Inhalation of poorly soluble particles. II. Influence Of particle surface area on inflammation and clearance

In this article the volumetric overload hypothesis, which predicts the impairment of clearance of particles deposited in the lung in terms of particle volume, is reevaluated. The degree to which simple expressions of retained lung burden explain pulmonary responses to overload was investigated using data from a series of chronic inhalation experiments on rats with two poorly soluble dusts, titanium dioxide and barium sulfate. The results indicated that the difference between the dusts in the level of inflammation and translocation to the lymph nodes could be explained most simply when the lung burden was expressed as total particle surface area. The shape of the statistical relationship for both lung responses indicated the presence of a threshold at approximately 200-300 cm(2) of lung burden. On the basis of this and other similar results, a hypothesis regarding a generic mechanism for the impairment of clearance and associated lung responses is proposed for such "low-toxicity" dusts.     

The cytotoxic activity of amorphous silica nanoparticles is mainly influenced by surface area and not by aggregation

The aggregation state of NP has been a significant source of difficulty for assessing their toxic activity and great efforts have been done to reduce aggregation of and/or to disperse NP in experimental systems. The exact impact of aggregation on toxicity has, however, not been adequately assessed.Here we compared in vitro the cytotoxic activity of stable monodisperse and aggregated silicon-based nanoparticles (SNP) without introducing a dispersing agent that may affect NP properties.SNP aggregates (180 nm) were produced by controlled electrostatic aggregation through addition of KCl to a Ludox SM sol (25 nm) followed by stabilization and extensive dialysis. The size of the preparations was characterized by TEM and DLS; specific surface area and porosity were derived from N₂ sorption measurements. Macrophage (J774) and fibroblast (3T3) cell lines were exposed to monodisperse or aggregate-enriched suspensions of SNP in DMEM in absence of serum. The cytotoxic activity of the different preparations was assessed by the WST1 assay after 24 h of exposure. Parameters that determined the cytotoxic activity were traced by comparing the doses of the different preparations that induced half a maximal reduction in WST1 activity (ED₅₀) in both cell lines.We found that ED₅₀ (6-9 μg/ml and 15-22 μg/ml, in J774 and 3T3, respectively) were hardly affected upon aggregation, which was consistent with the fact that the specific surface area of the SNP, a significant determinant of their cytotoxic activity, was unaffected upon aggregation (283-331 m²/g). Thus studying small aggregated NP could be as relevant as studying disperse primary NP, when aggregates keep the characteristics of NP, i.e. a high specific surface area and a nanosize dimension. This conclusion does, however, not necessarily hold true for other toxicity endpoints for which the determinants may be different and possibly modified by the aggregation process.     

Pulmonary bioassay studies with nanoscale and fine-quartz particles in rats: toxicity is not dependent upon particle size but on surface characteristics.

Pulmonary toxicology studies in rats demonstrate that nanoparticles are more toxic than fine-sized particles of similar chemistry. This study, however, provides evidence to contradict this theory. The aims of the study were (1) to compare the toxicity of synthetic 50 nm nanoquartz I particles versus (mined) Min-U-Sil quartz ( approximately 500 nm); the toxicity of synthetic 12 nm nanoquartz II particles versus (mined) Min-U-Sil ( approximately 500 nm) versus (synthetic) fine-quartz particles (300 nm); and (2) to evaluate the surface activities among the samples as they relate to toxicity. Well-characterized samples were tested for surface activity and hemolytic potential. In addition, groups of rats were instilled with either doses of 1 or 5 mg/kg of carbonyl iron (CI) or various alpha-quartz particle types in phosphate-buffered saline solution and subsequently assessed using bronchoalveolar lavage fluid biomarkers, cell proliferation, and histopathological evaluation of lung tissue at 24 h, 1 week, 1 month, and 3 months postexposure. Exposures to the various alpha-quartz particles produced differential degrees of pulmonary inflammation and cytotoxicity, which were not always consistent with particle size but correlated with surface activity, particularly hemolytic potential. Lung tissue evaluations of three of the quartz samples demonstrated "typical" quartz-related effects--dose-dependent lung inflammatory macrophage accumulation responses concomitant with early development of pulmonary fibrosis. The various alpha-quartz-related effects were similar qualitatively but with different potencies. The range of particle-related toxicities and histopathological effects in descending order were nanoscale quartz II = Min-U-Sil quartz > fine quartz > nanoscale quartz I > CI particles. The results demonstrate that the pulmonary toxicities of alpha-quartz particles appear to correlate better with surface activity than particle size and surface area.     

Pulmonary instillation studies with nanoscale TiO2 rods and dots in rats: toxicity is not dependent upon particle size and surface area.

Pulmonary toxicology studies in rats demonstrate that nanoparticles administered to the lung are more toxic than larger, fine-sized particles of similar chemistry at identical mass concentrations. The aim of this study was to evaluate the acute lung toxicity in rats of intratracheally instilled pigment-grade TiO2 particles (rutile-type particle size = approximately 300 nm) versus nanoscale TiO2 rods (anatase = 200 nm x 35 nm) or nanoscale TiO2 dots (anatase = approximately 10 nm) compared with a positive control particle type, quartz. Groups of rats were instilled with doses of 1 or 5 mg/kg of the various particle types in phosphate-buffered saline (PBS). Subsequently, the lungs of PBS- and particle-exposed rats were assessed using bronchoalveolar lavage fluid biomarkers, cell proliferation methods, and by the histopathological evaluation of lung tissue at 24 h, 1 week, 1 month, and 3 months postinstillation exposure. Exposures to nanoscale TiO2 rods or nanoscale TiO2 dots produced transient inflammatory and cell injury effects at 24 h postexposure (pe) and were not different from the pulmonary effects of larger sized TiO2 particle exposures. In contrast, pulmonary exposures to quartz particles in rats produced a dose-dependent lung inflammatory response characterized by neutrophils and foamy lipid-containing alveolar macrophage accumulation as well as evidence of early lung tissue thickening consistent with the development of pulmonary fibrosis. The results described herein provide the first example of nanoscale particle types which are not more cytotoxic or inflammogenic to the lung compared to larger sized particles of similar composition. Furthermore, these findings run counter to the postulation that surface area is a major factor associated with the pulmonary toxicity of nanoscale particle types.     

Retrospective analysis of 4-week inhalation studies in rats with focus on fate and pulmonary toxicity of two nanosized aluminum oxyhydroxides (boehmite) and pigment-grade iron oxide (magnetite): The key metric of dose is particle mass and not particle surface area

This paper compares the pulmonary toxicokinetics and toxicodynamics of three different types of poorly soluble dusts examined in repeated rat inhalation bioassays (6 h/day, 5 days/week, 4 weeks). In these studies the fate of particles was studied during a 3–6-month postexposure period. This retrospective analysis included two types of aluminum oxyhydroxides (AlOOH, boehmite), high purity calcined, and agglomerated nanosized aluminas of very low solubility with primary isometric particles of 10 or 40 nm, and synthetic iron oxide black (Fe₃O₄ pigment grade). Three metrics of dose (actual mass concentration, surface area concentration, mass-based lung burden) were compared with pulmonary toxicity characterized by bronchoalveolar lavage. The results of this analysis provide strong evidence that pulmonary toxicity (inflammation) corresponds best with the mass-based cumulative lung exposure dose. The inhalation study with a MMAD of ≈0.5 μm yielded a higher pulmonary dose than MMADs in the range of 1–2 μm, a range most commonly used in repeated exposure inhalation studies. Hence, a key premise for the dosimetric adjustment across species is that comparable lung tissue doses should cause comparable effects. From that perspective, the determination of mass-based pulmonary lung burdens appears to be amongst the most important and critical nominator of dose and dose-related pulmonary toxicity.     

Development of behavioral sensitization to the cocaine-like fungicide triadimefon is prevented by AMPA, NMDa, DA D1 but not DA D2 receptor antagonists.

Triadimefon (TDF) is a triazole fungicide that blocks the reuptake of dopamine (DA) and leads to increased locomotor activity levels in mice and rats, effects similar to those of indirect DA agonists such as cocaine. We recently found in mice that intermittent TDF administration led to robust locomotor sensitization, a phenomenon reflecting neuronal plasticity, following challenge with the same TDF dose after a 2-week withdrawal period. The current study sought to determine whether antagonists to DA D1-like receptors (SCH 23390; SCH), DA D2-like receptors (remoxipride; Rem), ionotropic glutamate n-methyl-d-aspartate (NMDA) receptors (CPP), or ionotropic glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (NBQX) could prevent the development of TDF behavioral sensitization, therefore indicating their mechanistic involvement in TDF sensitization. Mice were treated with either vehicle, SCH (0.015 mg/kg), remoxipride (Rem, 0.3 mg/kg), CPP (2.5 mg/kg) or NBQX (10.0 mg/kg), followed 30 min later by vehicle or 75 mg/kg TDF (TDF), twice a week for 7 weeks, with locomotor activity measured post-dosing once a week. After a 2-week withdrawal period, mice were challenged with 75 mg/kg TDF or vehicle, to test for the presence of behavioral sensitization. Pretreatment with SCH, CPP, or NBQX, but not Rem, blocked the development of behavioral sensitization to TDF specifically for vertical activity. Antagonists that blocked TDF vertical sensitization also attenuated the increase in extracellular DA turnover (homovanillic acid [HVA]/DA) normally associated with this behavioral response. Therefore, DA D1, NMDA and AMPA receptors appear to be necessary for the development of behavioral sensitization to TDF. As such, TDF may be considered an environmental risk factor for behavioral dysfunctions linked to glutamatergic and dopaminergic systems.     

Pulmonary toxicity of inhaled nanoscale and fine zinc oxide particles: mass and surface area as an exposure metric

The total surface area is known to be an effective exposure metric for predicting the lung toxicity of low solubility nanoparticles (NPs). However, if NPs are dissolved quickly enough in the lungs, the mass may be correlated with the toxicity. Recent studies have found that the toxicity of zinc oxide (ZnO) NPs was caused by the release of zinc ions. Thus, we hypothesized that mass could be used as an exposure metric for the toxicity of ZnO NPs. Healthy Sprague-Dawley rats were exposed to a low, moderate, or high dose of 35 and 250?nm ZnO particles or filtered air. Bronchoalveolar lavage fluid was collected to determine lung inflammation, injury and oxidative stress. The lung inflammation induced by ZnO particles according to different concentration metrics, including number, mass and surface area, was compared. The mass concentration was significantly correlated with the percentage of neutrophils (R(2)?=?0.84), number of neutrophils (R(2)?=?0.84) and total cells (R(2)?=?0.73). Similarly, surface area concentration was significantly correlated with the percentage of neutrophils (R(2)?=?0.94), number of neutrophils (R(2)?=?0.81) and total cells (R(2)?=?0.76). There was no correlation between the number and lung inflammation. We found that both mass and surface area were effective as metrics for the toxicity of ZnO NPs, although only surface area was previously indicated to be an effective metric. Our results are also consistent with recent study results that ZnO NPs and released zinc ions may play a role mediating the toxicity of NPs.     

Anti-phencyclidine monoclonal antibody binding capacity is not the only determinant of effectiveness, disproving the concept that antibody capacity is easily surmounted.

The effectiveness of a high-affinity monoclonal antibody (mAb) antagonist against chronic phencyclidine (PCP) use has been demonstrated in rats. In this study, we tested the hypothesis that intravenous doses of PCP in excess of the binding capacity of an anti-PCP mAb cannot easily surmount the beneficial effects of the mAb, even in the presence of a high body burden of the drug. One day after steady-state PCP concentrations were achieved in male rats by continuous s.c. infusion (18 mg/kg/day), a single i.v. dose of saline or the anti-PCP mAb (KD = 1.3 nM; at one-third the molar dose of the PCP body burden), treatment was administered. In an attempt to further surmount the effects of the mAb, rats were challenged with a single 1.0 mg/kg i.v. bolus PCP dose (along with a [3H]PCP tracer) 3 days after the mAb or saline treatment. Total (i.v. bolus + s.c. infusion) PCP concentrations were measured in serum, brain, and testis by radioimmunoassay before and after the challenge, and [3H]PCP concentrations were measured by liquid scintillation spectrometry. The anti-PCP mAb protected against adverse health effects, significantly increased the serum total and bolus PCP concentrations (p < 0.05), and significantly decreased brain total and bolus PCP concentrations (p < 0.05) after the i.v. challenge. These results showed the antibody can counteract extreme and potentially fatal PCP challenges and disproved the hypothesis that attempts to surmount the effects of the antibody with extremely high PCP doses would have immediate adverse health effects.     

The associative type of sensitization to d-amphetamine is expressed as an NO-dependent dramatic increase in extracellular dopamine in the nucleus accumbens

After repeated administration of psychomotor-stimulant drugs such as d-amphetamine, a sensitization to its stimulant effects such as locomotor activity and stereotyped behaviour can be observed depending on the treatment schedule. It was the aim of the present study to test whether (1) associative (conditioning) and non-associative (pseudoconditioning) types of sensitization differ in the corresponding alterations in extracellular dopamine in the nucleus accumbens, and (2) the inhibitor of nitric oxide synthase (NOS), N(G)-nitro-L-arginine methyl ester (L-NAME), influences the two types of sensitization in a different way. Rats were treated with d-amphetamine (1 mg/kg i.p.) on days 1, 3, 5 and 7 and alternatively with saline on days 2, 4 and 6 under associative or non-associative conditions, respectively. A third (saline control) group was treated with saline daily for 7 days. Subsequently, probes for microdialysis were implanted into the nucleus accumbens. After a drug-free period of 10 days, on day 17, the increase in extracellular dopamine produced by 1 mg/kg d-amphetamine was much more pronounced in associative (conditioned) than in non-associative (pseudoconditioned) or naive rats. Pretreatment with L-NAME (100 mg/kg i.p.) on day 17 did not significantly alter the baseline concentrations of dopamine, but it inhibited the dopamine increase much more in associative than in naive rats and did not significantly affect it in non-associative rats. The results suggest that (1) associative sensitization to d-amphetamine leads to the most pronounced increase in extracellular dopamine in the nucleus accumbens, and (2) NO is very much involved in the expression of the associative increase in extracellular dopamine in the nucleus accumbens.     

Modulation by the dorsal, but not the ventral, hippocampus of the expression of behavioural sensitization to amphetamine

Although the dorsal hippocampus (DH) and the ventral hippocampus (VH) densely innervate the nucleus accumbens, which mediates the expression of behavioural sensitization, the respective and specific contribution of DH and VH in the expression of behavioural sensitization to amphetamine has not been investigated. In the present study, we investigated how lidocaine infused in DH or VH modulated behavioural locomotor sensitization induced by repeated administration of systemic amphetamine. Rats, well habituated to their environmental conditions and experimental protocol, were given repeated administration of systemic amphetamine. Once behavioural sensitization was developed, rats were challenged with amphetamine and infused with saline (controls) or lidocaine into DH or VH. We found that reversible inhibition by lidocaine of DH, but not VH, blocks the expression of behavioural sensitization to amphetamine. Control animals injected with saline solution do express behavioural sensitization. Our results bring new insights on the role of the hippocampus complex in the expression of behavioural sensitization, indicating that, in individuals well habituated to the drug-associated context, DH but not VH would play a key role. The results provide experimental evidence for clinical studies in human addicts that have demonstrated that exposure to environmental stimuli associated with drug-taking behaviour elicits craving and can promote relapse, and further suggest that in drug abusers, once addiction has occurred, the contextual and spatial conditions that are associated with drug consumption may play a critical role in the maintenance of drug abuse.     

The association between pre- and postnatal tobacco smoke exposure and allergic sensitization during early childhood. Multicentre Allergy Study Group, Germany.

AIM: of our study was to investigate the effect of pre- and postnatal passive tobacco smoke exposure on the incidence of allergic sensitization. PATIENTS AND METHODS: Specific sensitization to food, outdoor and indoor allergens was determined in 342 children at the age of 1, 2 and 3 years. Parents were asked about their smoking habit at the birth of their children, at 18 months and 3 years of age. RESULTS: Multivariate regression analysis indicated, that during the first 3 years of life, pre- and postnatally exposed children had a significantly higher risk for sensitization to food allergens compared to children never exposed to tobacco smoke (OR 2.2, 95% CI 1.1-4.2; P = 0.02). With respect to inhalant allergens no significant influence of tobacco smoke exposure on specific sensitization could be demonstrated. CONCLUSION: During early childhood both pre- and postnatal tobacco smoke exposure has an adjuvant effect on allergic sensitization to food allergens.     

Characterization of liposomes. The influence of extrusion of multilamellar vesicles through polycarbonate membranes on particle size,particle size distribution and number of bilayers

The influence of extrusion on number of bilayers, particle size (ps) and particle size distribution (psd) of multilamellar vesicles (composition on a molar basis: phosphatidylcholine/phosphatidylserine/cholesterol, 10:1:4) is investigated. Ps and psd are investigated with freeze-fracture electron microscopy, average number of bilayers 〈N〉, with small angle X-ray scattering and ³¹P-NMR. The encapsulated volume (E) is determined using carboxyfluorescein as marker. From E and the volume/surface ratio of the liposomes, determined from the psd, 〈N〉 was also calculated. Determined and calculated number of bilayers are in reasonable agreement. Extrusion reduces 〈N〉.     

The low-chloride cough response is not inhibited by a single, high dose of aspirin.

The effect of a single, high dose of aspirin has been assessed against low chloride cough challenge. The drug does not affect the cough response, suggesting that airway prostaglandin generation is not responsible for the tussive activity of low chloride solution.     

Early social isolation, but not maternal separation, affects behavioral sensitization to amphetamine in male and female adult rats.

Early life stressful manipulations, such as maternal separation (MS) or social isolation (SI), can influence the neurobiological development of rats and alter the response of adult animals to drugs of abuse. The present study examined the acute and sensitized behavioral responses (locomotor activity (LMA) and stereotypy) induced by amphetamine after MS or SI in male and female rats. In addition, the hypothesis that the combination of SI and MS could lead to additional effects on the behavioral response to amphetamine was tested. After the repetitive, intermittent administration of 1.5 mg/kg D-amphetamine over five consecutive days, the behavioral expression of sensitization to a challenge injection was assessed following a 2-day withdrawal period. In both sexes, MS and SI did not alter the acute locomotor activating effects of amphetamine as measured in the open-field environment after the first administration of the drug. Whereas SI altered the expression of sensitization to amphetamine in both sexes, MS did not affect it. Finally, in none of the behavioral variables measured did MS and SI interact to further modify the behavioral profile of the animals. The present results suggest that a postweaning manipulation of the environment (SI) is more effective than a preweaning manipulation (MS) in modifying the expression of sensitization to amphetamine.     

The clinical stage of allergic rhinitis is correlated to inflammation as detected by nasal cytology

Allergic rhinitis (AR) is the most common allergic disease. The Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines classify AR according to its duration and severity and suggest recommended treatments, but there is evidence that these guidelines are insufficiently followed. Considering the validity of histopathological data, physicians are more likely to be persuaded by such information on AR. Thus, we attempted to define the severity of AR by nasal cytology on the basis of the ARIA classification. We examined 64 patients with AR caused by sensitization to grass pollen. We clinically defined AR according to the ARIA classification and performed nasal cytology by Rhino-probe sampling, staining and reading by optical microscopic observation. Clinically, 22 (34.4%), 21 (32.8%), 10 (15.6%), and 11 (17.2%) patients had mild intermittent, moderate-to-severe intermittent, mild persistent, and moderate-to-severe persistent AR, respectively. Nasal cytology detected neutrophils in 49 patients, eosinophils in 41 patients, mast cells in 21 patients, and lymphocytes or plasma cells in 28 patients. The patients with moderate-to-severe AR had significantly more mast cells and lymphocytes/ plasma cells than those with mild AR. Our findings demonstrate that the ARIA classification of AR severity is associated with different cell counts in nasal cytology; especially, moderate-to-severe AR shows significantly increased counts of mast cells and lymphocyte or plasma cells. The ease of performing nasal cytology ensures is feasibility as an office AR diagnostic procedure for primary care physicians, able to indicate when anti-inflammatory treatments, such as intranasal corticosteroids and subcutaneous or sublingual allergen immunotherapy, are needed.     

The effect of carrier surface and bulk properties on drug particle detachment from crystalline lactose carrier particles during inhalation, as function of carrier payload and mixing time.

The effect of carrier payload and mixing time on the redispersion of drug particles from adhesive mixtures during inhalation for two different drugs (budesonide and disodium cromoglycate) has been investigated. A special test inhaler which retains carrier crystals during inhalation was used at 30 and 60 l/min. The special inhaler enabled the analysis of residual drug on the carrier yielding so called carrier residue (CR) values. Mixtures with carrier size fractions of 32-45; 150-200 and 250-355 microm, derived from marketed lactose brands, with increasing carrier payload (0.4-6.0% w/w of drug) were prepared. It was found that with increasing carrier payload, the CR increases for the coarse carrier fraction, decreases for the fine fraction and remains roughly constant for the intermediate fraction at 30 l/min. At 60 l/min, the CR decreased for all carrier fractions with increasing payload. The effect of powder bulk properties on the adhesive forces between drug and carrier (during mixing) as well as changes in the balance between adhesion and separation forces (during inhalation) explain the results found. An improved understanding of the different effects is obtained through the recently introduced force distribution concept. The ratio of (mean) separation force to (mean) adhesion force increases with the flow rate. The adhesive forces (during mixing) increase with increasing carrier diameter (higher press-on and kneading forces) and longer mixing time.     

Behavioral sensitization to 3,4-methylenedioxymethamphetamine is long-lasting and modulated by the context of drug administration

To begin to characterize the temporal profile of behavioral sensitization to the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA), rats were treated with either saline or MDMA (5.0 mg/kg) twice daily for 5 days, followed by a challenge injection of MDMA (2.5 mg/kg) either 15 or 100 days later. Because we found previously that contextual drug associations are important for the expression of behavioral sensitization to MDMA following relatively short withdrawal periods, rats received the repeated injections either in their home cages (unpaired group) or in the activity monitors that were used for testing sensitization on challenge day (paired group). Locomotor sensitization was evident at 15 days of withdrawal but only in the paired MDMA-treated group. Interestingly, however, sensitization was apparent at 100 days of withdrawal in both paired and unpaired rats but the form of sensitization differed between groups. Thus, sensitization in paired rats was expressed as an increase in stereotypy, whereas sensitization in unpaired rats was expressed as an increase in locomotion, paralleling locomotion levels in paired animals at 15 days of withdrawal. These results suggest that the neural changes that underlie behavioral sensitization to MDMA are quite enduring but involve an interaction between withdrawal time and the context of drug administration.     

The total effective xenoestrogen burden, a biomarker of exposure to xenoestrogen mixtures, is predicted by the (anti)estrogenicity of its components

Appropriate biomarkers of human exposure are required for epidemiological studies of endocrine disruption. We addressed this issue by improving a standardized method to assess the total effective xenoestrogen burden (TEXB), a biomarker of xenoestrogen exposure. Extensive separation of xenoestrogens from endogenous hormones was made in 20 adipose tissue samples by HPLC, and two eluates were separated and tested in the E-Screen bioassay. An extensive fractionation protocol was also developed. The objective of this study was to investigate predictors of TEXB by using a multiple regression model after adjusting by confounding factors. The final model included the estrogenicity of 8 out of 11 individual 1-min fractions into which the xenoestrogen eluate was split and the marital status of patients, and it explained 97% of TEXB variability, and variables. Our results indicate that TEXB of complex mixtures can be accurately predicted from the estrogenicity of a small number of components.