Krishnan Sriram

Mouse pulmonary dose- and time course-responses induced by exposure to multi-walled carbon nanotubes

Carbon nanotubes (CNT) come in a variety of types, but one of the most common forms is multi-walled carbon nanotubes (MWCNT). MWCNT have potential applications in many diverse commercial processes, and thus human exposures are considered to be likely. In order to investigate the pulmonary toxicity of MWCNT, we conducted an in vivo dose-response and time course study of MWCNT in mice in order to assess their ability to induce pulmonary inflammation, damage, and fibrosis using doses that approximate estimated human occupational exposures. MWCNT were dispersed in dispersion medium (DM) and male C57BL/6J mice (7 weeks old) received either DM (vehicle control), 10, 20, 40 or 80mug MWCNT by aspiration exposure. At 1, 7, 28 and 56 days post-exposure, MWCNT-induced pulmonary toxicity was investigated. Bronchoalveolar lavage (BAL) studies determined pulmonary inflammation and damage was dose-dependent and peaked at 7 days post-exposure. By 56 days post-exposure, pulmonary inflammation and damage markers were returning to control levels, except for the 40mug MWCNT dose, which was still significantly higher than vehicle control. Histopathological studies determined that MWCNT exposure caused rapid development of pulmonary fibrosis by 7 days post-exposure, that granulomatous inflammation persisted throughout the 56-day post-exposure period, and also demonstrated that MWCNT can reach the pleura after pulmonary exposure. In summary, the data reported here indicate that MWCNT exposure rapidly produces significant adverse health outcomes in the lung. Furthermore, the observation that MWCNT reach the pleura after aspiration exposure indicates that more extensive investigations are needed to fully assess if pleural penetration results in any adverse health outcomes.

Minocycline attenuates microglial activation but fails to mitigate striatal dopaminergic neurotoxicity: role of tumor necrosis factor‐α

Activated microglia are implicated in the pathogenesis of disease‐, trauma‐ and toxicant‐induced damage to the CNS, and strategies to modulate microglial activation are gaining impetus. A novel action of the tetracycline derivative minocycline is the ability to inhibit inflammation and free radical formation, factors that influence microglial activation. Minocycline is therefore being tested as a neuroprotective agent to alleviate CNS damage, although findings so far have yielded mixed results. Here, we showed that administration of a single low dose of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) or methamphetamine (METH), a paradigm that causes selective degeneration of striatal dopaminergic nerve terminals without affecting the cell body in substantia nigra, increased the expression of mRNAs encoding microglia‐associated factors F4/80, interleukin (IL)‐1α, IL‐6, monocyte chemoattractant protein‐1 (MCP‐1, CCL2) and tumor necrosis factor (TNF)‐α. Minocycline treatment attenuated MPTP‐ or METH‐mediated microglial activation, but failed to afford neuroprotection. Lack of neuroprotection was shown to be due to the inability of minocycline to abolish the induction of TNF‐α and its receptors, thereby failing to modulate TNF signaling. Thus, TNF‐α appeared to be an obligatory component of dopaminergic neurotoxicity. To address this possibility, we examined the effects of MPTP or METH in mice lacking genes encoding IL‐6, CCL2 or TNF receptor (TNFR)1/2. Deficiency of either IL‐6 or CCL2 did not alter MPTP neurotoxicity. However, deficiency of both TNFRs protected against the dopaminergic neurotoxicity of MPTP. Taken together, our findings suggest that attenuation of microglial activation is insufficient to modulate neurotoxicity as transient activation of microglia may suffice to initiate neurodegeneration. These findings support the hypothesis that TNF‐α may play a role in the selective vulnerability of the nigrostriatal pathway associated with dopaminergic neurotoxicity and perhaps Parkinsons disease.

Glial fibrillary acidic protein and related glial proteins as biomarkers of neurotoxicity.

A variety of ‘-omic’ technologies are being increasingly applied in preclinical safety assessments. Such approaches, however, have not been implemented in neurotoxicity safety evaluations. Current regulatory guidelines for assessing neurotoxicity emphasise reliance on traditional histopathological stains and behavioural testing batteries. Although these methods may be sufficient to detect some neurotoxic effects, they lack both the sensitivity and specificity required for broad-scale neurotoxicity screening. The glial reaction to nervous system damage, often termed gliosis, represents a hallmark of all types of nervous system injury. As such, the development and implementation of gliosis biomarkers represents a broadly applicable approach for neurotoxicity safety assessment. Using a panel of known neurotoxic agents, the authors have shown that the astroglial protein, glial fibrillary acidic protein (GFAP), can serve as one such biomarker of neurotoxicity. Qualitative and quantitative analysis of GFAP has shown this biomarker to be a sensitive and specific indicator of the neurotoxic condition. The implementation of GFAP and related glial biomarkers in neurotoxicity screens may serve as the basis for further development of molecular signatures predictive of adverse effects on the nervous system

A biocompatible medium for nanoparticle dispersion

Our laboratory has reported that rat bronchoalveolar lavage (BAL) fluid is an effective nanoparticle (NP) dispersant. However, its utility is constrained by its cost and the lack of standardization to control for intra- and inter-laboratory variability in BAL fluid. In this study, we report the efficacy and biocompatibility of a dispersion medium (DM), which is a ‘lung fluid mimic’. In vitro studies, which used dynamic light scattering and transmission electron microscopy, determined that ultrafine titanium dioxide and ultrafine carbon black are equally well dispersed by DM or BAL fluid. We also determined that DM was effective at dispersing multi-walled carbon nanotubes. In vivo, when used as a vehicle, DM per se did not elicit toxicity and did not influence or alter toxic responses to crystalline silica in either the lung or brain. Overall, these studies indicate that DM is an effective, biocompatible, and economical vehicle for nanotoxicological studies.

Acute pulmonary dose–responses to inhaled multi-walled carbon nanotubes

Abstract This study investigated the in vivo pulmonary toxicity of inhaled multi-walled carbon nanotubes (MWCNT). Mice-inhaled aerosolized MWCNT (10 mg/m3, 5 h/day) for 2, 4, 8 or 12 days. MWCNT lung burden was linearly related to exposure duration. MWCNT-induced pulmonary inflammation was assessed by determining whole lung lavage (WLL) polymorphonuclear leukocytes (PMN). Lung cytotoxicity was assessed by WLL fluid LDH activities. WLL fluid albumin concentrations were determined as a marker of alveolar air–blood barrier integrity. These parameters significantly increased in MWCNT-exposed mice versus controls and were dose-dependent. Histopathologic alterations identified in the lung included (1) bronciolocentric inflammation, (2) bronchiolar epithelial hyperplasia and hypertrophy, (3) fibrosis, (4) vascular changes and (5) rare pleural penetration. MWCNT translocated to the lymph node where the deep paracortex was expanded after 8 or 12 days. Acute inhalation of MWCNT induced dose-dependent pulmonary inflammation and damage with rapid development of pulmonary fibrosis, and also demonstrated that MWCNT can reach the pleura after inhalation exposure.

Nanotoxicology—A Pathologist’s Perspective

Advances in chemistry and engineering have created a new technology, nanotechnology, involving the tiniest known manufactured products. These products have a rapidly increasing market share and appear poised to revolutionize engineering, cosmetics, and medicine. Unfortunately, nanotoxicology, the study of nanoparticulate health effects, lags behind advances in nanotechnology. Over the past decade, existing literature on ultrafine particles and respirable durable fibers has been supplemented by studies of first-generation nanotechnology products. These studies suggest that nanosizing increases the toxicity of many particulates. First, as size decreases, surface area increases, thereby speeding up dissolution of soluble particulates and exposing more of the reactive surface of durable but reactive particulates. Second, nanosizing facilitates movement of particulates across cellular and intracellular barriers. Third, nanosizing allows particulates to interact with, and sometimes even hybridize with, subcellular structures, including in some cases microtubules and DNA. Finally, nanosizing of some particulates, increases pathologic and physiologic responses, including inflammation, fibrosis, allergic responses, genotoxicity, and carcinogenicity, and may alter cardiovascular and lymphatic function. Knowing how the size and physiochemical properties of nanoparticulates affect bioactivity is important in assuring that the exciting new products of nanotechnology are used safely. This review provides an introduction to the pathology and toxicology of nanoparticulates.

Obesity exacerbates chemically induced neurodegeneration.

Obesity is a major risk factor associated with a variety of human disorders. While its involvement in disorders such as diabetes, coronary heart disease and cancer have been well characterized, it remains to be determined if obesity has a detrimental effect on the nervous system. To address this issue we determined whether obesity serves as a risk factor for neurotoxicity. Model neurotoxicants, methamphetamine (METH) and kainic acid (KA), which are known to cause selective neurodegeneration of anatomically distinct areas of the brain, were evaluated using an animal model of obesity, the ob/ob mouse. Administration of METH and KA resulted in mortality among ob/ob mice but not among their lean littermates. While METH caused dopaminergic nerve terminal degeneration as indicated by decreased striatal dopamine (49%) and tyrosine hydroxylase protein (68%), as well as an increase in glial fibrillary acidic protein by 313% in the lean mice, these effects were exacerbated under the obese condition (96%, 86% and 602%, respectively). Similarly, a dosage of KA that did not increase glial fibrillary acidic protein in lean mice increased the hippocampal content of this protein (93%) in ob/ob mice. KA treatment resulted in extensive neuronal degeneration as determined by Fluoro-Jade B staining, decreased hippocampal microtubule-associated protein-2 immunoreactivity and increased reactive gliosis in ob/ob mice. The neurotoxic outcome in ob/ob mice remained exacerbated even when lean and ob/ob mice were dosed with METH or KA based only on a lean body mass. Administration of METH or KA resulted in up-regulation of the mitochondrial uncoupling protein-2 to a greater extent in the ob/ob mice, an effect known to reduce ATP yield and facilitate oxidative stress and mitochondrial dysfunction. These events may underlie the enhanced neurotoxicity seen in the obese mice. In summary, our results implicate obesity as a risk factor associated with chemical- and possibly disease-induced neurodegeneration.

Mitochondrial dysfunction and loss of Parkinson's disease-linked proteins contribute to neurotoxicity of manganese-containing welding fumes

Welding generates complex metal aerosols, inhalation of which is linked to adverse health effects among welders. An important health concern of welding fume (WF) exposure is neurological dysfunction akin to Parkinsons disease (PD), thought to be mediated by manganese (Mn) in the fumes. Also, there is a proposition that welding might accelerate the onset of PD. Our recent findings link the presence of Mn in the WF with dopaminergic neurotoxicity seen in rats exposed to manual metal arc-hard surfacing (MMA-HS) or gas metal arc-mild steel (GMA-MS) fumes. To elucidate the molecular mechanisms further, we investigated the association of PD-linked (Park) genes and mitochondrial function in causing dopaminergic abnormality. Repeated instillations of the two fumes at doses that mimic ∼1 to 5 yr of worker exposure resulted in selective brain accumulation of Mn. This accumulation caused impairment of mitochondrial function and loss of tyrosine hydroxylase (TH) protein, indicative of dopaminergic injury. A fascinating finding was the altered expression of Parkin (Park2), Uchl1 (Park5), and Dj1 (Park7) proteins in dopaminergic brain areas. A similar regimen of manganese chloride (MnCl(2)) also caused extensive loss of striatal TH, mitochondrial electron transport components, and Park proteins. As mutations in PARK genes have been linked to early-onset PD in humans, and because welding is implicated as a risk factor for parkinsonism, PARK genes might play a critical role in WF-mediated dopaminergic dysfunction. Whether these molecular alterations culminate in neurobehavioral and neuropathological deficits reminiscent of PD remains to be ascertained.

Mild steel welding fume causes manganese accumulation and subtle neuroinflammatory changes but not overt neuronal damage in discrete brain regions of rats after short-term inhalation exposure

Serious questions have been raised by occupational health investigators regarding a possible causal association between neurological effects in welders and the presence of manganese (Mn) in welding fume. Male Sprague-Dawley rats were exposed by inhalation to 40 mg/m(3) of gas metal arc-mild steel (MS) welding fume for 3 h/day for 10 days. Generated fume was collected in the animal chamber during exposure, and particle size, composition, and morphology were characterized. At 1 day after the last exposure, metal deposition in different organ systems and neurological responses in dopaminergic brain regions were assessed in exposed animals. The welding particles were composed primarily of a complex of iron (Fe) and Mn and were arranged as chain-like aggregates with a significant number of particles in the nanometer size range. Mn was observed to translocate from the lungs to the kidney and specific brain regions (olfactory bulb, cortex, and cerebellum) after MS fume inhalation. In terms of neurological responses, short-term MS fume inhalation induced significant elevations in divalent metal ion transporter 1 (Dmt1) expression in striatum and midbrain and significant increases in expression of proinflammatory chemokines (Ccl2, Cxcl2) and cytokines (IL1beta, TNFalpha) in striatum. In addition, mRNA and protein expression of glial fibrillary acidic protein (GFAP) was significantly increased in striatum after MS fume exposure. However, the 10-day MS welding fume inhalation did not cause any changes in dopamine and its metabolites or GABA in dopaminergic brain regions nor did it produce overt neural cell damage as assessed by histopathology. In summary, short-term MS welding fume exposure led to translocation of Mn to specific brain regions and induced subtle changes in cell markers of neuroinflammatory and astrogliosis. The neurofunctional significance of these findings currently is being investigated in longer, more chronic welding fume exposure studies.

Differential Mouse Pulmonary Dose and Time Course Responses to Titanium Dioxide Nanospheres and Nanobelts

Three anatase titanium dioxide (TiO(2)) nanoparticles (NPs) were prepared; nanospheres (NSs), short nanobelts (NB1), and long nanobelts (NB2). These NPs were used to investigate the effect of NP shape and length on lung toxicity. Mice were exposed (0-30 µg per mouse) by pharyngeal aspiration and pulmonary toxicity was assessed over a 112-day time course. Whole lung lavage data indicated that NB1- and NB2-exposed mice, but not NS-exposed mice, had significant dose- and time-dependent pulmonary inflammation and damage. Histopathological analyses at 112 days postexposure determined no interstitial fibrosis in any NS-exposed mice, an increased incidence in 30 µg NB1-exposed mice, and significant interstitial fibrosis in 30 µg NB2-exposed mice. At 112 days postexposure, lung burden of NS was decreased by 96.4% and NB2 by 80.5% from initial deposition levels. At 112 days postexposure, enhanced dark field microscopy determined that alveolar macro- phages were the dominant deposition site, but a fraction of NB1 and NB2 was observed in the alveolar interstitial spaces. For the 30 µg exposure groups at 112 days postexposure, confocal micro- scopy and immunofluorescent staining demonstrated that retained NB2 but not NS were present in the interstitium subjacent to the terminal bronchiole near the normal location of the smallest lymphatic capillaries in the lung. These lymphatic capillaries play a critical role in particle clearance, and the accumulation of NB2, but not NS, suggests possible impaired lymphatic clearance by the high aspect ratio particles. In summary, our data indicate that TiO(2) NP shape alters pulmonary responses, with severity of responses being ranked as NS < NB1 < NB2.