Graft cell expansion from hiPSC-RPE strip after transplantation in primate eyes with or without RPE damage.

Clinical studies using suspensions or sheets of human pluripotent cell-derived retinal pigment epithelial cells (hiPSC-RPE) have been conducted globally for diseases such as age-related macular degeneration. Despite being minimally invasive, cell suspension transplantation faces challenges in targeted cell delivery and frequent cell leakage. Conversely, although the RPE sheet ensures targeted delivery with correct cell polarity, it requires invasive surgery, and graft preparation is time-consuming. We previously reported hiPSC-RPE strips as a form of quick cell aggregate that allows for reliable cell delivery to the target area with minimal invasiveness. In this study, we used a microsecond pulse laser to create a local RPE ablation model in cynomolgus monkey eyes. The hiPSC-RPE strips were transplanted into the RPE-ablated and intact sites. The hiPSC-RPE strip stably survived in all transplanted monkey eyes. The expansion area of the RPE from the engrafted strip was larger at the RPE injury site than at the intact site with no tumorigenic growth. Histological observation showed a monolayer expansion of the transplanted RPE cells with the expression of MERTK apically and collagen type 4 basally. The hiPSC-RPE strip is considered a beneficial transplantation option for RPE cell therapy.

Histological differences between the central and peripheral areas of the testes of busulfan-administered mice.

Busulfan is an anticancer drug known to cause serious damage to seminiferous tubules in the testes and deplete germ cells in human and animal models. The testicular artery is anastomosed with deferential and cremasteric arteries and is divided into capsular arteries, which give rise to the centripetal arteries and then recurrent arteries. The arterial blood in the testicular tissue is supplied by such a consequent system of arterial vessels, in order from the peripheral to the central area. As anticancer drugs are generally distributed throughout the whole body via the bloodstream and the running and distribution of arteries differ among the testicular areas, we hypothesized that the efficacy of busulfan differs in different testicular areas, particularly between the central and peripheral areas. In this study, busulfan was intraperitoneally injected at 40 mg/kg body weight into C57BL/6J male mice. After 28 days, in busulfan-treated mice, the diameters of seminiferous tubules were significantly higher in the central than in the peripheral area of the testes. The seminiferous tubular areas also significantly decreased in the peripheral areas compared with the central areas. The number of germ cells per seminiferous tubule was significantly higher in the central than in the peripheral area. Sertoli cell nuclei were detached into the lumen in the peripheral area. The number of Leydig cells was significantly lower in the peripheral areas. These data suggest that the effects of busulfan differ between the central and peripheral areas of the testis at 4 weeks after busulfan administration.

Relationship between residual visual field and full-field stimulus testing in patients with late-stage retinal degenerative diseases.

This study aimed to investigate how the extent and central/peripheral location of the residual visual field (VF) in patients with late-stage inherited retinal diseases (IRDs) are related to retinal sensitivity detected using full-field stimulus testing (FST). We reviewed the results of Goldmann perimetry and FST from the medical records of patients with IRDs whose VF represents central (within 10°) and/or peripheral islands, or undetectable. In total, 19 patients (19 eyes) were analyzed in this study. The median value of residual VF area was 1.38%. The median values of rod and cone sensitivities were - 14.9 dB and 7.4 dB, respectively. Patients with only the peripheral island (- 33.9 dB) had better median rod sensitivity than other groups (only central, - 18.9 dB; both, - 3.6 dB). VF area significantly correlated with rod sensitivity (r = - 0.943, p = 0.005) in patients with only peripheral island, but not with cone sensitivity. Peripheral VF islands were significant contributors to FST results, especially rod sensitivity. With reduced or loss of central vision, the extent of residual peripheral VF significantly affected rod sensitivity, suggesting that FST can be useful in quantitatively estimating the overall remaining vision in patients with late-stage IRD.

A novel alternative method for long-term evaluation of male reproductive toxicity and its recovery using a pre-pubertal mouse testis organ culture system.

Successful treatment of pediatric cancers often results in long-term health complications, including potential effects on fertility. Therefore, assessing the male reproductive toxicity of anti-cancer drug treatments and the potential for recovery is of paramount importance. However, in vivo evaluations are time-intensive and require large numbers of animals. To overcome these constraints, we utilized an innovative organ culture system that supports long-term spermatogenesis by placing the testis tissue between a base agarose gel and a polydimethylsiloxane ceiling, effectively mirroring the in vivo testicular environment. The present study aimed to determine the efficacy of this organ culture system for accurately assessing testicular toxicity induced by cisplatin, using acrosin-green fluorescent protein (GFP) transgenic neonatal mouse testes. The testis fragments were treated with different concentrations of cisplatin-containing medium for 24 h and incubated in fresh medium for up to 70 days. The changes in tissue volume and GFP fluorescence over time were evaluated to monitor the progression of spermatogenesis, in addition to the corresponding histopathology. Cisplatin treatment caused tissue volume shrinkage and reduced GFP fluorescence in a concentration-dependent manner. Recovery from testicular toxicity was also dependent on the concentration of cisplatin received. The results demonstrated that this novel in vitro system can be a faithful replacement for animal experiments to assess the testicular toxicity of anti-cancer drugs and their reversibility, providing a useful method for drug development.

Let-7c-5p associate with inhibition of phenobarbital-induced cell proliferation in human palate cells.

Cleft palate (CP) is one of the most common congenital diseases, and is accompanied by a complicated etiology. Medical exposure in women is among one of the reasons leading to CP. Recently, it has been reported that microRNA (miRNA) plays a crucial role in palate formation and the disruption of miRNA that influence the development of CP. Although association with pharmaceuticals and miRNAs were suggested, it has remained largely unknow. The aim of the current investigation is to elucidate upon the miRNA associated with the inhibition of phenobarbital (PB)-induced cell proliferation in human embryonic palatal mesenchymal (HEPM) cells. We showed that PB inhibited HEPM cell viability in a dose-dependent manner. We demonstrated that PB treatment suppressed cyclin-D1 expression in HEPM cells. Furthermore, PB upregulated let-7c-5p expression and downregulated the expression of two downstream genes (BACH1 and PAX3). Finally, we demonstrated that the let-7c-5p inhibitor alleviated PB-induced inhibition of cell proliferation and altered BACH1 and PAX3 expression levels. These results suggest that PB suppresses cell viability by modulating let-7c-5p expression.

Involvement of microRNA-4680-3p against phenytoin-induced cell proliferation inhibition in human palate cells.

Cleft palate (CP) is one of the most common birth defects and is caused by a combination of genetic and/or environmental factors. Environmental factors such as pharmaceutical exposure in pregnant women are known to induce CP. Recently, microRNA (miRNA) was found to be affected by environmental factors. The aim of the present study was to investigate the involvement of miRNA against phenytoin (PHE)-induced inhibition of proliferation in human embryonic palatal mesenchymal (HEPM) cells. We demonstrated that PHE inhibited HEPM cell proliferation in a dose-dependent manner. We found that treatment with PHE downregulated cyclin-D1 and cyclin-E expressions in HEPM cells. Furthermore, PHE increased miR-4680-3p expression and decreased two downstream genes (ERBB2 and JADE1). Importantly, an miR-4680-3p-specific inhibitor restored HEPM cell proliferation and altered expression of ERBB2 and JADE1 in cells treated with PHE. These results suggest that PHE suppresses cell proliferation via modulation of miR-4680-3p expression.

Evaluation of photoreceptor features in retinitis pigmentosa with cystoid macular edema by using an adaptive optics fundus camera.

OBJECTIVE: Cystoid macular edema (CME) in retinitis pigmentosa (RP) is an important complication causing visual dysfunction. We investigated the effect of CME on photoreceptors in RP patients with previous or current CME, using an adaptive optics (AO) fundus camera. METHODS: We retrospectively observed the CME and ellipsoid zone (EZ) length (average of horizontal and vertical sections) by optical coherence tomography. The density and regularity of the arrangement of photoreceptor cells (Voronoi analysis) were examined at four points around 1.5° from superior to inferior and temporal to nasal. We also performed a multivariate analysis using CME duration, central macular thickness and transversal length of CME. RESULTS: We evaluated 18 patients with previous or current CME (18 eyes; age, 48.7 ± 15.6 years) and 24 patients without previous or current CME (24 eyes; age, 46.0 ± 14.5 years). There were no significant differences in age, logMAR visual acuity, or EZ length. In groups with and without CME, cell density was 11967 ± 3148 and 16239 ± 2935 cells/mm2, and sequence regularity was 85.5 ± 3.4% and 88.5 ± 2.8%, respectively; both parameters were significantly different. The correlation between photoreceptor density and age was more negative in group with CME. The CME group tended toward greater reductions in duration of CME. CONCLUSION: Complications of CME in RP patients may lead to a decrease in photoreceptor density and regularity. Additionally, a longer duration of CME may result in a greater reduction in photoreceptor density.

Safety and stable survival of stem-cell-derived retinal organoid for 2 years in patients with retinitis pigmentosa.

Transplantation of induced pluripotent stem cell (iPSC)-derived retinal organoids into retinal disease animal models has yielded promising results, and several clinical trials on iPSC-derived retinal pigment epithelial cell transplantation have confirmed its safety. In this study, we performed allogeneic iPSC-derived retinal organoid sheet transplantation in two subjects with advanced retinitis pigmentosa (jRCTa050200027). The primary endpoint was the survival and safety of the transplanted retinal organoid sheets in the first year post-transplantation. The secondary endpoints were the safety of the transplantation procedure and visual function evaluation. The grafts survived in a stable condition for 2 years, and the retinal thickness increased at the transplant site without serious adverse events in both subjects. Changes in visual function were less progressive than those of the untreated eye during the follow-up. Allogeneic iPSC-derived retinal organoid sheet transplantation is a potential therapeutic approach, and the treatment's safety and efficacy for visual function should be investigated further.

Copper-induced diurnal hepatic toxicity is associated with Cry2 and Per1 in mice.

BACKGROUND: This study aimed to investigate diurnal variations in copper-induced hepatic toxicity and the molecular mechanisms underlying this chronotoxicity. METHODS: Male C57BL/6J mice were intraperitoneally injected with copper chloride (CuCl2) at zeitgeber time 2 (ZT2) or 14 (ZT14), twice per week for 5 or 8 weeks. Seventy-two hours after the final CuCl2 injection, the mice were euthanized, and plasma samples were collected. The livers and kidneys were collected and weighed. In vitro experiments were performed to assess cell viability and fluctuations in clock gene expression levels in Hepa1-6 cells after CuCl2 treatment. We examined copper homeostasis- and apoptosis-related genes under clock genes overexpression. RESULTS: Repeated CuCl2 administration for 8 weeks resulted in more severe toxicity at ZT14 compared to ZT2. CuCl2 administration at ZT14 elevated plasma aspartate aminotransferase, hepatic tumor necrosis factor-α, and interleukin-6 for 5 weeks, whereas the toxic effects of CuCl2 administration at ZT2 were weaker. Moreover, CuCl2 treatment inhibited Hepa1-6 cell viability in a dose-dependent manner. We observed increased expression of three clock genes (Ciart, Cry2, and Per1) after CuCl2 treatment. Among them, overexpression of Cry2 and Per1 accelerated CuCl2-induced inhibition of Hepa1-6 cell viability. Moreover, we found that the overexpression of Cry2 and Per1 regulates cleaved caspase-3 by modulating the copper transporter genes ATP7B and CTR1. CONCLUSION: These results suggest that CuCl2-induced diurnal toxicity is associated with Cry2 and Per1 expression through the regulation of copper transporter genes in mice.

Effects of inhalation of multi-walled carbon nanotube (MWCNT) on respiratory syncytial virus (RSV) infection in mice.

Multi-walled carbon nanotubes (MWCNTs), a kind of nanomaterial, are widely used in battery electrodes and composite materials, but the adverse effects associated with their accumulation in the living body have not been sufficiently investigated. MWCNTs are a fibrous material with molecules similar to asbestos fibers, and there are concerns about its effects on the respiratory system. In this study, we conducted a risk assessment by exposing mice using a previously developed nanomaterial inhalation exposure method. We quantified the exposure in the lungs by a lung burden test, evaluated the deterioration due to pneumonia using respiratory syncytial virus (RSV) infection, and measured inflammatory cytokines in bronchoalveolar lavage fluid (BALF). As a result, in the lung burden test, the amount of MWCNT in the lung increased according to the inhalation dose. In the RSV infection experiment, CCL3, CCL5, and TGF-ß, which are indicators of inflammation and lung fibrosis, were elevated in the MWCNT-exposed group. Histological examination revealed cells phagocytosing MWCNT fibers. These phagocytic cells were also seen during the recovery period from RSV infection. The present study found that MWCNT remained in the lungs for about a month or more, suggesting that the fibers may continue to exert immunological effects on the respiratory system. Furthermore, the inhalation exposure method enabled the exposure of nanomaterials to the entire lung lobe, allowing a more detailed evaluation of the effects on the respiratory system.

Involvement of Bmal1 and Clock in Bromobenzene Metabolite-Induced Diurnal Renal Toxicity.

Circadian rhythms are endogenous oscillators that regulate 24 h behavioral and physiological processes. Our previous investigation demonstrated that bromobenzene metabolite (4-bromocatechol: 4-BrCA) exhibited chronotoxicity (i.e., the nephrotoxicity induced by 4-BrCA was observed during the dark phase, while not observed at light phase in mice). However, the molecular mechanism is still unknown. The aim of the present study is to investigate the cellular molecule(s) involved in the 4-BrCA-induced nephrotoxicity using mouse renal cortex tubular cell lines (MuRTE61 cells). We found that 4-BrCA showed dose dependent (0.01-1 mM) cell proliferation defect in MuRTE61 cells. By treating with 0.03 mM 4-BrCA, we demonstrated that major clock genes (Bmal1, Clock, Cry1, Cry2, Per1, and Per2) were significantly downregulated. Interestingly, the expression levels of two genes, Bmal1 and Clock, continued to decrease after 3 h of treatment with 4-BrCA, while Cry1, Per1, and Per2 were unchanged until 24 h of treatment. Moreover, BMAL1 and CLOCK levels are higher at light phase. We speculated that BMAL1 and CLOCK might function defensively against 4-BrCA-induced nephrotoxicity since the expression levels of Bmal1 and Clock were rapidly decreased. Finally, overexpression of Bmal1 and Clock restored 4-BrCA-induced cell proliferation defect in MuRTE61 cells. Taken together, our results suggest that Bmal1 and Clock have protective roles against 4-BrCA-induced nephrotoxicity.

Immunohistochemical analysis of the vimentin filaments in Sertoli cells is a powerful tool for the prediction of spermatogenic dysfunction.

The close interaction between male germ cells and Sertoli cells, a type of somatic cell found in the seminiferous tubules of mammalian testis, is essential for the normal progression of spermatogenesis in mammals. Vimentin is an intermediate filament protein that primarily provides mechanical support, preserves cell shape, and maintains the nuclear position, and it is often used as a marker to identify Sertoli cells. Vimentin is known to be involved in many diseases and aging processes; however, how vimentin is related to spermatogenic dysfunction and the associated functional changes is still unclear. In a previous study, we reported that vitamin E deficiency affected the testes, epididymis, and spermatozoa of mice, accelerating the progression of senescence. In this study, we focused on the Sertoli cell marker vimentin and explored the relationship between the cytoskeletal system of Sertoli cells and spermatogenic dysfunction using testis tissue sections that caused male reproductive dysfunction with vitamin E deficiency. The immunohistochemical analysis showed that the proportion of the vimentin-positive area in seminiferous tubule cross-sections was significantly increased in testis tissue sections of the vitamin E-deficient group compared with the proportion in the control group. The histological analysis of testis tissue sections from the vitamin E-deficient group showed that vimentin-positive Sertoli cells were greatly extended from the basement membrane, along with an increased abundance of vimentin. These findings suggest that vimentin may be a potential indicator for detecting spermatogenic dysfunction.

Sasa veitchii extracts protect phenytoin-induced cell proliferation inhibition in human lip mesenchymal cells through modulation of miR-27b-5p.

A cleft lip, with or without a cleft palate, is a common birth defect caused by environmental factors or genetic mutations. Environmental factors, such as pharmaceutical exposure in pregnant women, are known to induce cleft lip, with or without cleft palate in the child. This study aimed to investigate the protective effect of Sasa veitchii extract (SE) on phenytoin-induced inhibition of cell proliferation in human lip mesenchymal cells (KD cells) and human embryonic palatal mesenchymal cells (HEPM cells). We demonstrated that cell proliferation was inhibited by phenytoin in a dose-dependent manner in both KD and HEPM cells. Co-treatment with SE restored phenytoin-induced toxicity in KD cells but did not protect HEPM cells against phenytoin-induced toxicity. Several microRNAs (miR-27b, miR-133b, miR-205, miR-497-5p, and miR-655-3p) is reported to associate with cell proliferation in KD cells. We measured the seven kinds of microRNAs (miR27b-3p, miR-27b-5p, miR-133b, miR-205-3p, miR-205-5p, miR-497-5p, and miR-655-3p) and found that SE suppressed miR-27b-5p induced by phenytoin in KD cells. Furthermore, co-treatment with SE enhanced the expression of miR-27b-5p downstream genes (PAX9, RARA, and SUMO1). These results suggest that SE protects phenytoin-induced cell proliferation inhibition by modulating miR-27b-5p.

Kif5a Regulates Mitochondrial Transport in Developing Retinal Ganglion Cells In Vitro.

Purpose: Axon transport of organelles and neurotrophic factors is necessary for maintaining cellular function and survival of retinal ganglion cells (RGCs). However, it is not clear how trafficking of mitochondria, essential for RGC growth and maturation, changes during RGC development. The purpose of this study was to understand the dynamics and regulation of mitochondrial transport during RGC maturation using acutely purified RGCs as a model system. Methods: Primary RGCs were immunopanned from rats of either sex during three stages of development. MitoTracker dye and live-cell imaging were used to quantify mitochondrial motility. Analysis of single-cell RNA sequencing was used to identify Kinesin family member 5A (Kif5a) as a relevant motor candidate for mitochondrial transport. Kif5a expression was manipulated with either short hairpin RNA (shRNA) or exogenous expression adeno-associated virus viral vectors. Results: Anterograde and retrograde mitochondrial trafficking and motility decreased through RGC development. Similarly, the expression of Kif5a, a motor protein that transports mitochondria, also decreased during development. Kif5a knockdown decreased anterograde mitochondrial transport, while Kif5a expression increased general mitochondrial motility and anterograde mitochondrial transport. Conclusions: Our results suggested that Kif5a directly regulates mitochondrial axonal transport in developing RGCs. Future work exploring the role of Kif5a in vivo in RGCs is indicated.

Development of a non-invasive method for testicular toxicity evaluation using a novel compact magnetic resonance imaging system.

In non-clinical animal studies for drug discovery, histopathological evaluation is the most powerful tool to assess testicular toxicity. However, histological analysis is extremely invasive; many experimental animals are needed to evaluate changes in the pathology and anatomy of the testes over time. As an alternative, small animal magnetic resonance imaging (MRI) offers a non-invasive methodology to examine testicular toxicity without radiation. The present study demonstrated the suitability of a new, ready-to-use compact MRI platform using a high-field permanent magnet to assist with the evaluation of testicular toxicity. To validate the utility of the MRI platform, male mice were treated with busulfan (40 mg/kg, intraperitoneal injection). Twenty-eight days after treatment, both testes in busulfan-treated and control mice (n = 6/group) were non-invasively scanned in situ by MRI at 1 tesla. On a T1-weighted 3D gradient-echo MRI sequences (voxel size: 0.23 × 0.23 × 0.50 mm), the total testicular volume in busulfan-treated mice was significantly smaller than in controls. On T1-weighted images, the signal intensity of the testes was significantly higher in busulfan-treated mice than in controls. The mice were sacrificed, and the testes were isolated for histopathological analysis. The weight of the testes in busulfan-treated mice significantly decreased, similar to the results of the non-invasive analysis. Additionally, periodic acid-Schiff stain-positive effusions were observed in the interstitium of the busulfan-treated mouse testes, potentially explaining T1 shortening due to a high concentration of glycoproteinaceous content. The present data demonstrated a rapid evaluation of testicular toxicity in vivo by compact MRI.

Genotype and phenotype characteristics of RHO-associated retinitis pigmentosa in the Japanese population.

PURPOSE: To identify the genotypic and phenotypic characteristics of rhodopsin (RHO)-associated retinitis pigmentosa (RP) in the Japanese population. STUDY DESIGN: Cross-sectional, single-center study METHODS: The medical records of 1336 patients with RP who underwent genetic testing at our clinic between November 2008 and September 2021 were reviewed, and patients with RHO variants were included. The patients were divided into class A and class B to assess genotype-phenotype correlations based on previous reports. The clinical findings, including best-corrected visual acuity (BCVA), OCT parameters (ellipsoid zone [EZ] width and central retinal thickness [CRT]), and presence of macular degeneration, of the 2 groups were compared. RESULTS: The study included 28 patients diagnosed with RHO-associated RP (class A, 19; class B, 9). The BCVA was significantly worse in class A patients than in class B patients (P = 0.045). Superior EZ width was significantly shorter in class A than in class B patients (P = 0.016). Class A patients tended to have thinner CRT and shorter inferior EZ width than those of class B patients, although this difference was not significant. Macular degeneration was observed in 61.5% of class A and 12.5% of class B patients, demonstrating that macular degeneration can be a common complication in class A variants. CONCLUSION: Patients with class A variants presented with a severer form of RP than that of patients with class B variants in the Japanese population. These results suggest that the phenotype of RHO-associated RP is linked to the location of the variants and that such a genotype-phenotype correlation is less affected by ethnicities with different genetic backgrounds.

Neonatal corticosterone administration increases p27-positive Sertoli cell number and decreases Sertoli cell number in the testes of mice at prepuberty.

Cortisol and corticosterone (CORT) are steroid, antistress hormones and one of the glucocorticoids in humans and animals, respectively. This study evaluated the effects of CORT administration on the male reproductive system in early life stages. CORT was subcutaneously injected at 0.36 (low-), 3.6 (middle-), and 36 (high-dosed) mg/kg body weight from postnatal day (PND) 1 to 10 in ICR mice. We observed a dose-dependent increase in serum CORT levels on PND 10, and serum testosterone levels were significantly increased only in high-dosed-CORT mice. Triiodothyronine levels were significantly higher in the low-dosed mice but lower in the middle- and high-dosed mice. However, testicular weights did not change significantly among the mice. Sertoli cell numbers were significantly reduced in low- and middle-dosed mice, whereas p27-positive Sertoli cell numbers increased in low- and middle-dosed mice. On PND 16, significant increases in testicular and relative testicular weights were observed in all-dosed-CORT mice. On PND 70, a significant decrease in testicular weight, Sertoli cell number, and spermatozoa count was observed. These results revealed that increased serum CORT levels in early life stages could induce p27 expression in Sertoli cells and terminate Sertoli cell proliferation, leading to decreased Sertoli cell number in mouse testes.

Elk-1 regulates retinal ganglion cell axon regeneration after injury.

Adult central nervous system (CNS) axons fail to regenerate after injury, and master regulators of the regenerative program remain to be identified. We analyzed the transcriptomes of retinal ganglion cells (RGCs) at 1 and 5 days after optic nerve injury with and without a cocktail of strongly pro-regenerative factors to discover genes that regulate survival and regeneration. We used advanced bioinformatic analysis to identify the top transcriptional regulators of upstream genes and cross-referenced these with the regulators upstream of genes differentially expressed between embryonic RGCs that exhibit robust axon growth vs. postnatal RGCs where this potential has been lost. We established the transcriptional activator Elk-1 as the top regulator of RGC gene expression associated with axon outgrowth in both models. We demonstrate that Elk-1 is necessary and sufficient to promote RGC neuroprotection and regeneration in vivo, and is enhanced by manipulating specific phosphorylation sites. Finally, we co-manipulated Elk-1, PTEN, and REST, another transcription factor discovered in our analysis, and found Elk-1 to be downstream of PTEN and inhibited by REST in the survival and axon regenerative pathway in RGCs. These results uncover the basic mechanisms of regulation of survival and axon growth and reveal a novel, potent therapeutic strategy to promote neuroprotection and regeneration in the adult CNS.

In vivo genotoxicity assessment of a multiwalled carbon nanotube in a mouse ex vivo culture.

BACKGROUND: Multiwalled carbon nanotubes (MWCNTs) are suspected lung carcinogens because their shape and size are similar to asbestos. Various MWCNT types are manufactured; however, only MWNT-7 is classified into Group 2B by The International Agency for Research on Cancer. MWNT-7's carcinogenicity is strongly related to inflammatory reactions. On the other hand, inconsistent results on MWNT-7 genotoxicity have been reported. We previously observed no significant differences in both Pig-a (blood) and gpt (lung) mutant frequencies between MWNT-7-intratracheally treated and negative control rats. In this study, to investigate in vivo MWNT-7 genotoxicity on various endpoints, we attempted to develop a lung micronucleus assay through ex vivo culture targeting the cellular fraction of Clara cells and alveolar Type II (AT-II) cells, known as the initiating cells of lung cancer. Using this system, we analyzed the in vivo MWNT-7 genotoxicity induced by both whole-body inhalation exposure and intratracheal instillation. We also conducted an erythrocyte micronucleus assay using the samples obtained from animals under intratracheal instillation to investigate the tissue specificity of MWNT-7 induced genotoxicities. RESULTS:  We detected a significant increase in the incidence of micronucleated cells derived from the cellular fraction of Clara cells and AT-II cells in both MWNT-7-treated and positive control groups compared to the negative control group under both whole-body inhalation exposures and intratracheal instillation. Additionally, the erythrocyte micronucleus assay detected a significant increase in the incidence of micronucleated reticulocytes only in the positive control group. CONCLUSIONS: Our findings indicated that MWNT-7 was genotoxic in the lungs directly exposed by both the body inhalation and intratracheal instillation but not in the hematopoietic tissue.

Two-year intermittent exposure of a multiwalled carbon nanotube by intratracheal instillation induces lung tumors and pleural mesotheliomas in F344 rats.

BACKGROUND: A mounting number of studies have been documenting the carcinogenic potential of multiwalled carbon nanotubes (MWCNTs); however, only a few studies have evaluated the pulmonary carcinogenicity of MWCNTs in vivo. A 2-year inhalation study demonstrated that MWNT-7, a widely used MWCNT, was a pulmonary carcinogen in rats. In another 2-year study, rats administered MWNT-7 by intratracheal instillation at the beginning of the experimental period developed pleural mesotheliomas but not lung tumors. To obtain data more comparable with rats exposed to MWNT-7 by inhalation, we administered MWNT-7 to F344 rats by intratracheal instillation once every 4-weeks over the course of 2 years at 0, 0.125, and 0.5 mg/kg body weight, allowing lung burdens of MWNT-7 to increase over the entire experimental period, similar to the inhalation study. RESULTS: Absolute and relative lung weights were significantly elevated in both MWNT-7-treated groups. Dose- and time-dependent toxic effects in the lung and pleura, such as inflammatory, fibrotic, and hyperplastic lesions, were found in both treated groups. The incidences of lung carcinomas, lung adenomas, and pleural mesotheliomas were significantly increased in the high-dose group compared with the control group. The pleural mesotheliomas developed mainly at the mediastinum. No MWNT-7-related neoplastic lesions were noted in the other organs. Cytological and biochemical parameters of the bronchoalveolar lavage fluid (BALF) were elevated in both treated groups. The lung burden of MWNT-7 was dose- and time-dependent, and at the terminal necropsy, the average value was 0.9 and 3.6 mg/lung in the low-dose and high-dose groups, respectively. The number of fibers in the pleural cavity was also dose- and time-dependent. CONCLUSIONS: Repeated administration of MWNT-7 by intratracheal instillation over the 2 years indicates that MWNT-7 is carcinogenic to both the lung and pleura of rats, which differs from the results of the 2 carcinogenicity tests by inhalation or intratracheal instillation.