Adverse outcome pathway (AOP): α2u-globulin nephropathy and kidney tumors in male rats.

The National Research Council's vision of using adverse outcome pathways (AOPs) as a framework to assist with toxicity assessment for regulatory requirements of chemical assessment has continued to gain traction since its release in 2007. The need to expand the AOP knowledge base has gained urgency, with the U.S. Environmental Protection Agency's directive to eliminate reliance on animal toxicity testing by 2035. To meet these needs, our goal was to elucidate the AOP for male-rat-specific kidney cancer. Male-rat-specific kidney tumors occur through the ability of structurally diverse substances to induce α2u-globulin nephropathy (α2u-N), a well-studied mode of action (MoA) not relevant in humans that results in kidney tumor formation in male rats. An accepted AOP may help facilitate the differentiation from other kidney tumors MoAs. Following identification and review of relevant in vitro and in vivo literature, both the MIE and subsequent KEs were identified. Based on the weight of evidence from the various resources, the confidence in this AOP is high. Uses of this AOP include hazard identification, development of in vitro assays to determine if the MoA is through α2u-N and not relevant to humans resulting in decreased use of animals, and regulatory applications.

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.

Inhaled carbon nanotubes reach the subpleural tissue in mice.

Carbon nanotubes are shaped like fibres and can stimulate inflammation at the surface of the peritoneum when injected into the abdominal cavity of mice, raising concerns that inhaled nanotubes may cause pleural fibrosis and/or mesothelioma. Here, we show that multiwalled carbon nanotubes reach the subpleura in mice after a single inhalation exposure of 30 mg m(-3) for 6 h. Nanotubes were embedded in the subpleural wall and within subpleural 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. Subpleural fibrosis unique to this form of nanotubes increased after 2 and 6 weeks following inhalation. None of these effects was seen in mice that inhaled carbon black nanoparticles or a lower dose of nanotubes (1 mg m(-3)). This work 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.

STAT-1 signaling in human lung fibroblasts is induced by vanadium pentoxide through an IFN-beta autocrine loop.

The inhalation of vanadium pentoxide (V(2)O(5)) results in bronchitis and airway fibrosis. The lung fibrotic response to V(2)O(5) partially resolves where fibroblasts first proliferate and deposit collagen, but then undergo growth arrest and apoptosis. STAT-1 mediates fibroblast growth arrest and apoptosis. We previously reported that STAT-1 is a protective factor and mice lacking STAT-1 are more susceptible to lung fibrosis. We also reported that V(2)O(5)-induced STAT-1 phosphorylation in lung fibroblasts requires H(2)O(2) and de novo protein synthesis. In this study, we identified IFN-beta as the protein that mediates STAT-1 activation by V(2)O(5) in normal human lung fibroblasts and identified NADPH and xanthine oxidase systems as sources of H(2)O(2) that drive IFN-beta gene expression. STAT-1 phosphorylation was decreased with neutralizing Abs to IFN-beta as well as an inhibitor of JAK. V(2)O(5) also increased transcription of an IFN-inducible and STAT-1-dependent chemokine, CXCL10. Inhibition of H(2)O(2)-generating enzyme systems NADPH oxidase by apocynin and xanthine oxidase by allopurinol individually reduced STAT-1 phosphorylation. Apocynin and allopurinol also decreased V(2)O(5)-induced IFN-beta mRNA levels and CXCL10 expression. IFN-alpha transcription was inhibited only by allopurinol. Taken together, these data indicate that fibroblasts play a role in the innate immune response to vanadium-induced oxidative stress by synthesizing IFN-beta and activating STAT-1 to cause growth arrest and increase levels of CXCL10, a potent antifibrotic factor. This mechanism is postulated to counterbalance profibrogenic mechanisms that follow V(2)O(5) injury.

Variables influencing interactions of untargeted quantum dot nanoparticles with skin cells and identification of biochemical modulators.

Skin cells (NHEK) take up untargeted quantum dots (QD) with surface polyethylene glycol (PEG), amines, and carboxylic acids, but the mechanisms are unknown. Time courses of QD-NHEK interactions were determined and effects of QD surface coating, temperature, culture medium supplements and inhibitors of the cell cycle and endocytosis identified. The magnitude of QD-NHEK interactions was coating dependent. Low-temperature or unsupplemented medium decreased QD-NHEK interactions. Biochemical inhibitors were identified that attenuate and potentiate QD-NHEK interactions. These results are important for understanding and controlling interactions of untargeted QD with cells.

Effects of mechanical flexion on the penetration of fullerene amino acid-derivatized peptide nanoparticles through skin.

Dermatomed porcine skin was fixed to a flexing device and topically dosed with 33.5 mg.mL-1 of an aqueous solution of a fullerene-substituted phenylalanine (Baa) derivative of a nuclear localization peptide sequence (Baa-Lys(FITC)-NLS). Skin was flexed for 60 or 90 min or left unflexed (control). Confocal microscopy depicted dermal penetration of the nanoparticles at 8 h in skin flexed for 60 and 90 min, whereas Baa-Lys(FITC)-NLS did not penetrate into the dermis of unflexed skin until 24 h. TEM analysis revealed fullerene-peptide localization within the intercellular spaces of the stratum granulosum.

Surface coatings determine cytotoxicity and irritation potential of quantum dot nanoparticles in epidermal keratinocytes.

Quantum dot (QD) nanoparticles have potential applications in nanomedicine as drug delivery vectors and diagnostic agents, but the skin toxicity and irritation potential of QDs are unknown. Human epidermal keratinocytes (HEKs) were used to assess if QDs with different surface coatings would cause differential effects on HEK cytotoxicity, proinflammatory cytokine release, and cellular uptake. Commercially available QDs of two different sizes, QD 565 and QD 655, with neutral (polyethylene glycol (PEG)), cationic (PEG-amine), or anionic (carboxylic acid) coatings were utilized. Live cell imaging and transmission electron microscopy were used to determine that all QDs localized intracellularly by 24 hours, with evidence of QD localization in the nucleus. Cytotoxicity and release of the proinflammatory cytokines IL-1beta, IL-6, IL-8, IL-10, and tumor necrosis factor-alpha were assessed at 24 and 48 hours. Cytotoxicity was observed for QD 565 and QD 655 coated with carboxylic acids or PEG-amine by 48 hours, with little cytotoxicity observed for PEG-coated QDs. Only carboxylic acid-coated QDs significantly increased release of IL-1beta, IL-6, and IL-8. These data indicate that QD surface coating is a primary determinant of cytotoxicity and immunotoxicity in HEKs, which is consistent across size. However, uptake of QDs by HEKs is independent of surface coating.

Functional selectivity of dopamine D1 receptor agonists in regulating the fate of internalized receptors.

Recently, we demonstrated that D(1) agonists can cause functionally selective effects when the endpoints of receptor internalization and adenylate cyclase activation are compared. The present study was designed to probe the phenomenon of functional selectivity at the D(1) receptor further by testing the hypothesis that structurally dissimilar agonists with efficacies at these endpoints that equal or exceed those of dopamine would differ in ability to influence receptor fate after internalization, a functional endpoint largely unexplored for the D(1) receptor. We selected two novel agonists of therapeutic interest that meet these criteria (the isochroman A-77636, and the isoquinoline dinapsoline), and compared the fates of the D(1) receptor after internalization in response to these two compounds with that of dopamine. We found that dopamine caused the receptor to be rapidly recycled to the cell surface within 1h of removal. Conversely, A-77636 caused the receptor to be retained intracellularly up to 48 h after agonist removal. Most surprisingly, the D(1) receptor recovered to the cell surface 48 h after removal of dinapsoline. Taken together, these data indicate that these agonists target the D(1) receptor to different intracellular trafficking pathways, demonstrating that the phenomenon of functional selectivity at the D(1) receptor is operative for cellular events that are temporally downstream of immediate receptor activation. We hypothesize that these differential effects result from interactions of the synthetic ligands with aspects of the D(1) receptor that are distal from the ligand binding domain.

Penetration of intact skin by quantum dots with diverse physicochemical properties.

Skin is the largest organ of the body and is a potential route of exposure to engineered nanomaterials, but the permeability of the skin to these nanomaterials is unknown. We selected commercially available quantum dots (QD) of two core/shell sizes and shapes and three different surface coatings to determine if QD could penetrate intact skin in a size- or coating-dependent manner. Spherical 4.6 nm core/shell diameter QD 565 and ellipsoid 12 nm (major axis) by 6 nm (minor axis) core/shell diameter QD 655 with neutral (polyethylene glycol), anionic (carboxylic acids) or cationic (polyethylene glycol-amine) coatings were topically applied to porcine skin in flow-through diffusion cells at an occupationally relevant dose for 8 h and 24 h. Confocal microscopy revealed that spherical QD 565 of each surface coating penetrated the stratum corneum and localized within the epidermal and dermal layers by 8 h. Similarly, polyethylene glycol- and polyethylene glycol-amine-coated ellipsoid QD 655 localized within the epidermal layers by 8 h. No penetration of carboxylic acid-coated QD 655 was evident until 24 h, at which time localization in the epidermal layers was observed. This study showed that quantum dots of different sizes, shapes, and surface coatings can penetrate intact skin at an occupationally relevant dose within the span of an average-length work day. These results suggest that skin is surprisingly permeable to nanomaterials with diverse physicochemical properties and may serve as a portal of entry for localized, and possibly systemic, exposure of humans to QD and other engineered nanoscale materials.

Differential activation of adenylate cyclase and receptor internalization by novel dopamine D1 receptor agonists.

Structurally dissimilar dopamine D(1) receptor agonists were compared with dopamine in their ability to activate adenylate cyclase and to internalize hemagglutinin-tagged human D(1) receptors in a stably transfected human embryonic kidney cell line. Thirteen dopamine D(1) receptor agonists were selected rationally from three different structural classes: rigid fused ring compounds [dihydrexidine, dinapsoline, dinoxyline, apomorphine, and (5aR,11bS)-4,5,5a,6,7,11b-hexahydro-2-propyl-3-thia-5-azacyclopent-1-ena[c]-phenanthrene-9,10-diol (A86929)]; isochromans [(1R,3S)-3-(1'adamantyl)-1-aminomethyl-3,4-dihydo-5,6-dihydroxy-1H-2-benzopyran (A77636) and (1R,3S)-3-phenyl-1-aminomethyl-3,4-dihydo-5,6-dihydroxy-1H-2-benzopyran (A68930)]; and benzazepines [7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF38393), (+/-)-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF77434), 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF82958), 3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-]H-3-benzazepine (SKF83959), R(+)-6-chloro-7,8,-dihydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF82957), and R(+)-6-chloro-7,8,-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF81297)]. The working hypothesis was that some agonists have differential effects on adenylate cyclase versus receptor internalization that could be correlated to the structural class of the agonist. First, the affinity for the hemagglutinin-hD(1) receptor and the intrinsic activity and potency of adenylate cyclase activation were determined for each compound. The internalization time course and internalization efficacy were then determined for each agonist. It was surprising that internalization efficacy was found to be independent of either agonist structural class or affinity. Only agonists that had both high adenylate cyclase functional potency and high intrinsic activity caused internalization. In addition, four agonists from two structural classes were identified that were capable of fully activating adenylate cyclase without eliciting an internalization response. This study provides the first extensive characterization of D(1) receptor internalization in response to structurally diverse agonists and, at least for the D(1) receptor, shows that functional selectivity is not predictable by simple structural examination. These data are consistent with the hypothesis that functional selectivity reflects subtle ligand-induced conformational changes as opposed to simple agonist trafficking among discrete receptor active states.