Mark F. Cesta

Normal Structure, Function, and Histology of the Spleen:

The spleen is the largest secondary immune organ in the body and is responsible for initiating immune reactions to blood-borne antigens and for filtering the blood of foreign material and old or damaged red blood cells. These functions are carried out by the 2 main compartments of the spleen, the white pulp (including the marginal zone) and the red pulp, which are vastly different in their architecture, vascular organization, and cellular composition. The morphology of these compartments is described and, to a lesser extent, their functions are discussed. The variation between species and effects of aging and genetics on splenic morphology are also discussed.

Inhaled carbon nanotubes reach the subpleural tissue in mice

Summary Carbon nanotubes have fibre-like shape1 and stimulate inflammation at the surface of the peritoneum when injected into the abdominal cavity of mice2, raising concerns that inhaled nanotubes3 may cause pleural fibrosis and/or mesothelioma4. Here we show that multi-walled carbon nanotubes reach the sub-pleura in mice after a single inhalation exposure of 30 mg/m3 for 6 hours. Nanotubes were embedded in the sub-pleural wall and within sub-pleural macrophages. Mononuclear cell aggregates on the pleural surface increased in number and size after 1 day and nanotube-containing macrophages were observed within these foci. Sub-pleural fibrosis increased after 2 and 6 weeks following inhalation. None of these effects were seen in mice that inhaled carbon black nanoparticles or a lower dose of nanotubes (1 mg/m3). This work advances a growing literature on pulmonary toxicology of nanotubes5 and suggests that minimizing inhalation of nanotubes during handling is prudent until further long term assessments are conducted.

Inhaled Multiwalled Carbon Nanotubes Potentiate Airway Fibrosis in Murine Allergic Asthma

Carbon nanotubes are gaining increasing attention due to possible health risks from occupational or environmental exposures. This study tested the hypothesis that inhaled multiwalled carbon nanotubes (MWCNT) would increase airway fibrosis in mice with allergic asthma. Normal and ovalbumin-sensitized mice were exposed to a MWCNT aerosol (100 mg/m(3)) or saline aerosol for 6 hours. Lung injury, inflammation, and fibrosis were examined by histopathology, clinical chemistry, ELISA, or RT-PCR for cytokines/chemokines, growth factors, and collagen at 1 and 14 days after inhalation. Inhaled MWCNT were distributed throughout the lung and found in macrophages by light microscopy, but were also evident in epithelial cells by electron microscopy. Quantitative morphometry showed significant airway fibrosis at 14 days in mice that received a combination of ovalbumin and MWCNT, but not in mice that received ovalbumin or MWCNT only. Ovalbumin-sensitized mice that did not inhale MWCNT had elevated levels IL-13 and transforming growth factor (TGF)-beta1 in lung lavage fluid, but not platelet-derived growth factor (PDGF)-AA. In contrast, unsensitized mice that inhaled MWCNT had elevated PDGF-AA, but not increased levels of TGF-beta1 and IL-13. This suggested that airway fibrosis resulting from combined ovalbumin sensitization and MWCNT inhalation requires PDGF, a potent fibroblast mitogen, and TGF-beta1, which stimulates collagen production. Combined ovalbumin sensitization and MWCNT inhalation also synergistically increased IL-5 mRNA levels, which could further contribute to airway fibrosis. These data indicate that inhaled MWCNT require pre-existing inflammation to cause airway fibrosis. Our findings suggest that individuals with pre-existing allergic inflammation may be susceptible to airway fibrosis from inhaled MWCNT.

Normal Structure, Function, and Histology of Mucosa-Associated Lymphoid Tissue

The mucosa-associated lymphoid tissue (MALT) initiates immune responses to specific antigens encountered along all mucosal surfaces. MALT inductive sites are secondary immune tissues where antigen sampling occurs and immune responses are initiated. Effector sites, present as diffuse lymphoid tissue along all mucosal surfaces are the sites of IgA transport across the mucosal epithelium. Though there are many differences between inductive sites in various organs, they all contain the same basic compartments—follicles, interfollicular regions, subepithelial dome regions, and follicle-associated epithelium. The morphologic differences between MALT and other secondary lymphoid tissues, between the MALT sites of differing anatomic locations, and species differences among laboratory animals are described. The morphologic changes in MALT associated with aging, route of nutrition, and genetic mutation (i.e., the nude and SCID mutations) are also discussed. MALT tissues comprise the mucosal immune system which can function independently of the systemic immune system and are, therefore, an important and often overlooked aspect of immunopathology.

Bacterial Lipopolysaccharide Enhances PDGF Signaling and Pulmonary Fibrosis in Rats Exposed to Carbon Nanotubes

Engineered multi-walled carbon nanotubes (MWCNT) represent a possible health risk for pulmonary fibrosis due to their fiber-like shape and potential for persistence in the lung. We postulated that bacterial lipopolysaccharide (LPS), a ubiquitous agent in the environment that causes lung inflammation, would enhance fibrosis caused by MWCNT. Rats were exposed to LPS and then intratracheally instilled with MWCNT or carbon black (CB) nanoparticles 24 hours later. Pulmonary fibrosis was observed 21 days after MWCNT exposure, but not with CB. LPS alone caused no fibrosis but enhanced MWCNT-induced fibrosis. LPS plus CB did not significantly increase fibrosis. MWCNT increased platelet-derived growth factor-AA (PDGF-AA), a major mediator of fibrosis. PDGF-AA production in response to MWCNT, but not CB, was synergistically enhanced by LPS. Immunostaining showed PDGF-AA in bronchiolar epithelial cells and macrophages. Since macrophages engulfed MWCNT, were positive for PDGF-AA, and mediate fibroblast responses, experiments were performed with rat lung macrophages (NR8383 cells) and rat lung fibroblasts in vitro. LPS exposure increased PDGF-A mRNA levels in NR8383 cells and enhanced MWCNT-induced PDGF-A mRNA levels. Moreover, LPS increased MWCNT- or CB-induced PDGF receptor-alpha (PDGF-Ralpha) mRNA in fibroblasts. Our data suggest that LPS exacerbates MWCNT-induced lung fibrosis by amplifying production of PDGF-AA in macrophages and epithelial cells, and by increasing PDGF-Ralpha on pulmonary fibroblasts. Our findings also suggest that individuals with pre-existing pulmonary inflammation are at greater risk for the potential adverse effects of MWCNT.

Pulmonary thrombosis in the mouse following intravenous administration of quantum dot-labeled mesenchymal cells

Abstract Quantum dots (QDs) are emerging as novel diagnostic agents. Yet, only a few studies have examined the possible deleterious effects of QD-labeled stem cells. We assessed the potential toxic effects of QD-labeled human embryonic palatal mesenchymal (QD-HEPM) cells in male NOD/SCID mice for six months, following the administration of a single intravenous injection. Control animals were administered with non-labeled HEPM cells. No treatment-related clinical signs, hematological, or biochemical parameters were found in the QD-HEPM animals in comparison to control animals. Histologically, multifocal organizing thrombi were noted in the pulmonary arteries of all QD-HEPM animals from the one-week study group and in one animal from the one-month group. Additionally, increased severity of perivascular inflammation was noted at the injection sites of QD-HEPM animals from the one-week group. This is the first study reporting histopathological evidence for pro-thrombotic adverse effects mediated by QD labeling.

Report of Partial findings from the National Toxicology Program Carcinogenesis Studies of Cell Phone Radiofrequency Radiation in Hsd: Sprague Dawley® SD rats (Whole Body Exposure)

The U.S. National Toxicology Program (NTP) has carried out extensive rodent toxicology and carcinogenesis studies of radiofrequency radiation (RFR) at frequencies and modulations used in the U.S. telecommunications industry. This report presents partial findings from these studies. The occurrences of two tumor types in male Harlan Sprague Dawley rats exposed to RFR, malignant gliomas in the brain and schwannomas of the heart, were considered of particular interest and are the subject of this report. The findings in this report were reviewed by expert peer reviewers selected by the NTP and National Institutes of Health (NIH). These reviews and responses to comments are included as appendices to this report, and revisions to the current document have incorporated and addressed these comments. When the studies are completed, they will undergo additional peer review before publication in full as part of the NTPs Toxicology and Carcinogenesis Technical Reports Series. No portion of this work has been submitted for publication in a scientific journal. Supplemental information in the form of four additional manuscripts has or will soon be submitted for publication. These manuscripts describe in detail the designs and performance of the RFR exposure system, the dosimetry of RFR exposures in rats and mice, the results to a series of pilot studies establishing the ability of the animals to thermoregulate during RFR exposures, and studies of DNA damage. (1) Capstick M, Kuster N, Kuhn S, Berdinas-Torres V, Wilson P, Ladbury J, Koepke G, McCormick D, Gauger J, and Melnick R. A radio frequency radiation reverberation chamber exposure system for rodents; (2) Yijian G, Capstick M, McCormick D, Gauger J, Horn T, Wilson P, Melnick RL, and Kuster N. Life time dosimetric assessment for mice and rats exposed to cell phone radiation; (3) Wyde ME, Horn TL, Capstick M, Ladbury J, Koepke G, Wilson P, Stout MD, Kuster N, Melnick R, Bucher JR, and McCormick D. Pilot studies of the National Toxicology Program’s cell phone radiofrequency radiation reverberation chamber exposure system; (4) Smith-Roe SL, Wyde ME, Stout MD, Winters J, Hobbs CA, Shepard KG, Green A, Kissling GE, Tice RR, Bucher JR, and Witt KL. Evaluation of the genotoxicity of cell phone radiofrequency radiation in male and female rats and mice following subchronic exposure. SUMMARY The purpose of this communication is to report partial findings from a series of radiofrequency radiation (RFR) cancer studies in rats performed under the auspices of the U.S. National Toxicology Program (NTP).1 This report contains peer-reviewed, neoplastic and hyperplastic findings only in the brain and heart of Hsd:Sprague Dawley® SD® (HSD) rats exposed to RFR starting in utero and continuing throughout their lifetimes. These studies found low incidences of malignant gliomas in the brain and schwannomas in the heart of male rats exposed to RFR of the two types [Code Division Multiple Access (CDMA) and Global System for Mobile Communications (GSM)] currently used in U.S. wireless networks. Potentially preneoplastic lesions were also observed in the brain and heart of male rats exposed to RFR. The review of partial study data in this report has been prompted by several factors. Given the widespread global usage of mobile communications among users of all ages, even a very small increase in the incidence of disease resulting from exposure to RFR could have broad implications for public health. There is a high level of public and media interest regarding the safety of cell phone RFR and the specific results of these NTP studies. Lastly, the tumors in the brain and heart observed at low incidence in male rats exposed to GSM-and CDMA-modulated cell phone RFR in this study are of a type similar to tumors observed in some epidemiology studies of cell phone use. These findings appear to support the International Agency for Research on Cancer (IARC) conclusions regarding the possible carcinogenic potential of RFR.2 It is important to note that this document reviews only the findings from the brain and heart and is not a complete report of all findings from the NTP’s studies. Additional data from these studies in Hsd:Sprague Dawley® SD® (Harlan) rats and similar studies conducted in B6C3F1/N mice are currently under evaluation and will be reported together with the current findings in two forthcoming NTP Technical Reports.

Subchronic Pulmonary Pathology, Iron Overload, and Transcriptional Activity after Libby Amphibole Exposure in Rat Models of Cardiovascular Disease

Background: Surface-available iron (Fe) is proposed to contribute to asbestos-induced toxicity through the production of reactive oxygen species. Objective: Our goal was to evaluate the hypothesis that rat models of cardiovascular disease with coexistent Fe overload would be increasingly sensitive to Libby amphibole (LA)-induced subchronic lung injury. Methods: Male healthy Wistar Kyoto (WKY), spontaneously hypertensive (SH), and SH heart failure (SHHF) rats were intratracheally instilled with 0.0, 0.25, or 1.0 mg LA (with saline as the vehicle). We examined bronchoalveolar lavage fluid (BALF) and histological lung sections after 1 week, 1 month, or 3 months for pulmonary biomarkers and pathology. SHHF rats were also assessed at 6 months for pathological changes. Results: All animals developed concentration- and time-dependent interstitial fibrosis. Time-dependent Fe accumulation occurred in LA-laden macrophages in all strains but was exacerbated in SHHF rats. LA-exposed SHHF rats developed atypical hyperplastic lesions of bronchiolar epithelial cell origin at 3 and 6 months. Strain-related baseline differences existed in gene expression at 3 months, with persistent LA effects in WKY but not SH or SHHF rats. LA exposure altered genes for a number of pathways, including inflammation, immune regulation, and cell-cycle control. Cell-cycle control genes were inhibited after LA exposure in SH and SHHF but not WKY rats, whereas tumor suppressor genes were induced only in WKY rats. The inflammatory gene expression also was apparent only in WKY rats. Conclusion: These data show that in Fe-overload conditions, progressive Fe accumulation occurs in fiber-laden macrophages within LA-induced lesions. Fe overload does not appear to contribute to chronic inflammation, and its role in hyperplastic lesion development requires further examination.

Complex histopathologic response in rat kidney to oral β-myrcene: an unusual dose-related nephrosis and low-dose alpha2u-globulin nephropathy.

Oral gavage studies with β-myrcene in male F344 rats showed a complex renal pathology comprising both alpha2u-globulin (α2u-g) nephropathy, an unusual nephrosis involving the outer stripe of outer medulla (OSOM), and an increased incidence of renal tubule tumors by 2 years. In the 90-day and 2-year studies, respectively, α2u-g nephropathy and linear papillary mineralization were observed in males at the two lower doses but were absent from the high dose. Nephrosis was characterized by dilation of the S3 tubules, nuclear enlargement (including karyomegaly), and luminal pyknotic cells, all in the outermost OSOM. Nephrosis was minimal at the higher doses in the 90-day study, but progressed to a severe grade in males dosed with 1,000 mg/kg for 2 years. Renal tubule tumors developed in treated groups with incidences up to 30% in the 250 and 500 mg/kg male dose groups. Tumors at the lower doses in males may have been associated with α2u-g nephropathy, while those at higher doses in both sexes may have been due to the nephrosis. Because β-myrcene induced a complex spectrum of renal pathology, the α2u-g nephropathy mechanism cannot be the sole mechanism of carcinogenesis in these rats.

Respiratory toxicity and immunotoxicity evaluations of microparticle and nanoparticle C60 fullerene aggregates in mice and rats following nose-only inhalation for 13 weeks

Abstract C60 fullerene (C60), or buckminsterfullerene, is a spherical arrangement of 60 carbon atoms, having a diameter of approximately 1 nm, and is produced naturally as a by-product of combustion. Due to its small size, C60 has attracted much attention for use in a variety of applications; however, insufficient information is available regarding its toxicological effects. The effects on respiratory toxicity and immunotoxicity of C60 aggregates (50 nm [nano-C60] and 1 μm [micro-C60] diameter) were examined in B6C3F1/N mice and Wistar Han rats after nose-only inhalation for 13 weeks. Exposure concentrations were selected to allow for data evaluations using both mass-based and particle surface area-based exposure metrics. Nano-C60 exposure levels selected were 0.5 and 2 mg/m3 (0.033 and 0.112 m2/m3), while micro-C60 exposures were 2, 15 and 30 mg/m3 (0.011, 0.084 and 0.167 m2/m3). There were no systemic effects on innate, cell-mediated, or humoral immune function. Pulmonary inflammatory responses (histiocytic infiltration, macrophage pigmentation, chronic inflammation) were concentration-dependent and corresponded to increases in monocyte chemoattractant protein (MCP)-1 (rats) and macrophage inflammatory protein (MIP)-1α (mice) in bronchoalveolar lavage (BAL) fluid. Lung overload may have contributed to the pulmonary inflammatory responses observed following nano-C60 exposure at 2 mg/m3 and micro-C60 exposure at 30 mg/m3. Phenotype shifts in cells recovered from the BAL were also observed in all C60-exposed rats, regardless of the level of exposure. Overall, more severe pulmonary effects were observed for nano-C60 than for micro-C60 for mass-based exposure comparisons. However, for surface-area-based exposures, more severe pulmonary effects were observed for micro-C60 than for nano-C60, highlighting the importance of dosimetry when evaluating toxicity between nano- and microparticles.