have restricted the use of PTT in clinical use and hence they have not yet been tested in large clinical trials. In this review, we attempted to discuss the progress, properties, applications, and challenges of 2D materials in the field of PTT and their application in photomedicine.
Since its discovery in 2004, graphene is increasingly applied in various fields owing to its unique properties. Graphene application in the biomedical domain is promising and intriguing as an emerging 2D material with a high surface area, good mechanical properties, and unrivalled electronic and physical properties. This review summarizes six typical synthesis methods to fabricate pristine graphene (p‐G), graphene oxide (GO), and reduced graphene oxide (rGO), followed by characterization techniques to examine the obtained graphene materials. As bare graphene is generally undesirable in vivo and in vitro, functionalization methods to reduce toxicity, increase biocompatibility, and provide more functionalities are demonstrated. Subsequently, in vivo and in vitro behaviors of various bare and functionalized graphene materials are discussed to evaluate the functionalization effects. Reasonable control of dose (<20 mg kg⁻¹), sizes (50–1000 nm), and functionalization methods for in vivo application are advantageous. Then, the key biomedical applications based on graphene materials are discussed, coupled with the current challenges and outlooks of this growing field. In a broader sense, this review provides a comprehensive discussion on the synthesis, characterization, functionalization, evaluation, and application of p‐G, GO, and rGO in the biomedical field, highlighting their recent advances and potential.
The current study included the use of (48) white male laboratory rats, where the rats were divided into six groups, with 8 rats in each group. The second group with a concentration of 30 mg/kg, the third group with a concentration of 40 mg/kg, the fourth group with a concentration of 50 mg/kg, and the fifth group with a concentration of 60 mg/kg of nano graphene oxide. The sixth group is the control group. The results of the statistical analysis of the study showed a significant decrease in the average body weight. On the last day of the experiment when comparing between the control group and the treated groups, and the results showed a significant decrease in the average liver weight when comparing between the control group and the treated groups. Central vein, hepatic sinusoidal dilatation, hepatocyte nucleus replication, hepatocyte rupture, and nucleolysis As well as thickening of the nuclei in some hepatocytes, infiltration of inflammatory cells, hemorrhage, and dissolution and necrosis of hepatocytes. The results of the over-study by transmission electron microscope also showed changes at the cellular level, represented by the rupture of the nucleus and cytoplasm, in addition to the dissolution of the nucleus.
The article focuses on the data from Russian and foreign literature on the morphofunctional characteristics of the appendage of the testis in humans and mammalian animals. In the appendage of the testis, the head, body and tail are isolated. The head is formed by the efferent ductules, then they pass into the duct of the appendage forming the body and tail of the appendage. The epithelial cell classification in the appendage is debatable, which is due to both different methodological approaches and species differences in the epithelial lining of the testis appendage in different species. Species differences in the morphofunctional organization of the epithelium of the appendage of the testis are insignificant, this manifests the principle of parallelism in the evolutionary dynamics of the appendage epithelium. The appendage of the testis is a hormone-dependent organ, androgens play a leading role in regulating the development and functioning of the appendage; in addition, estrogens and prolactin take a significant part in regulating the appendage activity. Morphological equivalents of the negative influence of various damaging factors are basically similar, which evidences the presence of a nonspecific mechanism of reaction to the action of adverse factors formed during evolution. Despite a significant number of studies related to the morphology of the testis appendage, many aspects of morphofunctional, molecular and biochemical transformations occurring in the testis appendage still remain poorly understood and require further in-depth study. There are also insufficient number of comparative studies, and studies investigating the appendage of the testis of animals of natural biocenosis.
Numerous studies have shown that graphene oxide (GO) respiratory exposure led to severe lung injury, but whether pulmonary fibrosis caused by GO respiratory exposure is related to the activation of the caspase-1/p38MAPK/TGF-β1 remains unclear. In this study, rats were administrated GO by intratracheal instillation and fed for three months, and the molecular mechanisms of GO on the pulmonary fibrosis and other organ damage caused by GO respiratory exposure were examined. The results showed that the expression of caspase-1/p38MAPK/TGF-β1 pathway-related factors were significantly elevated with the increase of exposure concentrations of GO. Those data proved that the caspase-1/p38MAPK/TGF-β1 signaling pathway was involved in the pulmonary fibrosis caused by GO respiratory exposure. The trends of related factors also proved that the caspase-1/p38MAPK/TGF-β1 pathway was likely to play a dominant role in the sub-acute and sub-chronic stages. The other organ damage examination found that the liver and spleen were damaged initially by the GO respiratory exposure. Meanwhile for the testicle, although the acute injury was severe, signs of recovery were found during the three-month trial period.
Graphene oxide (GO), an engineered nanomaterial, has a two-dimensional structure with carbon atoms arranged in a hexagonal array. While it has been widely used in many industries, such as biomedicine, electronics, and biosensors, there are still concerns over its safety. Recently, many studies have focused on the potential toxicity of GO. Epigenetic toxicity is an important aspect of a material’s toxicological profile, since changes in gene expression have been associated with carcinogenicity and disease progression. In this review, we focus on the epigenetic alterations caused by GO, including DNA methylation, histone modification, and altered expression of non-coding RNAs. GO can affect DNA methyltransferase activity and disrupt the methylation of cytosine bases in DNA strands, leading to alteration of genome expression. Modulation of histones by GO, targeting histone deacetylase and demethylase, as well as dysregulation of miRNA and lncRNA expression have been reported. Further studies are required to determine the mechanisms of GO-induced epigenetic alterations.
Nano-copper has been increasingly employed in various products. In previous studies, we showed that nano-copper caused damage in the rat testis, but it remains unclear whether the toxic reaction can affect the reproductive function. In this study, following 28 d of exposure to nano-copper at a dose of 44, 88, and 175 mg/kg/day, there was a decrease in sperm quality, fructose content, and the secretion of sex hormones. Nano-copper also increased the level of oxidative stress, sperm malformation rate, and induced abnormal structural changes in testicular tissue. Moreover, Nano-copper upregulated the expression of apoptosis-related protein Bax and autophagy-related protein Beclin, and downregulated the expression of Bcl2 and p62. Furthermore, nano-copper (175 mg/kg) downregulated the protein expression of AMPK, p-AKT, mTOR, p-mTOR, p-4E-BP1, p70S6K, and p-p70S6K, and upregulated the protein expression of p-AMPK. Therefore, nano-copper induced damage in testicular tissues and spermatogenesis is highly related to cell apoptosis and autophagy by regulating the Akt/mTOR signaling pathway. In summary, excess exposure to nano-copper may induce testicular apoptosis and autophagy through AKT/mTOR signaling pathways, and damage the reproductive system in adult males, which is associated with oxidative stress in the testes.
Graphene possesses wider biomedical applications including drug delivery, photothermal ablation of tumors, biosensors, and also in the disease diagnosis. The accidental or intentional exposure of the environment including plants, ecosystem, and humans toward graphene is gradually increasing. Therefore, graphene toxicity becomes a critical issue to be addressed despite their diverse applications in multiple fields. In this situation, the scientific community as well as the general public must get awareness about the toxicity of graphene. This article, therefore, reviews the investigations on graphene toxicity. This review reveals the toxicity of graphene in vitro, in vivo models along with the environmental toxicity. The advantages of graphene toxicity in bacterial cells and cancer cells were also reviewed.
Sex-ratio is considered as an end point during endocrine disrupting chemicals (EDCs) evaluation. Many fish species including Japanese medaka have XX/XY sex determination mechanism, however, sex reversal (SR) can be induced by external and genetic factors. SR imposed an imbalance in natural sex ratio of a population living in any ecosystem. Considering SR as an end point, we aimed to investigate the potential EDC effects of graphene oxide (GO), a nanocarbon, using Japanese medaka as a model. One-day post-hatch (dph) medaka fries were exposed to GO (2.5, 5.0, 10.0 and 20 mg/L) for 96 h without food, followed by 6 weeks depuration in a GO-free environment with feeding. Phenotypic sex was determined by gonad histology; genotypic sex by genotyping Y-chromosome-specific male sex determining gene, dmy. Our data indicated testes in both XY and XX genotypes, while ovaries were only in XX females. Histopathology of XY and XX testis showed isogenic spermatocysts with active spermatogenesis. Distribution of spermatocytes (SPTs), not the spermatogonium (SPGs), showed enhancement in XY than XX testis. Female phenotypes had single ovary, either in stage 0 or 1. Ovo-testis/testis-ova were absent in XX or XY gonads. GO (2.5-20 mg/L) had inconsistent concentration-dependent effect in both SPGs and SPTs; however, no effect on ovarian follicles. Despite genotypic differences (XY/XX), in the histopathology/histochemistry of liver and kidneys GO effects was found to be minimum. Taken together, present study showed spontaneous induction of SR in some XX genotypes; however, exposure of fasting fries to GO had no apparent EDC effects.
Advancement in the field of nanotechnology has prompted the need to elucidate the deleterious effects of nanoparticles (NPs) on reproductive health. Many studies have reported on the health safety issues related to NPs by investigating their exposure routes, deposition and toxic effects on different primary and secondary organs but few studies have focused on NPs’ deposition in reproductive organs. Noteworthy, even fewer studies have dealt with the toxic effects of NPs on reproductive indices and sperm parameters (such as sperm number, motility and morphology) by evaluating, for instance, the histopathology of seminiferous tubules and testosterone levels. To date, the research suggests that NPs can easily cross the blood testes barrier and, after accumulation in the testis, induce adverse effects on spermatogenesis. This review aims to summarize the available literature on the risks induced by NPs on the male reproductive system.
Due to its unique properties, graphene oxide (GO) has potential for biomedical and electronic applications, however environmental contamination including aquatic ecosystem is inevitable. Moreover, potential risks of GO in aquatic life are inadequately explored. Present study was designed to evaluate GO as an endocrine disrupting chemical (EDC) using the model Japanese medaka (Oryzias latipes). GO was injected intraperitoneally (25–200 μg/g) once to breeding pairs and continued pair breeding an additional 21 days. Eggs laid were analyzed for fecundity and the fertilized eggs were evaluated for developmental abnormalities including hatching. Histopathological evaluation of gonads, liver, and kidneys was made 21 days post-injection. LD50 was found to be sex-dependent. Fecundity tended to reduce in a dose-dependent manner during early post-injection days; however, the overall evaluation showed no significant difference. The hatchability of embryos was reduced significantly in the 200 μg/g group; edema (yolk and cardiovascular) and embryo-mortality remained unaltered. Histopathological assessment identified black particles, probably agglomerated GO, in the gonads of GO-treated fish. However, folliculogenesis in stromal compartments of ovary and the composition of germinal elements in testis remained almost unaltered. Moreover, granulosa and Leydig cells morphology did not indicate any significant EDC-related effects. Although liver and kidney histopathology did not show GO as an EDC, some GO-treated fish accumulated proteinaceous fluid in hepatic vessels and induced hyperplasia in interstitial lymphoid cells (HIL) located in kidneys. GO agglomerated in medaka gonads after 21-days post-injection. However, gonad histopathology including granulosa and Leydig cells alterations were associated with GO toxicity rather than EDC effects.
Engineered nanomaterials (ENMs) are used in food additives, food packages, and therapeutic purposes owing to their useful properties, Therefore, human beings are orally exposed to exogenous nanomaterials frequently, which means the intestine is one of the primary targets of nanomaterials. Consequently, it is of great importance to understand the interaction between nanomaterials and the intestine. When nanomaterials enter into gut lumen, they inevitably interact with various components and thereby display different effects on the intestine based on their locations; these are known as location‐oriented effects (LOE). The intestinal LOE confer a new biological‐effect profile for nanomaterials, which is dependent on the involvement of the following biological processes: nano–mucus interaction, nano–intestinal epithelial cells (IECs) interaction, nano–immune interaction, and nano–microbiota interaction. A deep understanding of NM‐induced LOE will facilitate the design of safer NMs and the development of more efficient nanomedicine for intestine‐related diseases. Herein, recent progress in this field is reviewed in order to better understand the LOE of nanomaterials. The distant effects of nanomaterials coupling with microbiota are also highlighted. Investigation of the interaction of nanomaterials with the intestine will stimulate other new research areas beyond intestinal nanotoxicity.
Due to the increasing global population, growing contamination of water and air, and widespread of infectious diseases, antibiotics are extensively used as a major antibacterial drug. However, many microbes have developed resistance to antibiotics through mutation over time. As an alternative to antibiotics, antimicrobial nanomaterials have attracted great attention due to their advantageous properties and unique mechanisms of action toward microbes. They inhibit bacterial growth and destroy cells through complex mechanisms, making it difficult for bacteria to develop drug resistance, though some health concerns related to biocompatibility remain for practical applications. Among various antibacterial nanomaterials, carbon-based materials, especially graphene oxide (GO) and carbon dots (C-Dots), are promising candidates due to the ease of production and functionalization, high dispersibility in aqueous media, and promising biocompatibility. The antibacterial properties of these nanomaterials can be easily adjusted by surface modification. They are promising materials for future applications against multidrug-resistant bacteria based on their strong capacity in disruption of microbial membranes. Though many studies have reported excellent antibacterial activity of carbon nanomaterials, their impact on the environment and living organisms is of concern due to the accumulatory and cytotoxic effects. In this review, we disucss antimicrobial applications of the functional carbon nanomaterials (GO and C-Dots), their antibacterial mechanisms, factors affecting antibacterial activity, and concerns regarding cytotoxicity.
Graphene-based materials (GBMs) are a family of novel materials including graphene, few layer graphene (FLG), graphene oxide (GO), reduced graphene oxide (rGO) and graphene nanoplatelets (GNP). Currently, the risk posed by them to human health is associated mainly with the occupational exposure during their industrial and small-scale production or waste discharge. The most significant occupational exposure routes are inhalation, oral, cutaneous and ocular, inhalation being the majorly involved and most studied one. This manuscript presents a critical up-to-date review of the available in vivo toxicity data of the most significant GBMs, after using these exposure routes. The few in vivo inhalation toxicity studies (limited to 5-days of repeated exposure and only one to 5 days per week for 4 weeks) indicate inflammatory/fibrotic effects at the pulmonary level, not always reversible after 14/90 days. More limited in vivo data are available for the oral and ocular exposure routes, whereas the studies on cutaneous toxicity are at the initial stage. A long persistence of GBMs in rodents is recorded, while contradictory genotoxic data are reported. Data gap identification is also provided. Based on the available data, the occupational exposure limit cannot be determined. More experimental toxicity studies according to specific guidelines (tentatively validated for nanomaterials) and more information on the actual occupational exposure level to GBMs are needed. Furthermore, ADME (Absorption, Distribution, Metabolism, Excretion), genotoxicity, developmental and reproductive toxicity data related to the occupational exposure to GBMs have to be implemented. In addition, sub-chronic and/or chronic studies are still needed to completely exclude other toxic effects and/or carcinogenicity.
In this study, we investigated arsenic uptake and enzymatic activities in rice seedlings after the addition of nanoparticles. Hydroponic experiments were conducted to investigate the effects of different nanomaterials (high-quality graphene oxide, multilayer graphene oxide, 20 nm hydroxyapatite (HA20), 40 nm hydroxyapatite (HA40), nano-Fe3O4 (nFe3O4) and nano-zerovalent iron [nFe]) on the biomass, arsenic uptake, and enzyme activities in seedlings of the rice cultivars T705 and X24. Compared with the control, the addition of different nanomaterials increased seedling growth, with X24 rice growing better than T705 rice. Nanomaterials effectively reduced arsenic uptake in T705 rice seedlings under low and high arsenic concentrations; however, they were only effective at lower arsenic concentrations in X24 seedlings. nFe3O4 and nFe performed better than other nanomaterials in preventing arsenic from being transported to the aboveground parts of the rice seedlings. Different nanomaterials obviously influenced enzyme activities in the T705 seedlings at low arsenic concentrations (≤ 0.8 mg L-1). High-quality and multilayer graphene oxide decreased enzyme activities in the aboveground parts of the T705 seedlings, whereas, HA20 and HA40 increased the enzyme activities. nFe3O4 and nFe also reduced the effect of antioxidants in the aboveground parts of the T705 seedlings. Nanomaterials effectively reduced the arsenic uptake of T705 and X24 rice seedlings at low arsenic concentrations.
The purification of homogeneous glutathione S-transferases B and C from rat liver is described. Kinetic and physical properties of these enzymes are compared with those of homogeneous transferases A and E. The letter designations for the transferases are based on the reverse order of elution from carboxymethylcellulose, the purification step in which the transferases are separated from each other. Transferase B was purified on the basis of its ability to conjugate iodomethane with glutathione, whereas transferase C was purified on the basis of conjugation with 1,2-dichloro-4-nitrobenzene. Although each of the four enzymes can be identified by its reactivity with specific substrates, all of the enzymes are active to differing degrees in the conjugation of glutathione with p-nitrobenzyl chloride. Assay conditions for a variety of substrates are included.
All four glutathione transferases have a molecular weight of 45,000 and are dissociable into subunits of approximately 25,000 daltons. Despite the similar physical properties and overlapping substrate specificities of these enzymes, only transferases A and C are immunologically related.
Objectives:
The widely promising applications of graphene nanomaterials raise considerable concerns regarding their environmental and human health risk assessment. The aim of the current study was to evaluate the toxicity profiling of graphene family nananomaterials (GFNs) in alternative in vitro and in vivo toxicity testing models.
Methods:
The GFNs used in this study are graphene nanoplatelets ([GNPs]-pristine, carboxylate [COOH] and amide [NH2]) and graphene oxides (single layer [SLGO] and few layers [FLGO]). The human bronchial epithelial cells (Beas2B cells) as in vitro system and the nematode Caenorhabditis elegans as in vivo system were used to profile the toxicity response of GFNs. Cytotoxicity assays, colony formation assay for cellular toxicity and reproduction potentiality in C. elegans were used as end points to evaluate the GFNs' toxicity.
Results:
In general, GNPs exhibited higher toxicity than GOs in Beas2B cells, and among the GNPs the order of toxicity was pristine>NH2>COOH. Although the order of toxicity of the GNPs was maintained in C. elegans reproductive toxicity, but GOs were found to be more toxic in the worms than GNPs. In both systems, SLGO exhibited profoundly greater dose dependency than FLGO. The possible reason of their differential toxicity lay in their distinctive physicochemical characteristics and agglomeration behavior in the exposure media.
Conclusions:
The present study revealed that the toxicity of GFNs is dependent on the graphene nanomaterial's physical forms, surface functionalizations, number of layers, dose, time of exposure and obviously, on the alternative model systems used for toxicity assessment.
Abstract Graphene oxide (GO) has shown great potential for biological, medical, energy and electronic applications. As a consequence of these diverse applications, GO release into the ecosystem is inevitable; however, the corresponding risks are largely unknown, particularly with respect to the critical period of embryogenesis. This study revealed that GO adhered to and enveloped the chorion of zebrafish embryos mainly via hydroxyl group interactions, blocked the pore canals of the chorionic membrane, and caused marked hypoxia and hatching delay. Furthermore, GO spontaneously penetrated the chorion, entered the embryo via endocytosis, damaged the mitochondria and primarily translocated to the eye, heart and yolk sac regions, which are involved in the circulatory system of zebrafish. In these organs, GO induced excessive generation of reactive oxygen species and increased oxidative stress, DNA damage and apoptosis. Graphene oxide also induced developmental malformation of the eye, cardiac/yolk sac edema, tail flexure and heart rate reduction. In contrast to the common dose-effect relationships of nanoparticles, the adverse effects of GO on heart rate and tail/spinal cord flexure increased and then decreased as the GO concentration increased. These findings emphasize the specific adverse effects of GO on embryogenesis and highlight the potential ecological and health risks of GO.
In this review, we discuss the fundamental characterization of graphene oxide (GO) and its future application perspectives. Morphology is discussed through optical microscopy, fluorescence microscopy, scanning electron microscopy, and atomic force microscopy studies. Chemical, structural, and vibrational properties are discussed through x-ray photoemission spectroscopy and Raman spectroscopy studies. Two easy characterization strategies, based on the correlation between x-ray photoemission spectroscopy and contact angle/optical contrast measurements are reported. Sensing and nano-biotechnology applications are discussed with focus on practical gas sensing and optical sensing, on the one hand, and on the toxicity issue of GO, on the other hand. Synthesis and post-synthesis treatments are also discussed, these latter with emphasis on lithography.
Both in vitro and in vivo studies have demonstrated the toxic effects of graphene oxide (GO). However, molecular basis for translocation and toxicity of GO is still largely unclear. We here employed in vivo Caenorhabditis elegans assay system to identify molecular signals involved in control of translocation and toxicity of GO. We identified 7 genes whose mutations altered both translocation and toxicity of GO. Mutations of hsp-16.48, gas-1, sod-2, sod-3, or aak-2 gene caused more severe GO translocation into the body and toxic effects on both primary and secondary targeted organs compared with wild-type; however, mutations of isp-1 or clk-1 gene resulted in significantly decreased GO translocation into the body and toxicity on both primary and secondary targeted organs compared with wild-type. Moreover, mutations of hsp-16.48, gas-1, sod-2, sod-3, or aak-2 gene caused increased intestinal permeability and prolonged mean defecation cycle length in GO exposed nematodes, whereas mutations of isp-1 or clk-1 gene resulted in decreased intestinal permeability in GO exposed nematodes. Therefore, for the underlying mechanism, we hypothesize that both intestinal permeability and defecation behavior may have crucial roles in controlling functions of identified molecular signals. The molecular signals might further contribute to the control of transgenerational toxic effects of GO. Our results provide an important insight into understanding the molecular basis for in vivo translocation and toxicity of GO.
Graphene and its derivatives (for example, nanoscale graphene oxide (NGO)) have emerged as extremely attractive nanomaterials for a wide range of applications, including diagnostics and therapeutics. In this work, we present a systematic study on the in vivo distribution and pulmonary toxicity of NGO for up to 3 months after exposure. Radioisotope tracing and morphological observation demonstrated that intratracheally instilled NGO was mainly retained in the lung. NGO could result in acute lung injury (ALI) and chronic pulmonary fibrosis. Such NGO-induced ALI was related to oxidative stress and could effectively be relieved with dexamethasone treatment. In addition, we found that the biodistribution of 125I-NGO varied greatly from that of 125I ions, hence it is possible that nanoparticulates could deliver radioactive isotopes deep into the lung, which might settle in numerous ‘hot spots’ that could result in mutations and cancers, raising environmental concerns about the large-scale production of graphene oxide.
Understanding and controlling the interaction of graphene-based materials with cell membranes is key to the development of graphene-enabled biomedical technologies and to the management of graphene health and safety issues. Very little is known about the fundamental behavior of cell membranes exposed to ultrathin 2D synthetic materials. Here we investigate the interactions of graphene and few-layer graphene (FLG) microsheets with three cell types and with model lipid bilayers by combining coarse-grained molecular dynamics (MD), all-atom MD, analytical modeling, confocal fluorescence imaging, and electron microscopic imaging. The imaging experiments show edge-first uptake and complete internalization for a range of FLG samples of 0.5- to 10-μm lateral dimension. In contrast, the simulations show large energy barriers relative to kBT for membrane penetration by model graphene or FLG microsheets of similar size. More detailed simulations resolve this paradox by showing that entry is initiated at corners or asperities that are abundant along the irregular edges of fabricated graphene materials. Local piercing by these sharp protrusions initiates membrane propagation along the extended graphene edge and thus avoids the high energy barrier calculated in simple idealized MD simulations. We propose that this mechanism allows cellular uptake of even large multilayer sheets of micrometer-scale lateral dimension, which is consistent with our multimodal bioimaging results for primary human keratinocytes, human lung epithelial cells, and murine macrophages.
The chemistry of graphene oxide is discussed in this critical review. Particular emphasis is directed toward the synthesis of graphene oxide, as well as its structure. Graphene oxide as a substrate for a variety of chemical transformations, including its reduction to graphene-like materials, is also discussed. This review will be of value to synthetic chemists interested in this emerging field of materials science, as well as those investigating applications of graphene who would find a more thorough treatment of the chemistry of graphene oxide useful in understanding the scope and limitations of current approaches which utilize this material (91 references).
Herein, we report the effects of graphene oxides on human fibroblast cells and mice with the aim of investigating graphene
oxides' biocompatibility. The graphene oxides were prepared by the modified Hummers method and characterized by high-resolution
transmission electron microscope and atomic force microscopy. The human fibroblast cells were cultured with different doses
of graphene oxides for day 1 to day 5. Thirty mice divided into three test groups (low, middle, high dose) and one control
group were injected with 0.1, 0.25, and 0.4 mg graphene oxides, respectively, and were raised for 1 day, 7 days, and 30 days,
respectively. Results showed that the water-soluble graphene oxides were successfully prepared; graphene oxides with dose
less than 20 μg/mL did not exhibit toxicity to human fibroblast cells, and the dose of more than 50 μg/mL exhibits obvious
cytotoxicity such as decreasing cell adhesion, inducing cell apoptosis, entering into lysosomes, mitochondrion, endoplasm,
and cell nucleus. Graphene oxides under low dose (0.1 mg) and middle dose (0.25 mg) did not exhibit obvious toxicity to mice
and under high dose (0.4 mg) exhibited chronic toxicity, such as 4/9 mice death and lung granuloma formation, mainly located
in lung, liver, spleen, and kidney, almost could not be cleaned by kidney. In conclusion, graphene oxides exhibit dose-dependent
toxicity to cells and animals, such as inducing cell apoptosis and lung granuloma formation, and cannot be cleaned by kidney.
When graphene oxides are explored for in vivo applications in animal or human body, its biocompatibility must be considered.
Graphene oxide–Biocompatibility–Toxicity–Cell–Mice
Male reproductive health has deteriorated considerably in the last few decades. Nutritional, socioeconomic, lifestyle and environmental factors (among others) have been attributed to compromising male reproductive health. In recent years, a large volume of evidence has accumulated that suggests that the trend of decreasing male fertility (in terms of sperm count, quality and other changes in male reproductive health) might be due to exposure to environmental toxicants. These environmental contaminants can mimic natural oestrogens and target testicular spermatogenesis, steroidogenesis, and the function of both Sertoli and Leydig cells. Most environmental toxicants have been shown to induce reactive oxygen species, thereby causing a state of oxidative stress in various compartments of the testes. However, the molecular mechanism(s) of action of the environmental toxicants on the testis have yet to be elucidated. This review discusses the effects of some of the more commonly used environmental contaminants on testicular function through the induction of oxidative stress and apoptosis.
Oxidative stress in the male germ line is thought to affect male fertility and impact upon normal embryonic development. Accordingly, fertility specialists are actively exploring the diagnosis of such stress in spermatozoa and evaluating the possible use of antioxidants to ameliorate this condition. In this review, evidence for the presence of oxidative stress in human spermatozoa, the origins of this phenomenon, its clinical significance in the aetiology of male infertility and recent advances in methods for its diagnosis and treatment are re-examined. Moreover, an extensive review of the results presented in published clinical studies has been conducted to evaluate the overall impact of oral antioxidants on measures of sperm oxidative stress and DNA damage. Administration of antioxidants to infertile men has been assessed in numerous clinical studies with at least 20 reports highlighting its effect on measures of oxidative stress in human spermatozoa. A qualitative but detailed review of the results revealed that 19 of the 20 studies conclusively showed a significant reduction relating to some measure of oxidative stress in these cells. Strong evidence also supports improved motility, particularly in asthenospermic patients. However, of these studies, only 10 reported pregnancy-related outcomes, with 6 reporting positive associations. Adequately powered, placebo-controlled comprehensive clinical trials are now required to establish a clear role for antioxidants in the prevention of oxidative stress in the male germ line, such that the clinical utility of this form of therapy becomes established once and for all.
The high incidence of low sperm counts in young (European) men and evidence for declining sperm counts in recent decades mean that the environmental/lifestyle impact on spermatogenesis is an important health issue. This review assesses potential causes involving adverse effects on testis development in perinatal life (primarily effects on Sertoli cell number), which are probably irreversible, or effects on the process of spermatogenesis in adulthood, which are probably mainly reversible. Several lifestyle-related (obesity, smoking) and environmental (exposure to traffic exhaust fumes, dioxins, combustion products) factors appear to negatively affect both the perinatal and adult testes, emphasizing the importance of environmental/lifestyle impacts throughout the life course. Apart from this, public concern about adverse effects of environmental chemicals (ECs) (pesticides, food additives, persistent pollutants such as DDT, polychlorinated biphenyls) on spermatogenesis in adult men are, in general, not supported by the available data for humans. Where adverse effects of ECs have been shown, they are usually in an occupational setting rather than applying to the general population. In contrast, a modern Western lifestyle (sedentary work/lifestyle, obesity) is potentially damaging to sperm production. Spermatogenesis in normal men is poorly organized and inefficient so that men are poorly placed to cope with environmental/lifestyle insults.
Two-dimensional graphene offers interesting electronic, thermal, and mechanical properties that are currently being explored for advanced electronics, membranes, and composites. Here we synthesize and explore the biological applications of nano-graphene oxide (NGO), i.e., single-layer graphene oxide sheets down to a few nanometers in lateral width. We develop functionalization chemistry in order to impart solubility and compatibility of NGO in biological environments. We obtain size separated pegylated NGO sheets that are soluble in buffers and serum without agglomeration. The NGO sheets are found to be photoluminescent in the visible and infrared regions. The intrinsic photoluminescence (PL) of NGO is used for live cell imaging in the near-infrared (NIR) with little background. We found that simple physisorption via pi-stacking can be used for loading doxorubicin, a widely used cancer drug onto NGO functionalized with antibody for selective killing of cancer cells in vitro. Owing to its small size, intrinsic optical properties, large specific surface area, low cost, and useful non-covalent interactions with aromatic drug molecules, NGO is a promising new material for biological and medical applications.
The sperm of (C57BL X C3H)F1 mice were examined 1, 4, and 10 weeks after a subacute treatment with one of 25 chemicals at two or more dose levels. The fraction of sperm that were abnormal in shape was elevated above control values of 1.2-3.4% for methyl methanesulfonate, ethyl methanesulfonate, griseofulvin, benzo[a]pyrene, METEPA [tris(2-methyl-l-aziridinyl)phosphine oxide], THIO-TEPA [tris(l-aziridinyl)phosphine sulfide], mitomycin C, myleran, vinblastine sulphate, hydroxyurea, 3-methylcholanthrene, colchicine, actinomycin D, imuran, cyclophosphamide, 5-iododeoxyuridine, dichlorvos, aminopterin, and trimethylphosphate. Dimethylnitrosamine, urethane, DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane], 1,1-dimethylhydrazine, caffeine, and calcium cyclamate did not induce elevated levels of sperm abnormalities. The results suggest that sperm abnormalities might provide a rapid inexpensive mammalian screen for agents that lead to errors in the differentiation of spermatogenic stem cells in vivo and thus indicate agents which might prove to be mutagenic, teratogenic, or carcinogenic.
Soluble carbon nanotubes show promise as materials for in vivo delivery and imaging applications. Several reports have described the in vivo toxicity of carbon nanotubes, but their effects on male reproduction have not been examined. Here, we show that repeated intravenous injections of water-soluble multiwalled carbon nanotubes into male mice can cause reversible testis damage without affecting fertility. Nanotubes accumulated in the testes, generated oxidative stress and decreased the thickness of the seminiferous epithelium in the testis at day 15, but the damage was repaired at 60 and 90 days. The quantity, quality and integrity of the sperm and the levels of three major sex hormones were not significantly affected throughout the 90-day period. The fertility of treated male mice was unaffected; the pregnancy rate and delivery success of female mice that mated with the treated male mice did not differ from those that mated with untreated male mice.
The unique physicochemical properties of two dimensional (2D) graphene oxide (GO) could greatly benefit the biomedical field; however, recent research demonstrated that GO could induce in vitro and in vivo toxicity. We determined the mechanism of GO induced toxicity, and our in vitro experiments revealed that pristine GO could impair cell membrane integrity and functions including regulation of membrane- and cytoskeleton-associated genes, membrane permeability, fluidity and ion channels. Furthermore, GO induced platelet depletion, pro-inflammatory response and pathological changes of lung and liver in mice. To improve the biocompatibility of pristine GO, we prepared a series of GO derivatives including aminated GO (GO-NH2), poly(acrylamide)-functionalized GO (GO-PAM), poly(acrylic acid)-functionalized GO (GO-PAA) and poly(ethylene glycol)-functionalized GO (GO-PEG), and compared their toxicity with pristine GO in vitro and in vivo. Among these GO derivatives, GO-PEG and GO-PAA induced less toxicity than pristine GO, and GO-PAA was the most biocompatible one in vitro and in vivo. The differences in biocompatibility were due to the differential compositions of protein corona, especially IgG, formed on their surfaces that determine their cell membrane interaction and cellular uptake, the extent of platelet depletion in blood, thrombus formation under short-term exposure and the pro-inflammatory effects under long-term exposure. Overall, our combined data delineated the key molecular mechanisms underlying the in vivo and in vitro biological behaviors and toxicity of pristine GO, and identified a safer GO derivative that could be used for future applications.
Abstract In the past few years, much work has been performed to explore the biomedical applications and toxicity of nano-graphene and its derivatives. However, the reproductive toxicity of those carbon nanomaterials has been rarely studied. In this study, we report on the male reproductive toxicity of nanoscale graphene oxide (GO) using a mouse model. The results showed that the adult male mice injected with high dosages of GO (25 mg/kg mouse) via the tail vein exhibited normal sex hormone secretion and retained normal reproductive activity. All untreated female mice mated with the GO-treated male mice could produce healthy pups. There were no significant differences in pup numbers, sex ratio, weights, pup survival rates or pup growth over time between the GO-treated and control groups. Furthermore, these GO-treated male mice could produce a second, third, fourth and even fifth litter of healthy offspring when they lived with the untreated female mice. The testicular and epididymal histology as well as the activities of several important epididymal enzymes including α-glucosidase, lactate dehydrogenase, glutathione peroxidase and acid phosphatase were not affected by GO treatment. In addition, no damaging effects were seen at high dose rates of GO (total 300 mg/kg male mouse, 60 mg/kg every 24 h for 5 days) via intra-abdominal injection. Thus, GO showed very low or nearly no toxicity for male reproduction. This work will greatly enable future investigations of GO nanosheets for in vivo biomedical applications.
A graphene oxide (GO)-based multifunctional hybrid has been developed for loading and delivery of anticancer drugs. The GO was functionalized by magnetic/fluorescent SiO2 microsphere via an amidation process. Doxorubicin (DOX) was chosen as a model drug to be loaded onto the GO via π-π stacking and hydrophobic interaction. The loading capacities of DOX to the GO-based magnetic fluorescent hybrids were investigated. The release profiles of DOX from the hybrids were depicted. The fluorescence images of the GO-based magnetic fluorescent hybrids indicated that the hybrids would be an effective drug carrier and a potential optical imaging tool. The application of the developed GO-based magnetic fluorescent hybrids for HepG2 cells demonstrated an excellent therapeutic effect of the DOX loaded hybrids to the target tumor.
This study is focused on the crucial issue of biodegradability of graphene under in vivo conditions. We have used characteristic Raman signatures of graphene to three dimensionally (3D) image its localization in lung, liver, kidney and spleen of mouse and identified gradual development of structural disorder, happening over a period of 3 months, as indicated by the formation of defect-related D' band, line broadening of D and G bands, increase in ID /IG ratio and overall intensity reduction. Prior to injection, the carboxyl functionalized graphene of lateral size ∼200 nm is well dispersed in aqueous medium, but 24 hours post injection, larger aggregates of size up to 10 μm is detected in various organs. Using Raman cluster imaging method, temporal development of disorder is detected from day 8 onwards, which began from the edges and grew inwards over a period of 3 months. The biodegradation is found prominent in graphene phagocytosed by tissue-bound macrophages and the gene expression studies of pro-inflammatory cytokines indicated the possibility of phagocytic immune response. In addition, in vitro studies conducted on macrophage cell lines also showed development of structural disorder in the engulfed graphene, reiterating the role of macrophages in biodegradation. This is the first report providing clear evidence of in vivo biodegradation of graphene and these results may radically change the perspective on potential biomedical applications of graphene.
The autoxidation of pyrogallol was investigated in the presence of EDTA in the pH range 7.9–10.6.
The rate of autoxidation increases with increasing pH. At pH 7.9 the reaction is inhibited to 99% by superoxide dismutase, indicating an almost total dependence on the participation of the superoxide anion radical, O2·−, in the reaction. Up to pH 9.1 the reaction is still inhibited to over 90% by superoxide dismutase, but at higher alkalinity, O2·− -independent mechanisms rapidly become dominant.
Catalase has no effect on the autoxidation but decreases the oxygen consumption by half, showing that H2O2 is the stable product of oxygen and that H2O2 is not involved in the autoxidation mechanism.
A simple and rapid method for the assay of superoxide dismutase is described, based on the ability of the enzyme to inhibit the autoxidation of pyrogallol.
A plausible explanation is given for the non-competitive part of the inhibition of catechol O-methyltransferase brought about by pyrogallol.
Two-dimensional carbon-based nanomaterials, including graphene oxide and graphene, are potential candidates for biomedical applications such as sensors, cell labeling, bacterial inhibition, and drug delivery. Herein, we explore the biocompatibility of graphene-related materials with controlled physical and chemical properties. The size and extent of exfoliation of graphene oxide sheets was varied by sonication intensity and time. Graphene sheets were obtained from graphene oxide by a simple (hydrazine-free) hydrothermal route. The particle size, morphology, exfoliation extent, oxygen content, and surface charge of graphene oxide and graphene were characterized by wide-angle powder X-ray diffraction, atomic force microscopy, X-ray photoelectron spectroscopy, dynamic light scattering, and zeta-potential. One method of toxicity assessment was based on measurement of the efflux of hemoglobin from suspended red blood cells. At the smallest size, graphene oxide showed the greatest hemolytic activity, whereas aggregated graphene sheets exhibited the lowest hemolytic activity. Coating graphene oxide with chitosan nearly eliminated hemolytic activity. Together, these results demonstrate that particle size, particulate state, and oxygen content/surface charge of graphene have a strong impact on biological/toxicological responses to red blood cells. In addition, the cytotoxicity of graphene oxide and graphene sheets was investigated by measuring mitochondrial activity in adherent human skin fibroblasts using two assays. The methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, a typical nanotoxicity assay, fails to predict the toxicity of graphene oxide and graphene toxicity because of the spontaneous reduction of MTT by graphene and graphene oxide, resulting in a false positive signal. However, appropriate alternate assessments, using the water-soluble tetrazolium salt (WST-8), trypan blue exclusion, and reactive oxygen species assay reveal that the compacted graphene sheets are more damaging to mammalian fibroblasts than the less densely packed graphene oxide. Clearly, the toxicity of graphene and graphene oxide depends on the exposure environment (i.e., whether or not aggregation occurs) and mode of interaction with cells (i.e., suspension versus adherent cell types).
Graphene has emerged as interesting nanomaterials with promising applications in a range of fields including biomedicine. In this work, for the first time we study the long-term in vivo biodistribution of (125)I-labeled nanographene sheets (NGS) functionalized with polyethylene glycol (PEG) and systematically examine the potential toxicity of graphene over time. Our results show that PEGylated NGS mainly accumulate in the reticuloendothelial system (RES) including liver and spleen after intravenous administration and can be gradually cleared, likely by both renal and fecal excretion. PEGylated NGS do not cause appreciable toxicity at our tested dose (20 mg/kg) to the treated mice in a period of 3 months as evidenced by blood biochemistry, hematological analysis, and histological examinations. Our work greatly encourages further studies of graphene for biomedical applications.
There is intense interest in graphene in fields such as physics, chemistry, and materials science, among others. Interest in graphene's exceptional physical properties, chemical tunability, and potential for applications has generated thousands of publications and an accelerating pace of research, making review of such research timely. Here is an overview of the synthesis, properties, and applications of graphene and related materials (primarily, graphite oxide and its colloidal suspensions and materials made from them), from a materials science perspective.
The reaction of lipid peroxides in animal tissues with thiobarbituric acid was dependent on pH of the reaction mixture as was the case for linoleic acid hydroperoxide. The optimum pH was found to be 3.5. Taking this fact into consideration, a standard procedure for the assay of lipid peroxide level in animal tissues by their reaction with thiobarbituric acid was developed as follows. Ten percent ( tissue homogenate was mixed with sodium dodecyl sulfate, acetate buffer (pH 3.5), and aqueous solution of thiobarbituric acid. After heating at 95°C for 60 min, the red pigment produced was extracted with n-butanol-pyridine mixture and estimated by the absorbance at 532nm. As an external standard, tetramethoxy-propane was used, and lipid peroxide level was expressed in terms of nmol malondialdehyde. Using this method, the liped peroxide level in the liver of rats suffering from carbon tetrachloride intoxication was investigated. The results were in good agreement with previously reported data obtained by measuring diene content.
The purification of homogeneous glutathione S transferases B and C from rat liver is described. Kinetic and physical properties of these enzymes are compared with those of homogeneous transferases A and E. The letter designations for the transferases are based on the reverse order of elution from carboxymethylcellulose, the purification step in which the transferases are separated from each other. Transferase B was purified on the basis of its ability to conjugate iodomethane with glutathione, whereas transferase C was purified on the basis of conjugation with 1,2 dichloro 4 nitrobenzene. Although each of the 4 enzymes can be identified by its reactivity with specific substrates, all of the enzymes are active to differing degrees in the conjugation of glutathione with p nitrobenzyl chloride. Assay conditions for a variety of substrates are included. All four glutathione transferases have a molecular weight of 45,000 and are dissociable into subunits of approximately 25,000 daltons. Despite similar physical properties and overlapping substrate specificities of these enzymes, only transferases A and C are immunologically related.
To evaluate the effect of oxidative stress on the spermatogenesis and lactate dehydrogenase-X (LDH-X) activity in mouse testis.
For creating different levels of oxidative stress in mice, three selenium (Se) level diets were fed in separate groups for 8 weeks. Group 1 animals were fed yeast-based Se-deficient (0.02 ppm) diet. Group 2 and Group 3 animals were fed with the same diet supplemented with 0.2 ppm and 1 ppm Se as sodium selenite, respectively. After 8 weeks, biochemical and histopathological observations of the testis were carried out. LDH-X levels in the testis were analyzed by western immunoblot and ELISA.
A significant decrease in testis Se level was observed in Group 1 animals, whereas it was enhanced in Group 3 as compared to Group 2. The glutathione peroxidase (GSH-Px) activity was significantly reduced in both the liver and testis in Group 1, but not in Group 2 and 3. A significant increase in the testis glutathione-S-transferase (GST) activity was observed in Group 1, whereas no significant change was seen in Groups 2 and 3. Histological analysis of testis revealed a normal structure in Group 2. A significant decrease in the germ cell population in Group 1 was observed as compared to Group 2 with the spermatids and mature sperm affected the most. Decrease in the lumen size was also observed. In the Se-excess group (Group 3), displacement of germ cell population was observed. Further, a decrease in the LDH-X level in testis was observed in Group 1.
Excessive oxidative stress in the Se deficient group, as indicated by changes in the GSH-Px/GST activity, affects the spermatogenic process with a reduction in mature sperm and in turn the LDH-X level.
To examine the effect of torsion on subsequent testicular venous plasma testosterone concentrations (TVT) and to determine the relationship between the TVT values 30 days after torsion repair and testicular reperfusion immediately after torsion repair, because testicular torsion followed by repair induces an ischemia/reperfusion injury of the testis.
Adult male rats were subjected to 1 hour of 720 degrees testicular torsion, a time and degree of torsion that has been shown to cause severe impairment of spermatogenesis. Testicular microvascular perfusion before torsion, during torsion, and 5 minutes after torsion repair was determined by laser Doppler flowmetry. The animals were evaluated 3 days and 30 days later for microvascular perfusion and TVT.
Experimental torsion significantly reduced testicular vascular perfusion. Five minutes after torsion repair, the mean flow values had returned to approximately 70% of the pretorsion values. Testicular torsion significantly reduced TVT at both 3 and 30 days after torsion repair. TVT 30 days after torsion repair was significantly, but inversely, related to reperfusion values immediately after torsion repair.
These results demonstrate that the minimal duration and degree of torsion known to cause loss of spermatogenesis in the rat also causes a significant reduction in testicular androgen production in the long term. This effect was inversely related to the reperfusion values immediately after torsion repair. This suggests that reperfusion/oxidative stress may play a role in Leydig cell dysfunction, as well as by acting directly in germ cell apoptosis.
To evaluate the immunohistopathological changes in the contralateral testis of rats after an experimental spermatic cord torsion.
Male Sprague-Dawley rats of 45-50 days old were subjected to a 720 degree unilateral spermatic cord torsion for 10, 30 and 80 days (experimental group, E), respectively or sham operation (control group, C). Histopathology of the contralateral testis as well as germ cell apoptosis were studied using the Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) technique. The number of testicular lymphocytes, mast cells and macrophages, and the expression of tumor necrosis factor-alpha (TNF-alpha) and its receptor (TNFR1) in testicular cells of the contralateral testis were quantified by histochemistry and immunohistochemistry. TNF-alpha concentration in testicular fluid was determined by ELISA.
In the contralateral testis of rats from the E group, the maximal degree of damage of the germinal epithelium was seen 30 days after torsion. At this time we observed in the E group vs. the C group increases: (i) the number of testicular T-lymphocytes; (ii) the number of testicular mast cells and macrophages; (iii) the percentage of macrophages expressing TNF-alpha; (iv) TNF-a concentration in testicular fluid; (v) the number of apoptotic germ cells; and (vi) the number of TNFR1+ germ cells.
Experimental spermatic cord torsion induces, in the contralateral testis, a focal damage of seminiferous tubules characterized by apoptosis and sloughing of germ cells. Results suggest humoral and cellular immune mediated testicular cell damage in which macrophages and mast cells seem to be involved in the induction of germ cell apoptosis through the TNF-alpha/TNFR1 system and in the modulation of the inflammatory process.
Protocols for evaluating antioxidant defence and oxidative stress parameters in rat testis
- Sahoo DK
Sahoo DK. Protocols for evaluating antioxidant defence and oxidative stress parameters in rat testis. WebmedCentral Biochem
2013;4:WMC004265.