David C. Dorman

OLFACTORY TRANSPORT: A DIRECT ROUTE OF DELIVERY OF INHALED MANGANESE PHOSPHATE TO THE RAT BRAIN

Experiments examining the dosimetry of inhaled manganese generally focus on pulmonary deposition and subsequent delivery of manganese in arterial blood to the brain. Growing evidence suggests that nasal deposition and transport along olfactory neurons represents another route by which inhaled manganese is delivered to certain regions of the rat brain. The purpose of this study was to evaluate the olfactory uptake and direct brain delivery of inhaled manganese phosphate ( 54 MnHPO 4 ). Male, 8-wk-old, CD rats with either both nostrils patent or the right nostril occluded underwent a single, 90-min, nose-only exposure to a 54 MnHPO 4 aerosol (0.39 mg 54 Mn/m 3 ; MMAD 1.68 w m, σ g 1.42). The left and right sides of the nose, olfactory pathway, striatum, cerebellum, and rest of the brain were evaluated immediately after the end of the 54 MnHPO 4 exposure and at 1, 2, 4, 8, and 21 d postexposure with gamma spectrometry and autoradiography. Rats with two patent nostrils had equivalent 54 Mn concentrations on both sides of the nose, olfactory bulb, and striatum, while asymmetrical 54 Mn delivery occurred in rats with one occluded nostril. High levels of 54 Mn activity were observed in the olfactory bulb and tubercle on the same side (i.e., ipsilateral) to the open nostril within 1-2 d following 54 MnHPO 4 exposure, while brain and nose samples on the side ipsilateral to the nostril occlusion had negligible levels of 54 Mn activity. Our results demonstrate that the olfactory route contributes to 54 Mn delivery to the rat olfactory bulb and tubercle. However, this pathway does not significantly contribute to striatal 54 Mn concentrations following a single, short-term inhalation exposure to 54 MnHPO 4 .

Neurotoxicity of manganese chloride in neonatal and adult CD rats following subchronic (21-day) high-dose oral exposure.

The purpose of this study was to evaluate the relative sensitivity of neonatal and adult CD rats to manganese‐induced neurotoxicity. Identical oral manganese chloride (MnCl2) doses (0, 25, or 50 mg kg−1 body wt. day−1) were given to neonatal rats throughout lactation (i.e. from postnatal day (PND) 1 through 21) and to adult male rats for 21 consecutive days. The MnCl2 doses administered to neonates were ca. 100‐fold higher than those resulting from the consumption of an equivalent volume of rats milk. Rats were assessed using similar behavioral and neurochemical evaluations. Several statistically significant changes occurred in Mn‐exposed rats relative to control animals. Neonates given the high dose of MnCl2 had reduced body weight gain. An increased pulse‐elicited acoustic startle response amplitude was observed in neonates from both MnCl2 treatment groups on PND 21. Increased striatal, hippocampal, hindbrain and cortical Mn concentrations were observed in all Mn‐exposed neonates on PND 21. Increased hypothalamic and cerebellar Mn concentrations were also observed on PND 21 in neonates from the high‐dose group only. Increased striatal, cerebellar and brain residue Mn concentrations were observed in adult rats from the high‐dose group. Increased striatal dopamine and 3,4‐dihydroxyphenylacetic acid levels were observed only in PND 21 neonates from the high‐dose group. No treatment‐related changes were observed in clinical signs, motor activity (assessed in neonates on PND 13, 17, 21 ± 1 and in adults), passive avoidance (assessed in neonates on PND 20 ± 1 and in adults) or neuropathology (assessed in PND 21 neonates only). The results of our experiment suggest that neonates may be at greater risk for Mn‐induced neurotoxicity when compared to adults receiving similar high oral levels of Mn. Copyright

The speciation of metals in mammals influences their toxicokinetics and toxicodynamics and therefore human health risk assessment.

Chemical form (i.e., species) can influence metal toxicokinetics and toxicodynamics and should be considered to improve human health risk assessment. Factors that influence metal speciation (and examples) include: (1) carrier-mediated processes for specific metal species (arsenic, chromium, lead and manganese), (2) valence state (arsenic, chromium, manganese and mercury), (3) particle size (lead and manganese), (4) the nature of metal binding ligands (aluminum, arsenic, chromium, lead, and manganese), (5) whether the metal is an organic versus inorganic species (arsenic, lead, and mercury), and (6) biotransformation of metal species (aluminum, arsenic, chromium, lead, manganese and mercury). The influence of speciation on metal toxicokinetics and toxicodynamics in mammals, and therefore the adverse effects of metals, is reviewed to illustrate how the physicochemical characteristics of metals and their handling in the body (toxicokinetics) can influence toxicity (toxicodynamics). Generalizing from mercury, arsenic, lead, aluminum, chromium, and manganese, it is clear that metal speciation influences mammalian toxicity. Methods used in aquatic toxicology to predict the interaction among metal speciation, uptake, and toxicity are evaluated. A classification system is presented to show that the chemical nature of the metal can predict metal ion toxicokinetics and toxicodynamics. Essential metals, such as iron, are considered. These metals produce low oral toxicity under most exposure conditions but become toxic when biological processes that utilize or transport them are overwhelmed, or bypassed. Risk assessments for essential and nonessential metals should consider toxicokinetic and toxicodynamic factors in setting exposure standards. Because speciation can influence a metals fate and toxicity, different exposure standards should be established for different metal species. Many examples are provided which consider metal essentiality and toxicity and that illustrate how consideration of metal speciation can improve the risk assessment process. More examples are available at a website established as a repository for summaries of the literature on how the speciation of metals affects their toxicokinetics. This article is based on a workshop entitled “Metal Speciation in Toxicology: Determination and Importance for Risk Assessment” presented at the 42nd Annual Meeting of the Society of Toxicology, March, 2003, Salt Lake City, UT.

Olfactory Neuron Loss in Adult Male CD Rats Following Subchronic Inhalation Exposure to Hydrogen Sulfide

Dysosmia and anosmia are reported to occur following human exposure to hydrogen sulfide (H2S) gas. The clinical association between H2S exposure and olfactory dysfunction in humans necessitates evaluation of the nasal cavity and olfactory system in experimental animals used to study H2S toxicity. The purpose of this study was to subchronically expose 10-week-old male CD rats to relatively low concentrations of H2S and to histologically evaluate the nasal cavity for exposure-related lesions. Rats (n = 12/ group) were exposed via inhalation to 0, 10, 30, or 80 ppm H2S 6 h/d and 7 d/wk for 10 weeks. Following exposure to 30 and 80 ppm H2S, a significant increase in nasal lesions limited to the olfactory mucosa was observed. The lesions, which consisted of olfactory neuron loss and basal cell hyperplasia, were multifocal, bilaterally symmetrical, and had a characteristic rostrocaudal distribution pattern. Regions of the nasal cavity affected included the dorsal medial meatus and the dorsal and medial portions of the ethmoid recess. The no observed adverse effect level for olfactory lesions in this study was 10 ppm. For perspective, the American Conference of Governmental Industrial Hygienists threshold limit value (TLV) recommendation for H 2S is currently 10 ppm (proposed revision: 5 ppm), so the concentrations employed in the present study were 3 and 8 times the TLV. These findings suggest that subchronic inhalation exposure to a relatively low level of H2 S (30 ppm) can result in olfactory toxicity in rats. However, because of differences in the breathing style and nasal anatomy of rats and humans, additional research is required to determine the significance of these results for human health risk assessment.

Nasal Toxicity of Manganese Sulfate and Manganese Phosphate in Young Male Rats Following Subchronic (13-Week) Inhalation Exposure

Growing evidence suggests that nasal deposition and transport along the olfactory nerve represents a route by which inhaled manganese and certain other metals are delivered to the rodent brain. The toxicological significance of olfactory transport of manganese remains poorly defined. In rats, repeated intranasal instillation of manganese chloride results in injury to the olfactory epithelium and neurotoxicity as evidenced by increased glial fibrillary acidic protein (GFAP) concentrations in olfactory bulb astrocytes. The purpose of the present study was to further characterize the nasal toxicity of manganese sulfate (MnSO4) and manganese phosphate (as hureaulite) in young adult male rats following subchronic (90-day) exposure to air, MnSO4 (0.01, 0.1, and 0.5 mg Mn/m3), or hureaulite (0.1 mg Mn/m3). Nasal pathology, brain GFAP levels, and brain manganese concentrations were assessed immediately following the end of the 90-day exposure and 45 days thereafter. Elevated end-of-exposure olfactory bulb, striatum, and cerebellum manganese concentrations were observed following MnSO4 exposure to ≥0.01, ≥0.1, and 0.5 mg Mn/m3, respectively. Exposure to MnSO4 or hureaulite did not affect olfactory bulb, cerebellar, or striatal GFAP concentrations. Exposure to MnSO4 (0.5 mg Mn/m3) was also associated with reversible inflammation within the nasal respiratory epithelium, while the olfactory epithelium was unaffected by manganese inhalation. These results confirm that high-dose manganese inhalation can result in nasal toxicity (irritation) and increased delivery of manganese to the brain; however, we could not confirm that manganese inhalation would result in altered brain GFAP concentrations.

Application of Physiological Computational Fluid Dynamics Models to Predict Interspecies Nasal Dosimetry of Inhaled Acrolein

Acrolein is a highly soluble and reactive aldehyde and is a potent upper-respiratory-tract irritant. Acrolein-induced nasal lesions in rodents include olfactory epithelial atrophy and inflammation, epithelial hyperplasia, and squamous metaplasia of the respiratory epithelium. Nasal uptake of inhaled acrolein in rats is moderate to high, and depends on inspiratory flow rate, exposure duration, and concentration. In this study, anatomically accurate three-dimensional computational fluid dynamics (CFD) models were used to simulate steady-state inspiratory airflow and to quantitatively predict acrolein tissue dose in rat and human nasal passages. A multilayered epithelial structure was included in the CFD models to incorporate clearance of inhaled acrolein by diffusion, blood flow, and first-order and saturable metabolic pathways. Kinetic parameters for these pathways were initially estimated by fitting a pharmacokinetic model with a similar epithelial structure to time-averaged acrolein nasal extraction data and were then further adjusted using the CFD model. Predicted air:tissue flux from the rat nasal CFD model compared well with the distribution of acrolein-induced nasal lesions from a subchronic acrolein inhalation study. These correlations were used to estimate a tissue dose-based no-observed-adverse-effect level (NOAEL) for inhaled acrolein. A human nasal CFD model was used to extrapolate effects in laboratory animals to human exposure conditions on the basis of localized tissue dose and tissue responses. Assuming that equivalent tissue dose will induce similar effects across species, a NOAEL human equivalent concentration for inhaled acrolein was estimated to be 8 ppb.

Duration of airborne-manganese exposure in rhesus monkeys is associated with brain regional changes in biomarkers of neurotoxicity

Juvenile (20-24-month-old) rhesus monkeys were exposed to airborne-manganese sulfate (MnSO(4)) 1.5 mg Mn/m(3) (6h/day, 5 days/week) for 15 or 33 days, or for 65 days followed by a 45 or 90 days post-exposure recovery period, or air. We assessed biochemical endpoints indicative of oxidative stress and excitotoxicity in the cerebellum, frontal cortex, caudate, globus pallidus, olfactory cortex, and putamen. Glutamine synthetase (GS), glutamate transporters (GLT-1 and GLAST) and tyrosine hydroxylase (TH) protein levels, metallothionein (MT), GLT-1, GLAST, TH and GS mRNA levels, and total glutathione (GSH) levels were determined for all brain regions. Exposure to Mn significantly decreased MT mRNA in the caudate (vs. air-exposed controls). This depression persisted at least 90 days post-exposure. In contrast, putamen MT mRNA levels were unaffected by Mn exposure. GLT-1 and GLAST were relatively unaffected by short term Mn exposure, except in the globus pallidus where exposure for 33 days led to decreased protein levels, which persisted after 45 days of recovery for both proteins and 90 days of recovery in the case of GLAST. Exposure to 1.5 mg Mn/m(3) caused a significant decrease in GSH levels in the caudate and increased GSH levels in the putamen of monkey exposed for 15 and 33 days with both effects persisting at least 90 days post-exposure. Finally, TH protein levels were significantly lowered in the globus pallidus of the monkeys exposed for 33 days but mRNA levels were significantly increased in this same region. Overall, the nonhuman primate brain responds to airborne Mn in a heterogeneous manner and most alterations in these biomarkers of neurotoxicity are reversible upon cessation of Mn exposure.

Fertility and developmental neurotoxicity effects of inhaled hydrogen sulfide in Sprague–Dawley rats

In this study, we examined whether perinatal exposure by inhalation to hydrogen sulfide (H2S) had an adverse impact on pregnancy outcomes, offspring prenatal and postnatal development, or offspring behavior. Virgin male and female Sprague-Dawley rats (12 rats/sex/concentration) were exposed (0, 10, 30, or 80 ppm H2S; 6 h/day, 7 days/week) for 2 weeks prior to breeding. Exposures continued during a 2-week mating period (evidence of copulation = gestation day 0 = GD 0) and then from GD 0 through GD 19. Exposure of dams and their pups (eight rats/litter after culling) resumed between postnatal day (PND) 5 and 18. Adult male rats were exposed for 70 consecutive days. Offspring were evaluated using motor activity (PND 13, 17, 21, and 60+/-2), passive avoidance (PND 22+/-1 and 62+/-3), functional observation battery (PND 60+/-2), acoustic startle response (PND 21 and 62+/-3), and neuropathology (PND 23+/-2 and 61+/-2). There were no deaths and no adverse physical signs observed in F0 male or female rats during the study. A statistically significant decrease in feed consumption was observed in F0 male rats from the 80-ppm H2S exposure group during the first week of exposure. There were no statistically significant effects on the reproductive performance of the F0 rats as assessed by the number of females with live pups, litter size, average length of gestation, and the average number of implants per pregnant female. Exposure to H2S did not affect pup growth, development, or performance on any of the behavioral tests. The results of our study suggest that H2S is neither a reproductive toxicant nor a behavioral developmental neurotoxicant in the rat at occupationally relevant exposure concentrations (< or =10 ppm).

Manganese: pharmacokinetics and molecular mechanisms of brain uptake.

Manganese is an essential mineral that is found at low levels in virtually all diets. Manganese ingestion represents the principal route of human exposure, although inhalation also occurs, predominantly in occupational cohorts. Regardless of intake, animals generally maintain stable tissue manganese levels as a result of homeostatic mechanisms that tightly regulate the absorption and excretion of this metal. However, high-dose exposures are associated with increased tissue manganese levels, causing adverse neurological, reproductive and respiratory effects. In humans, manganese-induced neurotoxicity is associated with a motor dysfunction syndrome, commonly referred to as manganism or Parkinsonism, which is of paramount concern and is considered to be one of the most sensitive endpoints. This article focuses on the dosimetry of manganese with special focus on transport mechanisms of manganese into the CNS. It is not intended to be an exhaustive review of the manganese literature; rather it aims to provide a useful synopsis of contemporary studies from which the reader may progress to other research citations as desired. Specific emphasis is directed towards recent published literature on manganese transporters systemic distribution of manganese upon inhalation exposure as well as the utility of magnetic resonance imaging in quantifying brain manganese distribution.

Oxidative stress is induced in the rat brain following repeated inhalation exposure to manganese sulfate

Eight-week-old rats inhaled manganese (Mn) in the form of MnSO4 at 0, 0.03, 0.3, or 3.0 mg Mn/m3 for 6 h/d for 7 d/wk (14 consecutive exposures). Brain manganese concentrations in these animals were reported by Dorman et al. in 2001, noting the following rank order: olfactory bulb>striatum>cerebellum. We assessed biochemical end points indicative of oxidative stress in these three brain regions, as well as the hypothalamus and hippocampus. Glutamine synthetase (GS) protein levels and total glutathione (GSH) levels were determined for all five regions. GS mRNA and metallothionein (MT) mRNA levels were also evaluated for the cerebellum, hypothalamus, and hippocampus. Statistically significant increases (p<0.05) in GS protein were observed in the olfactory bulb upon exposure to the medium and high manganese doses. In the hypothalamus, statistically significant (p<0.05) but more modest increases were also noted in the medium and high manganese dose. Total GSH levels significantly (p<0.05) decreased only in the hypothalamus (high manganese dose), and MT mRNA significantly increased in the hypothalamus (medium manganese dose). No significant changes were noted in any of the measured parameters in the striatum, although manganese concentrations in this region were also increased. These results demonstrate that the olfactory bulb and hypothalamus represent potentially sensitive areas to oxidative stress induced by exceedingly high levels of inhaled manganese sulfate and that other regions, and especially the striatum, are resistant to manganese-induced oxidative stress despite significant accumulation of this metal.