Intratracheal instillation methods and the distribution of administered material in the lung of the rat.

Intratracheal instillation is widely used for respiratory toxicity tests in experimental animals. However, there are wide variations in the techniques used for instillation, and it is thus difficult to compare the results obtained using different techniques. To examine the effect of instillation methods, we compared the distribution of a test substance in the lungs of rats after intratracheal instillations under various conditions. Rats received an intratracheal instillation of 0.3 mL of india ink suspension under different conditions as follows: 3 different angles of body restraint, 0° (supine horizontal), 45° (supine head up) and 90° (vertical head up); 2 instillation speeds, high (40 mL/min) and low (4 mL/min); and 2 different devices, a standard bulb-tipped gavage needle and an aerosolizing microsprayer designed for intratracheal instillation. One hour after treatment under these various conditions, rats were sacrificed, and the local distribution of the suspension in the lungs was observed. No animal restrained in the supine head-up or vertical head-up position died from the treatment; however, fatalities were observed when rats were restrained in the supine horizontal position except under high-speed dosing conditions with a microsprayer. Better distribution of the suspension in the lungs was observed in the rats restrained in the supine head-up position after instillation at high speed when compared with other conditions. These results indicated that high-speed instillation to the subject restrained in the supine head-up position is an appropriate condition for performing intratracheal instillation.

Effective dispersal of titanium dioxide nanoparticles for toxicity testing.

Currently, protocols for the dispersal of titanium dioxide (TiO2) nanoparticles are not standardized and often yield non-uniform particles and/or insufficient dispersal in liquid medium. Our study aimed to improve dispersal so that TiO2 nanoparticles are of uniform size, making nanotoxicity testing more reliable. Various combinations of vehicles, sonication durations, and sonication volumes were assessed for optimizing preparations of TiO2 nanoparticles. We tested each of five vehicles: ultrapure water (UPW), 0.2% disodium hydrogen phosphate (DSP), Dulbecco's phosphate-buffered saline (PBS), 0.9% saline (S), or S containing 0.05% Tween 80 (ST). We also assessed two sonication durations and three sonication volumes. Each suspension underwent ultrasonication and centrifugation; the supernatants were then analyzed. Particle size was measured by dynamic light scattering. P25 nanoparticles (~100 nm; the type of TiO2 nanoparticles used in our study) in UPW and 0.2% DSP were effectively dispersed; however, those in PBS, S, or ST were not. Relevant duration time and volume for sonication were examined with 0.2% DSP. A sonication time of 30 min and volume of 10 mL for each vial were determined to be optimal sonication conditions as determined with our dispersal assay. Under these optimal conditions, P25 nanoparticles sonicated/centrifuged in UPW or 0.2% DSP remained dispersed and exhibited long-term stability (90 days). We thus have developed a reliable procedure for preparing TiO2 nanoparticles in liquid-phase dispersions for toxicity testing.

Testicular development in growth-retarded mice.

To assess delayed fertility in male growth-retarded (grt) mice with congenital primary hypothyroidism, their testes were chronologically examined. The testicular weight in grt mice was significantly lower than age-matched normal mice until 8 weeks but was comparable at 13 and 26 weeks. While normal mice had mature sperm cells in both testes and epididymides at 5 weeks, age-matched grt mice did not. The size of the seminiferous tubules in testes of grt mice was smaller than that of normal mice before 13 weeks but was comparable at 26 weeks. These findings suggest that male grt mice might need more than 13 weeks to develop mature testes.

Increased expression of GRP94 protein is associated with decreased sensitivity to X-rays in cervical cancer cell lines.

PURPOSE: Radiation therapy is one of the standard treatments for cervical cancer. Glucose regulated protein 94 (GRP94) is a molecular chaperone, which increases in amount after X-ray irradiation. This study examined the involvement of GRP94 in radio-resistance in human cervical cancer cells. MATERIALS AND METHODS: Seven human cervical carcinoma cell lines (HeLa, SKG-I, SKG-IIIb, QG-U, Caski, SiHa and C33A) were examined for basal levels of GRP94 protein by western blotting analysis. Sensitivity to X-ray irradiation of these cell lines was determined with a colony survival assay. The suppression of GRP94 expression was performed using specific small-interfering RNA (siRNA) in HeLa and Caski cells. RESULTS: HeLa cells and QG-U cells, with higher basal levels of GRP94, exhibited a low sensitivity to X-ray cell killing. In HeLa cells, the sensitivity increased when protein GRP94 levels were reduced by specific siRNA transfection. However, a reduction in GRP94 protein had little effect on the X-ray sensitivity of Caski cells, which expressed low basal GRP94 protein levels but showed a low sensitivity to X-rays. CONCLUSIONS: High basal protein levels of GRP94 were correlated with a modest decrease in sensitivity to X-ray cell death in some cervical cancer cell lines. These results suggest that higher GRP94 protein expression is one of the molecular mechanisms causing resistance to radiation, and therefore GRP94 siRNA might be useful in tumor-specific gene therapy by reversing radio-resistance prior to radiation in cervical cancer.

Electron microscopic study of subcapsular cell hyperplasia in the adrenal glands of IQI/Jic mice.

Hyperplastic cells in subcapsular cell hyperplasia (SCH) lesion in adrenal glands of female IQI/Jic mice were examined by electron microscopy. These cells were small and polygonal, and had irregular nuclei, elongated mitochondria with lamellar cristae and dense lipid droplets. While these cells showed different features, some of them had desmosomes and basement membranes, and a few round mitochondria with tubular cristae as endocrine cells. These findings suggest that hyperplastic cells in SCH lesions might originate from endocrine blastemic cells.

Comparative investigation of several sperm analysis methods for evaluation of spermatotoxicity of industrial chemical: 2-bromopropane as an example.

Reproductive toxicity of 2-bromopropane (2BP), a substitute for ozone layer-depleting chloro-fluorocarbon, was found among the workers in an electronics factory in Korea in 1995. Furthermore the importance of testicular toxicity has been realized since the problem of endocrine disruptors arose all over the world, but manual methods must rely on subjective assessment. Recently, computer-assisted sperm analysis (CASA) was proposed but this system requires vast investment. We then investigated the applicability of the MTT method with a microplate and sperm quality analyzer (SQA) as simple, rapid, and economic instrumental methods for the examination of sperm quality in rats, comparing it with the manual microscopic method and CASA. Epididymal fluid derived from male F344/N Slc (Fischer) rats intraperitoneally injected with 2BP in the dose range of 125-1,000 mg/kg/d twice a week (total 8 times) were examined by these methods as a model experiment. Sperm count measured by the manual method and CASA in the epididymal fluid, absorbance by the MTT method and sperm motility index value by the SQA method were significantly lower in the 2BP 1,000 mg/kg administered group than in the control group. This result suggests that the MTT method can detect oligospermia. With the microplate and microplate reader, the efficiency of detection becomes much better. Sperm analyses by the MTT method with the microplate reader and the SQA method are available for reproductive toxicity study in rats.

Dose-dependent effects of perinatal hypothyroidism on postnatal testicular development in rat offspring.

The role of thyroid hormones in gonad development remains incompletely understood. We examined the dose-related effects of perinatal hypothyroidism induced by a reversible goitrogen, 6-propyl-2-thiouracil (PTU), on reproductive development in male rat offspring. Timed-pregnant Sprague-Dawley rats were orally administered PTU (0, 0.5, 1.0, or 2.0 mg/kg/day) by gavage from gestational day 15 through postnatal day 20. We observed a significant dose-dependent decrease in body weight in offspring with PTU exposure up to 13 weeks of age, but body weight became comparable among groups by 26 weeks of age. Testicular weight tended to be lower up to 7 weeks but was higher after 13 weeks of age. Epididymis weight was not different among the groups at any age. Plasma concentrations of thyroxine and triiodothyronine in the PTU groups were significantly lower at 3 weeks of age but recovered to normal levels by 26 weeks of age. No dose-related trend in plasma testosterone concentrations was found. Seminiferous tubules were larger at 13 and 26 weeks of age with PTU exposure. The number of Sertoli cells was significantly higher from 3 through 26 weeks of age. The number of Leydig cells was significantly lower up to 7 weeks of age but was comparable among groups from 13 weeks of age onwards. Thus, transient gestational and lactational thyroid hormone suppression induced small testes in early life but led to paradoxical dose-dependent testicular enlargement in adults as indicated partly by larger seminiferous tubules with numerous Sertoli cells in male rat offspring.

Construction of Artificial Hepatic Lobule-Like Spheroids on a Three-Dimensional Culture Device.

One major purpose of cell culture is the reconstruction of physiological structures. Using bovine aortic epithelium cell line HH (JCRB0099) as feeder cells and rat primary hepatocytes, we constructed hepatic lobule-like spheroids on a cell array plate designed for three-dimensional (3D) culture. Microfabricated patterning of the cell array with poly(ethyleneglycol) brushes promotes the formation of spheroids at 100-µm diameter at 100-µm intervals. Our standard protocol is to seed with feeder HH cells and then seed with primary hepatic parenchymal cells. The composite cell spheroids thus obtained are called heterospheroids. Feeder cells that were attached to the plate migrated and encompassed the spheroidal hepatocyte mass. Electron microscopy revealed Disse space-like structures characterized by hepatocyte-rooted microvilli rooted between hepatocyte and feeder epithelial HH cells. Differentiated hepatic functions such as albumin synthesis and cytochrome P450 subfamily CYP3A activities were maintained for 28 days in the heterospheroid versus monospheroid and monolayer cultures. In addition, glucuronide conjugation activity was maintained at a high level in heterospheroids. These results indicate that structurally similar hepatic lobules were formed in a microfabricated cell array coculture system and that the culture conditions are beneficial for maintaining differentiated hepatic functions.

Lack of effects for dietary exposure of bisphenol A during in utero and lactational periods on reproductive development in rat offspring.

The potential for health effects on humans with exposure to bisphenol A (BPA) has raised concerns, and the adverse effects of low-dose exposure to BPA on reproduction have been controversial. The purpose of the present study was to investigate the effects of low-dose exposure to BPA on reproductive development in F(1) rat offspring. Pregnant female Sprague-Dawley rats (F(0)) were fed a diet containing low doses of BPA (0, 0.33, 3.3, or 33 ppm) from gestational day (GD) 6 through postnatal day (PND) 21. The weanlings (F(1)) from all dose groups were fed a normal diet ad libitum after weaning and then were subjected to necropsy at 5 weeks or 3 months of age. No BPA-related changes were observed in body weight or weight of any of the major reproductive organs in F(1) males and females. Epididymis weight was significantly lower only in 3-month-old F(1) males exposed to 33 ppm BPA. Anogenital distance (AGD), the ratio of AGD to the cube root of body weight, and relative ovary weight were significantly lower in 5-week-old F(1) females exposed to 3.3 and 33 ppm BPA, but significant differences were not observed in 3-month-old females. There were no BPA-related effects on cauda epididymal sperm motility in 3-month-old F(1) males. Plasma reproductive steroid hormone concentrations were not altered among groups in either sex. These outcomes indicate that low-dose exposure to BPA in the diet does not adversely affect reproductive development in F(1) rat offspring.

Evaluation of exposure risk in the weaving process of MWCNT-coated yarn with real-time particle concentration measurements and characterization of dust particles.

Various applications of multiwalled carbon nanotubes (MWCNT) have been developed. One of these applications is an efficient sheet heating element that is woven from MWCNT-coated yarn. In this research, we assessed the exposure to MWCNT and/or the probability of particle release from broken MWCNT-coated yarn during the weaving process. This was accomplished using particle concentrations, microscopic observation, and carbon analysis. In the weaving process, neither an increase in the number of particles nor a difference in particle-size distribution was observed. In the scanning electron micrographic observation, nanosize MWCNT particles were not detected, but there were micron-size particles containing MWCNT as fragments of the yarn. Carbon analysis showed the concentration of micron-size particles containing MWCNT did not exceed 0.0053 mg-C/m(3) around the loom. This value was much lower than the respirable dust mass concentration. Most of micron-size particles seemed to originate from polyester yarn without MWCNT coating. It is recommended that workers use conventional (even not specialized for nanoparticles) personal protective equipment such as respirators and gloves to prevent exposure to respirable-size MWCNT-containing particles. The probability of MWCNT fall-off from the MWCNT-coated yarn was not detected by transmission electron microscopic observation of MWCNT-coated yarn before or after the weaving process.

Translocation of intratracheally instilled multiwall carbon nanotubes to lung-associated lymph nodes in rats.

In order to assess the extrapulmonary effects of multiwall carbon nanotubes (MWCNT), deposition of MWCNT and histopathologic changes in lung-associated lymph nodes (LALN) were examined in MWCNT-administered rats. At the age of 13 wk, male F344 rats were intratracheally instilled with MWCNT at a dose of 0 (vehicle), 40 or 160 µg/rat. The rats were sacrificed on Day 1, 7, 28 or 91 after instillation and light microscopic examinations were performed on LALN tissues. MWCNT was translocated to right and left posterior mediastinal lymph nodes and parathymic lymph nodes. Deposition of MWCNT was greater in the posterior mediastinal lymph node than in the parathymic lymph node, and the amount of MWCNT deposited in these two lymph nodes increased gradually and dose-dependently with time. MWCNT was phagocytosed by nodal macrophages, and some of the MWCNT-laden macrophages were aggregated. Transmission electron microscopic (TEM) observation confirmed the presence of MWCNT fibers with a characteristic multi-walled cylindrical structure.

Dietary exposure to low doses of bisphenol A: effects on reproduction and development in two generations of C57BL/6J mice.

The present study was conducted to examine the effects of low-dose exposure to bisphenol A on reproduction and development in two generations of mice. Pregnant female C57BL/6J mice (F(0)) were fed a diet containing low doses of bisphenol A (0, 0.33, 3.3, or 33 ppm) from gestational day 6 through postnatal day 22, and the weanlings (F(1) and F(2)) from each F(0) and F(1) dam group, respectively, were also fed these same concentrations of bisphenol A ad libitum until sacrifice. There were no treatment-related changes in body weight, body weight gain, food consumption, gestation length, or the number of live births on postnatal day 1 in F(0) dams between the control group and bisphenol A groups. Sex ratio and viability were similar in all F(1) pups. No treatment-related changes were observed in body weight, food consumption, developmental parameters, anogenital distance, or weight of any of the organs (liver, kidney, heart, spleen, thymus, testis, ovary, or uterus) in F(1) and F(2) adults in either sex. The epididymis weight was slightly higher with 0.33 and 3.3 ppm in F(1) males, but this slight increase was neither dose dependent nor seen across generations. There were no treatment-related effects of bisphenol A on cauda epididymal sperm count or sperm motility in F(1) or F(2) males. These findings indicate that dietary exposure to bisphenol A between 0.33 and 33 ppm does not adversely affect reproduction or development as assessed in two generations of mice.

Fluoroquinolone antibacterial eye drops: effects on normal human corneal epithelium, stroma, and endothelium.

BACKGROUND: In vitro studies have suggested the corneal cytotoxicity of third-generation fluoroquinolone levofloxacin (LVFX) and fourth-generation fluoroquinolone moxifloxacin hydrochloride (MFLX) among fluoroquinolone antibacterial eye drops. This study investigated the effects of these two eye drops on the human cornea in vivo. METHODS: We evaluated 30 healthy adults (19 men and 11 women, 38.3 ± 6.3 years old). Each subject received an LVFX ophthalmic solution 0.5% in one eye and an MFLX ophthalmic solution 0.5% in the other eye three times daily for 7 days. Functional and morphological corneal changes before and after instillation were evaluated through ophthalmic examinations including breakup time of tear film (BUT) as measured by fluorescein staining and DR-1, Schirmer I test, Heidelberg Retina Tomograph II Rostock Cornea Module (HRTII-RCM), specular microscope, and Pentacum examination. RESULTS: Both the LVFX and MFLX groups had no significant change in each examination before and after instillation. There was also no statistically significant difference in measurements after the 7-day instillation between the groups. CONCLUSION: Our study results suggest that as with LVFX, MFLX used in a normal clinical setting is unlikely to cause any obvious adverse effects on human normal cornea.

Characteristics of multiwall carbon nanotubes for an intratracheal instillation study with rats.

Much concern has been raised over the health consequences of workers exposed to carbon nanotubes. In order to characterize multi-wall carbon nanotubes (MWCNT) suspended in a phosphate-buffered saline containing 0.1% Tween 80 for an intratracheal instillation study. Length and width distributions of the MWCNT fibers, dispersion of MWCNT in the suspension and in the lung tissue and the MWCNT contents of metal impurities were investigated. Arithmetic mean length and width of the MWCNT fibers as measured on scanning electron microscope (SEM) photographs were 5.0 microm and 88 nm, respectively, and fibers longer than 5.0 microm were 38.9% of all fibers measured. Dynamic light scattering size measurement revealed that 5-min ultrasonication, together with addition of Tween 80 into the suspension, decreased the hydrodynamic diameters of the agglomerated MWCNT to those of finer particles below 1.0 microm. SEM observation showed good dispersion of MWCNT in the suspension, and in the alveoli on Day 1 after instillation. Concentration of iron, chromium and nickel in the MWCNT were 4,400, 48 and 17 ppm (wt/wt), respectively, all of which were below levels that would elicit positive pulmonary toxic responses to these metals. The results suggest that well-dispersed, long and thin MWCNT fibers exhibit asbestos-like pathogenicity in the lung.

Identification of a quantitative trait locus regulating B cell-dominant infiltration into autoimmune sialitis lesions of the IQI mouse model of primary Sjögren's syndrome.

Sjögren's syndrome (SS) is caused by an autoimmune sialodacryoadenitis, and up to 5% of patients with SS develop malignant B cell growth. The IQI mouse is a spontaneous model of primary SS in which B cells are the dominant cellular subpopulation among mononuclear infiltrates in sialitis lesions. Understanding the genetic control of aberrant B cell growth in IQI mice may help elucidate the genetic mechanisms involved in B-lineage hyperplasia leading to malignant transformation in human SS. B cell-dominant infiltration in the submandibular glands of 6-month-old IQI and C57BL/6 (B6) mice and their F1 and F2 progenies was quantified as B-lymphocytic sialitis score, and a genome-wide scan of 179 (IQI x B6) F2 females was performed to identify a quantitative trait locus (QTL) controlling this phenotype. A QTL significantly associated with variance in B-lymphocytic sialitis score was mapped to the D6Mit138 marker (position of 0.68cM) on proximal chromosome 6, with a logarithm of odds score of 4.3 (p = 0.00005). This QTL, named autoimmune sialitis in IQI mice, associated locus 1 (Asq1), colocalized with Islet cell autoantigen 1 (Ica1), which encodes a target protein of the immune processes that define the pathogenesis of primary SS in humans and in the nonobese diabetic mouse model.

Kinetics of biglycan, decorin and thrombospondin-1 in mercuric chloride-induced renal tubulointerstitial fibrosis.

We investigated the kinetics of decorin, biglycan and thrombospondin-1 in mercuric chloride-treated Brown Norway (BN) rats. BN rats were injected subcutaneously with 1 mg/kg b.w. of mercuric chloride one or three times. The kidney was examined histopathologically and the kinetics of decorin, biglycan and thrombospondin-1 was also examined using immunohistochemistry and real time RT-PCR. As a result, mercuric chloride induced tubular injury and subsequent tubulointerstitial fibrosis. In this lesion, the expression of thrombospondin-1 mRNA was most prominently elevated. The expression of decorin mRNA was next, but biglycan mRNA expression was not elevated. Moreover, decorin and thrombospondin-1 proteins were localized in tubular epithelial cells and peritubular interstitium. Moreover, kinetics of their mRNA expressions was relatively similar to the kinetics of TGF-beta1 mRNA expression previously reported. The present findings suggest that decorin and thrombospondin-1 may participate in the development of tubulointerstitial fibrosis and may have some relation with TGF-beta1 in mercuric chloride-treated BN rats.