Malathion-induced testicular toxicity in male rats and the protective effect of vitamins C and E

Sexually mature male Wistar rats (weighing 300–320 g and each group 6 animals) were given malathion (27 mg/kg; 1/50 of the LD₅₀ for an oral dose) and/or vitamin C (200 mg/kg) + vitamin E (200 mg/kg) daily via gavage for 4 weeks. The sperm counts, sperm motility, sperm morphology, FSH, LH, and testosterone levels, and histopathological changes in the testes of these rats, were investigated at the end of the 4th week. By the end of 4th week, rats given malathion alone, or in combination with vitamins C and E, had significantly lower sperm counts and sperm motility, and significantly higher abnormal sperm numbers, than the untreated control rats. The rats given malathion alone or in combination with vitamins also had significantly lower plasma FSH, LH and testosterone levels than the control rats. Co-treatment of malathion-exposed rats with vitamins E and C had a protective effect on sperm counts, sperm motility and abnormal sperm numbers, but not on plasma FSH, LH and testosterone levels. Light microscopic investigations revealed that 4 weeks of malathion exposure was associated with necrosis and edema in the seminiferous tubules and interstitial tissues. Degenerative changes in the seminiferous tubules were also observed in the rats which received malathion and supplemented with vitamins C and E, but milder histopathological changes were observed in the interstitial tissues. Thus, it appears that vitamins C and E ameliorate malathion testicular toxicity but are not completely protective.     

Time‐dependent biodistribution and excretion of silver nanoparticles in male Wistar rats

Silver nanoparticles (AgNPs) are the most commonly used nanoparticles owing to their antimicrobial properties. The motivation of the present study was (1) to analyze the effect of silver particle size on rat tissue distribution at different time points, (2) to determine the accumulation of AgNPs in potential rat target organs, (3) to analyze the intracellular distribution of AgNPs and (4) to examine the excretion of AgNPs by urine and feces. AgNPs were characterized by dynamic light scattering (DLS), zeta potential measurements, BET surface area measurements, transmission and scanning electron microscopy. AgNPs (20 and 200 nm) were administered intravenously (i.v.) to male Wistar rats at a dose of 5 mg kg^–1 of body weight. Biological material was sampled 24 h, 7 and 28 days after injection. Using inductively coupled plasma‐mass spectrometry (ICP‐MS) and transmission electron microscopy (TEM) it was observed that AgNPs translocated from the blood to the main organs and the concentration of silver in tissues was significantly higher in rats treated with 20 nm AgNPs as compared with 200 nm AgNPs. The highest concentration of silver was found in the liver after 24 h. After 7 days, a high level of silver was observed in the lungs and spleen. The silver concentration in the kidneys and brain increased during the experiment and reached the highest concentration after 28 days. Moreover, the highest concentration of AgNPs was observed in the urine 1 day after the injection, maintained high for 14 days and then decreased. The fecal level of silver in rats was the highest within 2 days after AgNPs administration and then decreased. Copyright © 2012 John Wiley & Sons, Ltd.     

Reproductive toxicity of methomyl insecticide in male rats and protective effect of folic acid

The acute toxicity (LD₅₀) of insecticide methomyl and its effects on male reproduction in rats were carried out. Methomyl was given orally to male rats daily for 65 successive days at two doses (0.5 and 1.0 mg kg⁻¹ b.wt., corresponding to 1/40 and 1/20 LD₅₀) alone and in combination with folic acid (1.1 mg kg⁻¹ b.wt., corresponding to acceptable daily intake, ADI). Fertility index, weight of sexual organs, semen picture, serum testosterone level and histopathology of testes were the parameters used to evaluate the reproductive efficiency of treated rats. The reversibility of methomyl effects was also studied after 65 days post-administration. The oral LD₅₀ of methomyl was 20.0 mg kg⁻¹ b.wt. in male rats. Methomyl significantly decreased the fertility index, weight of testes and accessory male sexual glands, serum testosterone level and sperm motility and count, but increased sperm cell abnormality. It induced testicular lesions characterized by moderate to severe degenerative changes of seminiferous tubules and incomplete arrest of spermatogenesis. These toxic effects were not persistent (reversible). Coadministration of folic acid with methomyl decreased its reproductive toxicity. A great attention should be taken during field application of methomyl to avoid its deleterious effects in farm animals and occupationally exposed humans.     

Particle size-dependent and surface charge-dependent biodistribution of gold nanoparticles after intravenous administration

Gold nanoparticles (GNP) provide many opportunities in imaging, diagnostics, and therapies of nanomedicine. Hence, their biokinetics in the body are prerequisites for specific tailoring of nanomedicinal applications and for a comprehensive risk assessment.We administered ¹⁹⁸Au-radio-labelled monodisperse, negatively charged GNP of five different sizes (1.4, 5, 18, 80, and 200 nm) and 2.8 nm GNP with opposite surface charges by intravenous injection into rats. After 24 h, the biodistribution of the GNP was quantitatively measured by gamma-spectrometry.The size and surface charge of GNP strongly determine the biodistribution. Most GNP accumulated in the liver increased from 50% of 1.4 nm GNP to >99% of 200 nm GNP. In contrast, there was little size-dependent accumulation of 18-200 nm GNP in most other organs. However, for GNP between 1.4 nm and 5 nm, the accumulation increased sharply with decreasing size; i.e. a linear increase with the volumetric specific surface area. The differently charged 2.8 nm GNP led to significantly different accumulations in several organs.We conclude that the alterations of accumulation in the various organs and tissues, depending on GNP size and surface charge, are mediated by dynamic protein binding and exchange. A better understanding of these mechanisms will improve drug delivery and dose estimates used in risk assessment.     

Genotoxicity of polyvinylpyrrolidone-coated silver nanoparticles in BEAS 2B cells

Silver nanoparticles (AgNPs) are widely utilized in various consumer products and medical devices, especially due to their antimicrobial properties. However, several studies have associated these particles with toxic effects, such as inflammation and oxidative stress in vivo and cytotoxic and genotoxic effects in vitro. Here, we assessed the genotoxic effects of AgNPs coated with polyvinylpyrrolidone (PVP) (average diameter 42.5 ± 14.5 nm) on human bronchial epithelial BEAS 2B cells in vitro. AgNPs were dispersed in bronchial epithelial growth medium (BEGM) with 0.6 mg/ml bovine serum albumin (BSA). The AgNP were partially well-dispersed in the medium and only limited amounts (ca. 0.02 μg Ag⁺ ion/l) could be dissolved after 24 h. The zeta-potential of the AgNPs was found to be highly negative in pure water but was at least partially neutralized in BEGM with 0.6 mg BSA/ml. Cytotoxicity was measured by cell number count utilizing Trypan Blue exclusion and by an ATP-based luminescence cell viability assay. Genotoxicity was assessed by the alkaline single cell gel electrophoresis (comet) assay, the cytokinesis-block micronucleus (MN) assay, and the chromosomal aberration (CA) assay. The cells were exposed to various doses (0.5–48 μg/cm² corresponding to 2.5–240 μg/ml) of AgNPs for 4 and 24 h in the comet assay, for 48 h in the MN assay, and for 24 and 48 h in the CA assay. DNA damage measured by the percent of DNA in comet tail was induced in a dose-dependent manner after both the 4-h and the 24-h exposures to AgNPs, with a statistically significant increase starting at 16 μg/cm² (corresponding to 60.8 μg/ml) and doubling of the percentage of DNA in tail at 48 μg/cm². However, no induction of MN or CAs was observed at any of the doses or time points. The lack of induction of chromosome damage by the PVP-coated AgNPs is possibly due to the coating which may protect the cells from direct interaction with the AgNPs, either by reducing ion leaching from the particles or by causing extensive agglomeration of the nanoparticles, with a possible reduction of the cellular uptake.     

Propolis protection from reproductive toxicity caused by aluminium chloride in male rats

Different forms of aluminium (Al) are environmental xenobiotics that induce free radical-mediated cytotoxicity and reproductive toxicity. Propolis has been reported to be important antioxidant. Therefore, this study aimed at elucidating the protective effects of propolis against reproductive toxicity of aluminium chloride (AlCl₃) in male rats. The first group served as control. Group 2 received 34 mg AlCl₃/kg bw (1/25 LD₅₀). Group 3 was administered 50 mg propolis/kg bw/day. Group 4 was treated with AlCl₃ plus propolis. Treatment was continued for 70 days. AlCl₃ caused a decrease in testes, seminal vesicle and epididymis weights, sperm concentration, motility, testosterone level and the activities of 17-ketosteroid reductase, CAT and GST, and GSH content. While, dead and abnormal sperm and testes TBARS concentrations were increased. In the AlCl₃-treated group, histopathologic examinations revealed apparent alterations in the testes, where it induced marked lesions in seminiferous tubules. Propolis alone decreased dead and abnormal sperm and TBARS, and increased testosterone, GSH, 17-ketosteroid reductase, CAT and GST. Results showed that propolis antagonized the harmful effects of AlCl₃. This was proved histopathologically by the great improvement in testes. In conclusion propolis could be effective in the protection against the reproductive toxicity of AlCl₃.     

4-Nitrophenol induces Leydig cells hyperplasia,which may contribute to the differential modulation of the androgen receptor and estrogen receptor-α and -β expression in male rat testes

4-Nitrophenol (PNP) is generally regarded as an environmental endocrine disruptor capable of estrogenic and anti-androgenic activities. To investigate PNP-induced reproductive effects, immature male rats were injected subcutaneously with PNP (0.1, 1, 10 mg/kg body weight or vehicle) daily for 4 weeks. We assessed reproductive tract alterations, sex hormone balance in the serum and estrogen receptor (ER)-α, -β and androgen receptor (AR) expression in testes. Although no significant difference was observed in body weight or testes weights of PNP-treated rats compared with the controls, the serum concentrations of testosterone in the 10 mg/kg PNP-treated group were significantly elevated. This effect was accompanied by Leydig cells hyperplasia in the testes. Conversely, there was a significant decrease in estradiol concentration and aromatase expression in the testes of the 10 mg/kg PNP-treated group. Furthermore, we observed a significant increase in ERα expression in the testes of the 10 mg/kg PNP-treated group compared with the control group. Conversely, ERβ expression displayed a significant reduction. Moreover, AR expression was significantly increased in the 10 mg/kg PNP-treated group compared with the control group. The existence of AR, ER-α and -β in the testes suggests that estradiol and testosterone directly affect germ cells and that differential modulation of AR, ER-α and -β in the testis may be involved in the direct effects of PNP or either the indirect effects of PNP-induced disruption of the estradiol-to-testosterone balance or the Leydig cells hyperplasia. Thus, the measurement of many endpoints is necessary for good risk assessment.     

Studies on the testicular effects of vitamin A palmitate in the Sprague-Dawley rat

The purpose of the research was to investigate the mechanism of reported vitamin A-induced testicular degeneration. Three studies of vitamin A toxicity were conducted in male Sprague-Dawley rats; a 10-day study with daily ip injections of retinol palmitate at doses of 0, 115,000 and 230,000 IU/kg/day in adult rats; a 10-day study with juvenile rats treated with 115,000 IU/kg/day, pair-fed controls and ad lib.-fed controls; a 13-wk dietary study in which retinol palmitate beadlets were mixed in the food of juvenile rats at doses of 0, 60,000, 120,000 and 200,000 IU/kg/day; a second untreated group was pair-fed to the high-dose group. Even at doses that produced overt signs of hypervitaminosis A and mortality, minimal or no changes were observed in the testes. In the 10-day ip studies, only a 20% incidence of treated juvenile rats (115,000 IU/kg) and adult rats (230,000 IU/kg) showed sloughing germ cells in some of the tubule lumens of the testes, but the structure and integrity of the seminiferous epithelium was completely intact. No change in testicular morphology or spermatid counts was observed in the 13-wk dietary study. In all studies, testicular weights of treated rats were not significantly reduced when corrected for body weight or compared with pair-fed controls. In the 10-day ip studies, serum testosterone levels of treated rats did not differ from the respective pair-fed control rats, but in the 13-wk study, a dose-related reduction in testosterone occurred that was considered to be a direct effect of chronic vitamin A treatment. Seminal vesicle weights were decreased, as would be expected with decreased testosterone levels. Adrenal weights were increased in all studies. These findings suggest that the testes of rat are resistant to orally administered vitamin A palmitate and only slightly affected by ip administration.     

Chronic exposure to perfluorododecanoic acid disrupts testicular steroidogenesis and the expression of related genes in male rats

Perfluorododecanoic acid (PFDoA), a synthetic perfluorinated chemical, has been detected in environmental matrices, wildlife, and human serum. Its potential health risk for humans and animals has raised public concern. However, the effects of chronic PFDoA exposure on male reproduction remain unknown. The aim of this study was to determine the effects of chronic PFDoA exposure (110 days) on testosterone biosynthesis and the expression of genes related to steroidogenesis in male rats. In this study, we examined the serum levels of sex hormones, growth hormone, and insulin in male rats. Testicular morphology and the expression of key genes and proteins in testosterone biosynthesis were also analyzed. Markedly decreased serum testosterone levels were recorded after 110 days of PFDoA exposure at 0.2 mg PFDoA/kg/day and 0.5 mg PFDoA/kg/day, and cast-off cells were observed in some seminiferous tubules in testes exposed to 0.5 mg PFDoA/kg/day. PFDoA exposure resulted in significantly decreased protein levels of steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme (P450scc), along with significantly reduced mRNA levels of insulin-like growth factor I (IGF-I), insulin-like growth factor I receptor (IGF-IR), and interleukin 1α (IL-1α) in rat testes at 0.2 mg/kg/day and 0.5 mg/kg/day. In addition, PFDoA exposure also affected the expression of some genes in the hypothalamo-neurohypophyseal system. However, PFDoA did not affect the expression of 5α-reductase, 3α-hydroxysteroid dehydrogenase, or aromatase in testis and liver. These findings demonstrate that chronic PFDoA exposure disrupts testicular steroidogenesis and expression of related genes in male rats. Multiple factors may be involved in the inhibition of testosterone by PFDoA.     

Effects of maternal toluene exposure on testosterone levels in fetal rats

The goal of our study was to determine if toluene affected the synthesis and secretion of testosterone in fetal rats. Dams were exposed to atmospheres that contained 0.09 ppm, 0.9 ppm or 9 ppm of toluene for 90 min/day from gestational days (GDs) 14.5 to 18.5 via nasal inhalation. Fetal plasma testosterone concentrations determined by enzyme immunoassay were significantly reduced on GD18.5 after exposure to 0.9 and 9 ppm, but not to 0.09 ppm, of toluene in male, but not in female, fetuses. We measured, using real-time PCR methods, mRNA levels in fetal testes for several steroidogenic enzymes involved in testosterone synthesis and insulin-like 3 (Insl3), a maker of Leydig cell differentiation. The mRNA levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) were significantly reduced after exposure to 0.9-ppm toluene. However, the mRNA levels of cytochrome P450 cholesterol side-chain cleavage, cytochrome P450 17α-hydroxylase/c17-20 lyase, 17β-hydroxysteroid dehydrogenase, and Insl3 were not significantly altered by exposure to 0.9-ppm toluene. In addition, immunohistochemical analysis showed reduced 3β-HSD-immunoreactive areas in the interstitial region of fetal testes after exposure to 0.9 and 9 ppm, but not 0.09 ppm, toluene. These findings indicate that toluene reduced the synthesis and secretion of testosterone in fetal testes from rats possibly as a consequence of reduced 3β-HSD expression.     

Silver nanoparticles induced changes in the expression of NF-κB related genes are cell type specific and related to the basal activity of NF-κB

Silver nanoparticles (AgNPs) are widely used in industry and medicine but the recent evidence for their cytotoxicity rise a concern about the safety of their use. We have previously shown that human A549 cells are resistant to AgNPs cytotoxicity, as compared with similarly treated HepG2 cells. In order to check for the role of the NF-κB signaling pathway in response of A549 and HepG2 cell lines to the treatment with 20 nm and 200 nm AgNps, we analyzed the expression of 84 key genes related to the functionality of the NF-κB signaling pathway. We observed considerable alternations in gene expression in HepG2 cells treated with 20 nm AgNPs, and minor changes when exposed to 200 nm AgNPs. Surprisingly, no changes in gene expression were observed in A549 cells treated with both size AgNPs. Using the NF-κB luciferase reporter system, we further tested the basal activity and inducibility of the NF-κB pathway in both cell lines and found that the inducibility of NF-κB signaling in A549 cells is approximately 5 times lower than this of HepG2 cells, but the basal activity is approximately 3.5 times higher. In accordance, the NF-κB activation after AgNPs treatment was observed in HepG2 but not in A549. Altogether indicate that NF-kB mediated cellular response to AgNPs is cell type specific and related to the basal activity of NF-κB.     

The critical importance of defined media conditions in Daphnia magna nanotoxicity studies

Due to the widespread use of silver nanoparticles (AgNPs), the likelihood of them entering the environment has increased and they are known to be potentially toxic. Currently, there is little information on the dynamic changes of AgNPs in ecotoxicity exposure media and how this may affect toxicity. Here, the colloidal stability of three different sizes of citrate-stabilized AgNPs was assessed in standard strength OECD ISO exposure media, and in 2-fold (media2) and 10-fold (media10) dilutions by transmission electron microscopy (TEM) and atomic force microscopy (AFM) and these characteristics were related to their toxicity towards Daphnia magna. Aggregation in undiluted media (media1) was rapid, and after diluting the medium by a factor of 2 or 10, aggregation was reduced, with minimal aggregation over 24 h occurring in media10. Acute toxicity measurements were performed using 7 nm diameter particles in media1 and media10. In media10 the EC₅₀ of the 7 nm particles for D. magna neonates was calculated to be 7.46 μg L⁻¹ with upper and lower 95% confidence intervals of 6.84 μg L⁻¹ and 8.13 μg L⁻¹ respectively. For media1, an EC₅₀ could not be calculated, the lowest observed adverse effect concentration (LOAEC) of 11.25 μg L⁻¹ indicating a significant reduction in toxicity compared to that in media10. The data suggest the increased dispersion of nanoparticles leads to enhanced toxicity, emphasising the importance of appropriate media composition to fully assess nanoparticle toxicity in aquatic ecotoxicity tests.     

In vivo measurement of extravasation of silver nanoparticles into liver extracellular space by push–pull-based continuous monitoring system

With the increasing prevalence of silver nanoparticles (AgNPs) in various products, whether such AgNPs will introduce new injury mechanisms from new pathologies remains to be determined. From the toxicokinetic viewpoint, it is vital to have in-depth knowledge of their in vivo transport kinetics and extravasation phenomenon. By combining push–pull perfusion sampling, in-tube solid phase extraction, and inductively coupled plasma mass spectrometry, we used an in vivo push–pull-based continuous monitoring system to investigate in vivo transport kinetics of extracellular AgNPs in living rat liver with a detection limit and temporal resolution of 0.64 μg L⁻¹ and 10 min, respectively. Before administration into living rats, the pre-incubation in DMEM with 10% FBS for 8 h was adopted as the optimized exposure condition for the used AgNPs. After repeated-dose treatments, we observed a higher concentration of AgNPs in the liver extracellular space, suggesting that AgNP clearance by the reticuloendothelial system (RES) may be blocked by a prior administration of AgNPs. Future studies on AgNP distribution in different liver compartments (blood stream, extracellular space and Kupffer cells/hepatocytes) are necessary for defining the risks and benefits of AgNP applications.     

Vitamin E-supplementation protect chromium (VI)-induced spermatogenic and steroidogenic disorders in testicular tissues of rats

Excess chromium (Cr) exposure is associated with various pathological conditions including reproductive dysfunction. Generation of oxidative stress is one of the plausible mechanisms behind Cr induced cellular deteriorations. The efficacy of vitamin E to combat Cr induced oxidative damage in adult rat testis has investigated in the current study. Adult male rats exposed to hexavalent Cr (intraperitoneal injection with 0.4 mg K₂Cr₂O₇/kg bw/day) for 26 days resulted in decreased accessory sex organs weight compared to controls. Development of oxidative stress in testis was evidenced by increased lipid peroxidation along with decreased superoxide dismutase (SOD) and catalase activities than control animals. Marked reduction in the activities of testicular steroidogenic enzymes; Δ⁵3β-hydroxysteroid dehydrogenase (HSD), 17β-HSD, serum testosterone and Leutinizing Hormone (LH) levels were observed. However significant increase in serum Follicle Stimulating Hormone (FSH) level was observed with Cr treated group. Histological evaluation of testis revealed degeneration of stage VII spermatogenic cycle along with decrease in epithelial cell height in epididymis and seminiferous tubules; number of different germ cells per seminiferous tubule and seminiferous tubular diameter reduced after Cr exposure. Simultaneous oral supplementation of vitamin E (50 mg/kg bw/day) in Cr exposed rats showed less oxidative damage and restored the otherwise altered testicular activities. Epididymal sperm number was also restored in vitamin E-supplemented group than Cr induced rats. This study implicates vitamin E as a possible protective agent against Cr induced spermatogenic and steroidogenic alteration.     

Anti-inflammatory and anti-ulcer activity of the extract from Alhagi maurorum (camelthorn)

Gastric hyperacidity, gastro inflammation and ulcer are very common diseases causing human suffering these days. Gastric irritation mechanism is still very poorly understood as mentioned in many scientific articles. Alhagi maurorum (camelthorn) is considered a medicinal plant with its prospective potent flavonoids. GC–MS spectrum has found three flavone structures (2-phenyl-1,4-benzopyrone derivatives) with rate more than 50% in the ethanolic plant extract. In rat experiment, ethanolic A. maurorum extract (oral daily 100 mg/kg body weight) and ranitidine the standard ulcer drug (oral daily 100 mg/kg body weight) were treated rats to protect against administration of aspirin ASP (oral 200 mg/kg body weight) for two times through the 10 days. Some rats were sacrificed after first and second aspirin administrations and the rest were sacrificed in the end of the experiment. Gastro fluid volume has been decreased in ASP group, and acid output was decreased for plant extract followed by ranitidine. Ranitidine and plant extract protect liver enzymes, oxidation status (MDA and GSH), fucosidase tumor marker and risk lipid ratio. No ulcer patterns have been shown in the histopathological study, but some inflammation in the gastric wall and vascular change dilatation of blood vessels were detected. More studies should be demonstrated potent natural plant extracts and their active components against gastro inflammation and ulcers.     

Testicular toxicity of Nigerian bonny light crude oil in male albino rats

The purpose of this work was to investigate the testicular effects of Nigerian bonny light crude oil on male albino rats. Male albino rats were administered 200, 400, and 800 mg/kg body weight of bonny light crude oil dissolved in Tween 80 in their drinking water for 7 days, while the control group received Tween 80 in their drinking water only. After 7 days, the rats were sacrificed and testis excised, weighed, and processed for histological examination. Treatment with bonny light crude oil showed a dose-dependent decrease in the absolute weight of the testes, and a significant (P < 0.05) dose-dependent reduction in the epididymal sperm number (ESN). The final body weights of the animals treated with crude oil were also significantly (P < 0.05) decreased. Histological evaluation of the testes showed slight to severe degeneration or even complete absence of seminiferous tubules and necrosis of cells depending on the dose of the crude oil. This study suggests that the Nigerian bonny light crude oil is a testicular toxicant and its use as a folklore medicine may cause infertility.     

Silver nanoparticles alter zebrafish development and larval behavior: distinct roles for particle size, coating and composition

Silver nanoparticles (AgNPs) act as antibacterials by releasing monovalent silver (Ag⁺) and are increasingly used in consumer products, thus elevating exposures in human and wildlife populations. In vitro models indicate that AgNPs are likely to be developmental neurotoxicants with actions distinct from those of Ag⁺. We exposed developing zebrafish (Danio rerio) to Ag⁺ or AgNPs on days 0-5 post-fertilization and evaluated hatching, morphology, survival and swim bladder inflation. Larval swimming behavior and responses to different lighting conditions were assessed 24 h after the termination of exposure. Comparisons were made with AgNPs of different sizes and coatings: 10 nm citrate-coated AgNP (AgNP-C), and 10 or 50 nm polyvinylpyrrolidone-coated AgNPs (AgNP-PVP). Ag⁺ and AgNP-C delayed hatching to a similar extent but Ag⁺ was more effective in slowing swim bladder inflation, and elicited greater dysmorphology and mortality. In behavioral assessments, Ag⁺ exposed fish were hyperresponsive to light changes, whereas AgNP-C exposed fish showed normal responses. Neither of the AgNP-PVPs affected survival or morphology but both evoked significant changes in swimming responses to light in ways that were distinct from Ag⁺ and each other. The smaller AgNP-PVP caused overall hypoactivity whereas the larger caused hyperactivity. AgNPs are less potent than Ag⁺ with respect to dysmorphology and loss of viability, but nevertheless produce neurobehavioral effects that highly depend on particle coating and size, rather than just reflecting the release of Ag⁺. Different AgNP formulations are thus likely to produce distinct patterns of developmental neurotoxicity.     

General 4-week toxicity study with EMS in the rat

In this subacute toxicity study, ethyl methanesulfonate (EMS) was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 20, 60 and 180/120 mg/kg body weight (bw)/day for a period of 28 days (for 19 days in the high-dose group). A control group was treated similarly with the vehicle, bidistilled water, only. The groups comprised 10 animals per sex, which were sacrificed after 28 days, respectively 19 days in the high-dose group, of treatment. Additional five rats per sex and group were treated accordingly and then allowed a 14 days treatment-free recovery period. Additional six rats per sex and group (three rats per sex in the control group) were treated accordingly and used for hemoglobin adduct analysis after EMS exposure. All animals survived until their scheduled necropsy. Treatment with EMS had a direct dose-dependent effect on food consumption and consequently on body weight at doses ≥20 mg/kg bw/day in male rats and at ≥60 mg/kg bw/day in females rats. Hence, treatment with the high dose of 180 mg/kg bw/day had to be interrupted for 9 days after which, the animals were re-dosed at 120 mg/kg bw/day. This dose was also poorly tolerated over the remaining two treatment weeks causing again a marked reduction in food consumption and body weight. A dose of 60 mg/kg bw/day was moderately tolerated over 4 weeks treatment with mean daily food consumption and body weight distinctly lower than in controls. Primary targets of systemic toxicity were the hematopoietic system, thymolymphatic system and sexual organs. Characteristic changes in hematology parameters were decreased red blood cell counts, hematocrit, and hemoglobin concentration. White blood cell counts were also decreased due to reduced lymphocyte and granulocyte populations of each fraction. The corresponding histopathology findings were fatty atrophy of bone marrow and minimal hypocellularity of the white pulp of the spleen. Similarly, treatment with EMS caused an involution of the thymolymphatic system characterized by decreased organ weight of thymus, lymph nodes, and spleen microscopically associated with atrophy of the thymus and hypocellularity of Peyer's patches, lymph nodes and the white pulp of the spleen. The effects on sexual organs included lower organ weight/reduced size for testes, epididymides, seminal vesicles, prostate, and uterus. Tubular atrophy, single cell necrosis of the germ cells and in epididymides reduced spermatozoa were recorded microscopically. The described findings occurred at doses of 60 and 180/120 mg/kg bw/day and were dose-dependent with regard to incidence and severity. Other target organs were the pancreas (acinar cell vacuolation), thyroid gland (follicular cell hypertrophy), and salivary gland (secretory depletion of convoluted ducts). The systemic exposure to EMS was monitored by hemoglobin ethylvaline adduct measurement. The concentration of hemoglobin ethylvaline adducts was linear with the dose and accumulated 11–26-fold over the treatment period. In summary, decreases in food consumption and body weight were the dose-limiting effects of treatment with EMS. Organ toxicity was characterized by depression of cell proliferation (hematopoiesis and spermatogenesis) and changes suggestive of reduced metabolism and/or physiological imbalances (e.g. thymolymphatic system and thyroid gland) without signs of inflammatory or necrotic lesions. For some findings, especially the effects on the thymolymphatic system and sexual organs, it cannot be excluded that starvation-like condition contributed to the occurrence of such changes. The low dose of 20 mg/kg bw/day was basically free of adverse effects despite of a clear evidence for hemoglobin adducts.