Janet M. Carter

Principles for characterizing the potential human health effects from exposure to nanomaterials: Elements of a screening strategy

AbstractThe rapid proliferation of many different engineered nanomaterials (defined as materials designed and produced to have structural features with at least one dimension of 100 nanometers or less) presents a dilemma to regulators regarding hazard identification. The International Life Sciences Institute Research Foundation/Risk Science Institute convened an expert working group to develop a screening strategy for the hazard identification of engineered nanomaterials. The working group report presents the elements of a screening strategy rather than a detailed testing protocol. Based on an evaluation of the limited data currently available, the report presents a broad data gathering strategy applicable to this early stage in the development of a risk assessment process for nanomaterials. Oral, dermal, inhalation, and injection routes of exposure are included recognizing that, depending on use patterns, exposure to nanomaterials may occur by any of these routes. The three key elements of the toxicity screening strategy are: Physicochemical Characteristics, In Vitro Assays (cellular and non-cellular), and In Vivo Assays.There is a strong likelihood that biological activity of nanoparticles will depend on physicochemical parameters not routinely considered in toxicity screening studies. Physicochemical properties that may be important in understanding the toxic effects of test materials include particle size and size distribution, agglomeration state, shape, crystal structure, chemical composition, surface area, surface chemistry, surface charge, and porosity. In vitro techniques allow specific biological and mechanistic pathways to be isolated and tested under controlled conditions, in ways that are not feasible in in vivo tests. Tests are suggested for portal-of-entry toxicity for lungs, skin, and the mucosal membranes, and target organ toxicity for endothelium, blood, spleen, liver, nervous system, heart, and kidney. Non-cellular assessment of nanoparticle durability, protein interactions, complement activation, and pro-oxidant activity is also considered.Tier 1 in vivo assays are proposed for pulmonary, oral, skin and injection exposures, and Tier 2 evaluations for pulmonary exposures are also proposed. Tier 1 evaluations include markers of inflammation, oxidant stress, and cell proliferation in portal-of-entry and selected remote organs and tissues. Tier 2 evaluations for pulmonary exposures could include deposition, translocation, and toxicokinetics and biopersistence studies; effects of multiple exposures; potential effects on the reproductive system, placenta, and fetus; alternative animal models; and mechanistic studies.

Translocation of inhaled ultrafine manganese oxide particles to the central nervous system

Background Studies in monkeys with intranasally instilled gold ultrafine particles (UFPs; < 100 nm) and in rats with inhaled carbon UFPs suggested that solid UFPs deposited in the nose travel along the olfactory nerve to the olfactory bulb. Methods To determine if olfactory translocation occurs for other solid metal UFPs and assess potential health effects, we exposed groups of rats to manganese (Mn) oxide UFPs (30 nm; ~ 500 μg/m3) with either both nostrils patent or the right nostril occluded. We analyzed Mn in lung, liver, olfactory bulb, and other brain regions, and we performed gene and protein analyses. Results After 12 days of exposure with both nostrils patent, Mn concentrations in the olfactory bulb increased 3.5-fold, whereas lung Mn concentrations doubled; there were also increases in striatum, frontal cortex, and cerebellum. Lung lavage analysis showed no indications of lung inflammation, whereas increases in olfactory bulb tumor necrosis factor-α mRNA (~ 8-fold) and protein (~ 30-fold) were found after 11 days of exposure and, to a lesser degree, in other brain regions with increased Mn levels. Macrophage inflammatory protein-2, glial fibrillary acidic protein, and neuronal cell adhesion molecule mRNA were also increased in olfactory bulb. With the right nostril occluded for a 2-day exposure, Mn accumulated only in the left olfactory bulb. Solubilization of the Mn oxide UFPs was < 1.5% per day. Conclusions We conclude that the olfactory neuronal pathway is efficient for translocating inhaled Mn oxide as solid UFPs to the central nervous system and that this can result in inflammatory changes. We suggest that despite differences between human and rodent olfactory systems, this pathway is relevant in humans.

CLONING, EXPRESSION, AND FUNCTIONAL CHARACTERIZATION OF RAT MIP-2 : A NEUTROPHIL CHEMOATTRACTANT AND EPITHELIAL CELL MITOGEN

Macrophage inflammatory protein‐2 (MIP‐2) is a member of a family of cytokines that play roles in inflammatory, immune, and wound healing responses. To clone the cDNA for rat MIP‐2, RNA was isolated from the lungs of Fischer 344 rats after instillation of lipopolysaccharide. Reverse transcription‐polymerase chain reaction was performed by using synthetic oligonucleotide primers designed from the mouse MIP‐2 cDNA sequence. A cDNA containing the coding region of rat MIP‐2 was cloned and sequenced. Comparison to the mouse MIP‐2 cDNA demonstrated 90.3% homology at the nucleotide level and 86% homology at the amino acid level. The rat MIP‐2 cDNA was expressed in Escherichia coli and protein evaluated for bioactivity. The recombinant rat MIP‐2 was chemotactic for rat neutrophils but did not stimulate migration of rat alveolar macrophages or human peripheral blood eosinophils or lymphocytes. In addition, the recombinant rat MIP‐2 and the related rat chemokine, KC/CINC stimulated proliferation of rat alveolar epithelial cells but not fibroblasts in vitro.

TNFα and increased chemokine expression in rat lung after particle exposure

Abstract Macrophage inflammatory protein 2 (MIP-2) and CINC (Cytokine-Induced-Neutrophil-Chemoattractant) are members of the chemokine family of inflammatory and immunoregulatory cytokines. MIP-2 and CINC exhibit potent neutrophil chemotactic activity and are thought to be key mediators of inflammatory cell recruitment in response to tissue injury and infection. In the present studies, we examined the potential involvement of MIP-2 and CINC in particle-elicited inflammation in the rat lung and the role of TNFα in particle-induced chemokine expression. Acute intratracheal instillation exposure of F344 rats to α quartz or titanium dioxide was shown to markedly increase steady-state levels of MIP-2 and CINC mRNA in lung tissue; a response which was associated with a significant increase in neutrophils in the bronchoalveolar lavage fluid. Additional studies demonstrated that acute inhalation of crocidolite fibers by rats also induced increased MIP-2 and CINC expression. Since previous studies had demonstrated that TNFα stimulates MIP-2 and CINC expression in vitro and that particle exposure induces TNFα production in rat lung we examined the role of TNFα in a quartz-induced MIP-2 gene expression. We demonstrated that passive immunization of mice against TNFα markedly attenuated the increased lung MIP-2 mRNA seen in response to a quartz inhalation. Collectively, these findings suggest that the chemokines MIP-2 and CINC play a role in neutrophil recruitment to the rat lung after particle exposure and indicate that particle-induced expression of these chemokines is mediated, at least in part, by production of TNFα.

A comparative dose-related response of several key pro- and antiinflammatory mediators in the lungs of rats, mice, and hamsters after subchronic inhalation of carbon black

Objective: The objective of this study was to investigate mechanisms underlying species specificity in particle-induced lung inflammation. Methods: Rats, mice, and hamsters exposed to air, 1, 7, or 50 mg/m3 of carbon black for 13 weeks were killed at 1 day, 3 months, and 11 months after exposure. Bronchoalveolar lavage was performed and characterized for cell number, cell type, reactive oxygen and nitrogen species, and cytokine levels. Ex vivo mutational analysis of inflammatory cells was evaluated by coincubating with lung epithelial cells. Lung tissue was evaluated for gene expression of various antiinflammatory mediators. Results: There was a dose- and time-related effect with all the parameters. Rats demonstrated greater propensity for generating a proinflammatory response, whereas mice and hamsters demonstrated an increased antiinflammatory response. Conclusions: These differences in pro- and antiinflammatory responses may contribute to the apparent species differences in inflammation and tumorigenesis.

Antioxidant defense mechanisms and the toxicity of fibrous and nonfibrous particles.

A topic that continues to attract considerable interest in many areas ofbiomedical research, is that of the cell’s defense against oxidative stress.Various mechanisms exist to protect cells and tissues against oxidants, and itis conceivable that genetic and acquired variations in these systems maycontribute to, or account for, interindividual variation in the response tooxidative stress. Similarly, species differences in antioxidant defenses orthe capacity of various defenses may underlie differences in response toxenobiotics that act, in whole or in part, through oxidative mechanisms.Oxidative mechanisms of response to xenobiotics is especially rele-vant to the respiratory tract, which is directly and continually exposed toan external environment containing oxidant pollutants such as ozone andoxides of nitrogen; cigarette smoke, which includes a variety of oxidizingmolecules; and particles, which may generate oxidants as a result of theirchemical properties or by stimulating production of cell-derived oxidants(Halliwell, 1990). Moreover, exposure to particles or other pollutants mayproduce oxidative stress in the lung by stimulating the recruitment of in-flammatory cells. Regarding particulate exposure, there is considerableevidence that many pneumotoxic particles act, at least in part, throughmechanisms that involve production of reactive oxygen species. For exam-ple, the toxicity of asbestos fibers, crystalline silica, and transition-metal-containing ambient particulates likely involves production of oxidantssuch as hydroxyl radical, superoxide anion, and hydrogen peroxide (Kampet al., 1992; Quinlan et al., 1994). Studies have also shown that theseand other particulates may act by stimulating cellular production of reac-

Mitochondrial-derived oxidants and quartz activation of chemokine gene expression.

Macrophage inflammatory protein 2 (MIP-2) is a chemotactic cytokine which mediates neutrophil recruitment in the lung and other tissues. Pneumotoxic particles such as quartz increase MIP-2 expression in rat lung and rat alveolar type II epithelial cells. Deletion mutant analysis of the rat MIP-2 promoter demonstrated quartz-induction depended on a single NFkappaB consensus binding site. Quartz activation of NFkappaB and MIP-2 gene expression in RLE-6TN cells was inhibited by anti-oxidants suggesting the responses were dependent on oxidative stress. Consistent with anti-oxidant effects, quartz was demonstrated to increase RLE-6TN cell production of hydrogen peroxide. Rotenone treatment of RLE-6TN cells attenuated hydrogen peroxide production, NFkappaB activation and MIP-2 gene expression induced by quartz indicating that mitochondria-derived oxidants were contributing to these responses. Collectively, these findings indicate that quartz and crocidolite induction of MIP-2 gene expression in rat alveolar type II cells results from stimulation of an intracellular signaling pathway involving increased generation of hydrogen peroxide by mitochondria and subsequent activation of NFkappaB.

Characterizing Mutagenesis in the Hprt Gene of Rat Alveolar Epithelial Cells

A clonal selection assay was developed for mutation in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene of rat alveolar epithelial cells. Studies were conducted to establish methods for isolation and long-term culture of rat alveolar epithelial cells. When isolated by pronase digestion purified on a Nycodenz gradient and cultured in media containing 7.5% fetal bovine serum (FBS), pituitary extract, EGF, insulin, and IGF-1, rat alveolar epithelial cells could be maintained in culture for several weeks with cell doubling times of 2-4 days. The rat alveolar epithelial cell cultures were exposed in vitro to the mutagens ethylnitrosourea (ENU) and H2O2, and mutation in the hprt gene was selected for by culture in the presence of the toxic purine analog, 6-thioguanine (6TG). In vitro exposure to ENU or H2O2 produced a dose-dependent increase in hprt mutation frequency in the alveolar epithelial cells. To determine if the assay system could be used to evaluate mutagenesis in alveolar type II cells after in vivo mutagen or carcinogen exposure, cells were isolated from rats treated previously with ENU or alpha-quartz. A significant increase in hprt mutation frequency was detected in alveolar epithelial cells obtained from rats exposed to ENU or alpha-quartz; the latter observation is the first demonstration that crystalline silica exposure is mutagenic in vivo. In summary, these studies show that rat alveolar epithelial cells isolated by pronase digestion and Nycodenz separation techniques and cultured in a defined media can be used in a clonal selection assay for mutation in the hprt gene. This assay demonstrates that ENU and H2O2 in vitro and ENU and alpha-quartz in vivo are mutagenic for rat alveolar epithelial cells. This model should be useful for investigating the genotoxic effects of chemical and physical agents on an important lung cell target for neoplastic transformation.

Interleukin-10 regulates quartz-induced pulmonary inflammation in rats

Interleukin-10 (IL-10) can downregulate expression of several proinflammatory cytokines including chemokines. This study investigated the role of IL-10 in the acute response of the rat lung to quartz particles. Intratracheal instillation of rats with 1 mg of quartz produced an inflammatory and cytotoxic response demonstrated by increased bronchoalveolar lavage (BAL) fluid neutrophils, lactate dehydrogenase, and protein. IL-10 was detected in rat lung, but IL-10 levels were not altered by quartz. In contrast, quartz increased lung levels of the chemokine macrophage inflammatory protein-2 (MIP-2). Treatment with recombinant murine IL-10 (rmIL-10) attenuated quartz-induced pulmonary inflammation and injury. Pretreatment with anti-IL-10 antiserum enhanced inflammatory responses to quartz. Consistent with effects on quartz-induced inflammation, rmIL-10 and anti-IL-10 serum decreased and increased, respectively, lung MIP-2 mRNA and protein in response to quartz. Additionally, rmIL-10 reduced production of hydrogen peroxide, superoxide anion, and nitric oxide by BAL cells from quartz-exposed and control rats. These results demonstrate that IL-10 is expressed in rat lung and downregulates quartz-induced inflammation and cell activation. The mechanism of the anti-inflammatory action of IL-10 after quartz administration involves, at least in part, attenuation of MIP-2 expression.Interleukin-10 (IL-10) can downregulate expression of several proinflammatory cytokines including chemokines. This study investigated the role of IL-10 in the acute response of the rat lung to quartz particles. Intratracheal instillation of rats with 1 mg of quartz produced an inflammatory and cytotoxic response demonstrated by increased bronchoalveolar lavage (BAL) fluid neutrophils, lactate dehydrogenase, and protein. IL-10 was detected in rat lung, but IL-10 levels were not altered by quartz. In contrast, quartz increased lung levels of the chemokine macrophage inflammatory protein-2 (MIP-2). Treatment with recombinant murine IL-10 (rmIL-10) attenuated quartz-induced pulmonary inflammation and injury. Pretreatment with anti-IL-10 antiserum enhanced inflammatory responses to quartz. Consistent with effects on quartz-induced inflammation, rmIL-10 and anti-IL-10 serum decreased and increased, respectively, lung MIP-2 mRNA and protein in response to quartz. Additionally, rmIL-10 reduced production of hydrogen peroxide, superoxide anion, and nitric oxide by BAL cells from quartz-exposed and control rats. These results demonstrate that IL-10 is expressed in rat lung and downregulates quartz-induced inflammation and cell activation. The mechanism of the anti-inflammatory action of IL-10 after quartz administration involves, at least in part, attenuation of MIP-2 expression.

Effects of subchronic inhalation exposure to carbon black nanoparticles in the nasal airways of laboratory rats

The nose can be an efficient filter for inhaled gases, vapours and particles that may be harmful to the lung. Nasal airways may also be targets for injury caused by inhaled toxicants. To investigate the nasal toxicity of carbon black nanoparticles (CB), rats were exposed to 0, 1, 7 or 50 mg/m? of high surface area CB (HSCB; primary particle size 17 nm; particle surface area 300 m?/g) for 6 h/day, 5 days/week for 13 week. Additional rats were exposed to 50 mg/m? of low surface area CB (LSCB; primary particle size 70 nm; particle surface area 37 m?/g). Rats were sacrificed 1 day, 13 week, or 11 months postexposure (PE). Rats exposed to mid- or high-dose HSCB had nasal inflammatory and epithelial lesions at one day PE. HSCB-induced nasal inflammation resolved by 13 week PE, but some nasal epithelial lesions were still present in rats at 11 months after high-dose HSCB exposure. Low-dose HSCB or high-dose LSCB induced only minimal epithelial lesions that were resolved by 13 week PE. Results indicate that incidence, severity, and persistence of CB-induced nasal toxicity in rats is dependent on exposure concentration, particle surface area, and time PE. Effects of inhaled CB on human nasal airways are yet to be determined.