Cardiopulmonary toxicity of pulmonary exposure to ...

Nanotoxicology, 2013; Early Online, 1–12 © 2013 Informa UK, Ltd. ISSN: 1743-5390 print / 1743-5404 online DOI: 10.3109/17435390.2013.809809

Cardiopulmonary toxicity of pulmonary exposure to occupationally relevant zinc oxide nanoparticles Hsiao-Chi Chuang1,2, Hung-Tzu Juan3, Chun-Nung Chang3, Yuan-Horng Yan3,4, Tzu-Hsuen Yuan3, Jyh-Seng Wang5, Hao-Cheng Chen1,2, Yaw-Huei Hwang3,6, Chii-Hong Lee7, Tsun-Jen Cheng3,6

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1

School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan, 2Division of Pulmonary Medicine, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan, 3Institute of Occupational Medicine and Industrial Hygiene, College of Public Health, National Taiwan University, Taipei, Taiwan, 4Department of Medical Research, Ditmanson Medical Foundation Chia-Yi Christian Hospital, Chiayi, Taiwan, 5Department of Pathology and Laboratory Medicine, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, 6Department of Public Health, College of Public Health, National Taiwan University, Taipei, Taiwan and 7Department of Pathology, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan

Introduction

Abstract Exposure to zinc oxide (ZnO) metal fumes is linked to adverse human health effects; however, the hazards of ZnO nanoparticles (ZnONPs) remain unclear. To determine pulmonary exposure to occupationally relevant ZnONPs cause cardiopulmonary injury, Sprague-Dawley rats were exposed to ZnONPs via intratracheal (IT) instillation and inhalation. The relationship between intrapulmonary zinc levels and pulmonary oxidative-inflammatory responses 72 h after ZnONP instillation was determined in bronchoalveolar lavage fluid (BALF). Instilled ZnONPs altered zinc balance and increased the levels of total cells, neutrophils, lactate dehydrogenase (LDH) and total protein in BALF and 8-hydroxy-2¢deoxyguanosine (8-OHdG) in blood after 72 h. The ZnONPs accumulated predominantly in the lungs over 24 h, and trivial amounts of zinc were determined in the heart, liver, kidneys and blood. Furthermore, the inflammatory-oxidative responses induced by occupationally relevant levels of 1.1 and 4.9 mg/m3 of ZnONP inhalation for 2 weeks were determined in BALF and blood at 1, 7 and 30 days post-exposure. Histopathological examinations of the rat lungs and hearts were performed. Inhalation of ZnONP caused an inflammatory cytological profile. The total cell, neutrophil, LDH and total protein levels were acutely increased in the BALF, and there was an inflammatory pathology in the lungs. There were subchronic levels of white blood cells, granulocytes and 8-OHdG in the blood. Cardiac inflammation and the development of fibrosis were detected 7 days after exposure. Degeneration and necrosis of the myocardium were detected 30 days after exposure. The results demonstrate that ZnONPs cause cardiopulmonary impairments. These findings highlight the occupational health effects for ZnONP-exposed workers.

Metal fume fever is an acute occupational disease caused by the inhalation of metal fumes, particularly zinc oxide (ZnO) in nano-scaled size fraction (Donaldson et al. 2005). This disease leads to respiratory and systemic syndromes (Fine et al. 1997, 2000), which often occur in workers who are exposed to zinc metal fumes during the welding of galvanised metal or melting metal (Fine et al. 1997; Ahsan et al. 2009). Clinically, the symptoms of zinc metal fume fever are induced a few hours after ZnO exposure, with illness following within 24 h and acquired tolerance after successive exposures. The adverse health effects caused by zinc metal fume exposure have been well documented based on observations of galvanised metal workers; however, there is an emerging population, that is, workers exposed to ZnO nanomaterials, suffering from unknown health impacts caused by exposure to nano-scale zinc metal fumes. ZnO nanoparticles (ZnONPs) are widely used in the manufacture of paints, coatings and semiconductors. ZnONPs have also been shown to be biocompatible nanomaterials; therefore, they have a higher potential for use in biomedical applications. However, the safety of ZnONPs has not been adequately demonstrated. Increasing numbers of studies have reported adverse health effects in vivo and in vitro after exposure to ZnONPs (Cho et al. 2011, 2012), which may relate to ZnONPs and their dissolved fractions such as zinc ions (Akhtar et al. 2012; Park et al. 2011). Zinc homeostasis under conditions of ZnONP exposure may be important for cytotoxicity. A previous study showed that exposure to ZnONPs interfered with the homeostasis of zinc ion and that elevated zinc ion resulted in apoptosis (Kao et al. 2012). However, a paucity of studies has measured intrapulmonary zinc balance after exposure to ZnONPs. The

Keywords: cardiac injury, inflammation, oxidative stress, zinc ion, zinc oxide nanoparticle

Correspondence: Tsun-Jen Cheng, Institute of Occupational Medicine and Industrial Hygiene and Department of Public Health, College of Public Health, National Taiwan University, Taipei, Taiwan. Tel: +886 2 33668090. Fax: +886 2 23957845. E-mail: [email protected] (Received 24 January 2013; accepted 27 May 2013)

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 H.-C. Chuang et al. relevant guideline for ZnO exposure in occupational environments lists a 5 mg/m3 time-weighted average threshold limit value (TLV) for an 8-h exposure. Early results from animal and clinical human exposure studies have shown that the current threshold for ZnO fume exposure is insufficient to protect workers (Fine et al. 1997; Gordon & Fine 1993). ZnONPs are easily transported into alveolar space through inhalation and may lead to severe health effects due to their physicochemical characteristics. Therefore, it is important to evaluate the health effects that result from exposure to occupationally relevant doses of ZnONPs. The majority of in vivo and in vitro ZnONP toxicology studies are predominantly based on exposure to particle suspensions via intratracheal (IT) instillation or pulmonary aspiration. However, these approaches do not reflect the natural conditions of respiratory exposure. Landsiedel et al. (2012) suggested that inhalation studies are required to check the potential association between occupational nanoparticle exposure and adverse health effects such as cardiopulmonary diseases. Therefore, nanoparticle generation systems were developed for particle inhalation studies in animals. For in vivo toxicology evaluation, a long-term and stable system with flexible settings is important for understanding the role of various physical characteristics, such as size, mass and surface area, in regulating ZnONP toxicity. The ZnONP generation system used in the present study was originally designed by the Energy and Environment Research Laboratories of the Industrial Technology Research Institute of Taiwan with moderate modification (Ho et al. 2011); the principle behind this system is based on the description by Singh et al. (2002). Using a transmission electron microscope equipped with energy dispersive X-ray microanalysis, uncontaminated ZnONPs with broad range geometric mean diameter (GMD) and small geometric standard deviation (GSD) were observed (Ho et al. 2011). Epidemiological and clinical studies have postulated that zinc is an important component in particles that determines the response to cardiopulmonary toxicity (Chen & Lippmann 2009; Shannahan et al. 2012). ZnONP-induced adverse health effects have been linked to the formation of oxidative stress and inflammation (Ho et al. 2011). Therefore, inhalation exposure to occupationally relevant levels of ZnONP needs to be evaluated to understand the adverse human health effects that may impact workers. To achieve this objective, rats were exposed to ZnONPs via IT instillation (zinc distribution in bronchoalveolar lavage fluid (BALF) and tissues) and inhalation (occupationally relevant exposure). Associations between oxidative-inflammatory responses and intrapulmonary zinc balance after ZnONP exposure were determined. The authors also considered ZnONP-induced pulmonary-to-systemic oxidative stress and inflammation, as well as cardiopulmonary histopathology.

Materials and methods Animals All of the animal experiments were approved by the Laboratory Animal Centre of the National Taiwan University (Taiwan). Seven-week-old male Sprague-Dawley (SD) rats

(250–300 g) obtained from BiOLASCO (Taipei, Taiwan) were maintained under a 12-h light/dark cycle at a consistent temperature and relative humidity of 22 ± 2 C and 55 ± 10%, respectively, throughout the study. Animals were housed in plastic cages and provided with LabDiet 5001 (PMI Nutrition International, Brentwood, MO, USA) and water ad libitum during acclimation, pre-exposure and post-exposure.

Experimental design The experimental design is shown in Figure 1. To investigate the status of intrapulmonary zinc balance and the oxidativeinflammatory responses after ZnONP exposure, the rats were divided into eight groups (control (n = 4) and exposure (n = 6) at four different time points). The animals were administered 10 mg/ml ZnONPs (1 ml each rat) in phosphate buffered saline (PBS) with 5% bovine serum albumin (BSA) or particle-free PBS with 5% BSA as vehicle control (Figure 1A). IT instillation of the samples was performed under light anaesthesia induced by 3% Ultane vapour (Abbott Laboratories, UK) for 20 30 s. The ZnONPs (Sigma-Aldrich, St Louis, MO, USA) were suspended in PBS with 5% BSA. Immediately before IT instillation, the ZnONP suspension was sonicated for 10 min and then vortexed. To evaluate the consequent oxidative-inflammatory responses and corresponding intrapulmonary zinc concentrations over time, rats were euthanised at 6, 24, 48 and 72 h after instillation for BALF sample collection. To determine the tissue distribution of ZnONPs, the rats were divided into five groups (control (n = 3) and exposure (n = 3) at four different time points). The animals were administered 5 mg/ml ZnONPs (1 ml each rat) in PBS with 5% BSA or particle-free PBS with 5% BSA as vehicle control (Figure 1B). IT instillation of the samples was performed under light anaesthesia induced by 3% Ultane vapour for 20 30 s. The various groups of rat were euthanised at 0 (control), 3, 6, 12 and 24 h after IT instillation. The tissue samples were lyophilised to obtain the dry weight. The tissue samples collected at each time point included lungs, heart, liver, kidneys and blood. The zinc concentrations in the samples were determined using inductively coupled plasma-mass spectrometry (ICP-MS). To investigate the systemic effects of occupationally relevant ZnONP inhalation (Figure 1C), a total of 60 rats were randomly divided into two groups for the two exposure concentrations. The 30 grouped rats were then divided into two subgroups, and the subgrouped animals were exposed to either ZnONPs (n = 18; exposure) or highefficiency particulate air (HEPA)-filtered air (n = 12; control) for 2 weeks (5 h/day and 5 days/week). The animals were euthanised (6 rats from the ZnONP exposure group and 4 rats from the HEPA-filtered air control group) after 1 day, 7 days or 30 days of exposure, and BALF, peripheral blood, lungs and hearts were collected.

Hydrodynamic diameters of ZnONPs Commercial ZnONPs with an average diameter and surface area of 50 nm and 10.8 m2/g, respectively, were obtained from Sigma-Aldrich. ZnONPs were thoroughly dispersed in three media: deionised water (d-H2O), PBS or PBS

Cardiopulmonary injury by ZnONPs

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A Sacrifice

Intratracheal exposure ZnONP (10 mg/ml each rat) Five groups (n = 4–6 each group)

ZnONP: n = 6 per sacrifice PBS: n = 4 per sacrifice 6h

24 h

48 h

72 h

PBS

B Sacrifice

Intratracheal exposure ZnONP (5 mg/ml rat)


Five groups (n = 3 each group)

ZnONP: n = 3 per sacrifice PBS: n = 3 per sacrifice 3h

6h

12 h

24 h

PBS

C Sacrifice

Inhalation exposure Two modes (n = 4–6 each group) (1.1 & 4.9 mg/m3)

ZnONP exposure 2 weeks (5 h/day, 5 days/week)

ZnONP: n = 6 per sacrifice HEPA-filtered air: n = 4 per sacrifice 1 day

7 days

30 days

HEPA-filtered air Figure 1. Overview of the three experimental designs for ZnONP exposure in rats. (A) A dose 10 mg/ml ZnONPs were administered via intratracheal (IT) instillation to SD rats (n = 4 6 each group); 5% serum in phosphate buffered saline (PBS) was used as vehicle control. The animals were sacrificed 6, 24, 48 and 72 h after exposure. (B) A dose 5 mg/ml ZnONPs were administered via IT instillation to SD rats (n = 3 each group). The animals were sacrificed 0 (control), 3, 6, 12 and 24 h after exposure. The tissue samples collected at each time point included lungs, heart, liver, kidneys and blood. (C) A total of 60 rats were divided into groups for exposure to 1.1 or 4.9 mg/m3 ZnONPs (n = 4 6 each group). The animals were sacrificed 1, 7 and 30 days after exposure to occupationally relevant ZnONPs or HEPA-filtered air for 2 weeks.

supplemented with 5% BSA. These media were prepared at the final concentration of 10 mg/ml after 30 min sonication. Malvern dynamic light scattering (DLS; Malvern Zetasizer Nano-ZS, UK) was then used to determine the ZnONP hydrodynamic diameters in the three media.

ZnONP stream was mixed with 75 and 100 l/min HEPAfiltered air (containing 22% oxygen) before being introduced into animal whole-body exposure chambers with or without

Evaporation–condensation ZnONP generation system

Table I. Operation parameters of the two modes used to generate two different concentrations (number and mass) of ZnONPs with similar size fractions by the evaporation–coagulation ZnONP generation system. Mode 1 Mode 2 Operation conditions 550 550 Furnace temperature ( C) Carrier air (l/min) 1 1 Reaction air (l/min) 10 10 Dilution air (l/min) 100 75 Physical characteristics GMD (nm) 48 51 GSD 1.8 1.8 Number (#/cm3) 2.7 106 3.9 105 (1.4 105) (1.1 106) Mass (mg/m3) 1.1 (0.3) 4.9 (1.6) 4.2 107 8.1 105 Surface area (mm2/m3) (1.4 106) (1.3 108)

An evaporation–condensation method was employed to generate ZnONPs; the experimental setup was described in previous studies (Ho et al. 2011; Singh et al. 2002). Two setup modes were maintained by controlling dilution rates to produce two different ZnONP concentrations with similar characteristics (Table I). In summary, ~3 g zinc powder (>99%, Merck, Germany) was evaporated at the centre of a 550 C tube furnace (Model T11-301, SJ Ltd., Taiwan; Table I), and the generated zinc vapour was continuously transported by nitrogen with a flow rate of 1 l/min to interact with oxygen (10 l/min HEPA-filtered air) resulting in oxidised zinc product (ZnO). Because the outlets of the furnace were equipped with a chilling system, the ZnO vapour was then suddenly quenched and condensed to form ZnONPs. In this study, an additional condition section was introduced between the generation system and animal exposure chambers to further ensure the temperature, relative humidity and composition of the exposed ZnONP stream consistent with environmental conditions. The

The physical characteristics of the produced ZnONPs were 48 and 51 nm (GMD), with GSDs of 1.8. Significant differences in ZnONP number, mass concentrations and surface area were obtained by the two mode setups. Values are the mean (SD). Abbreviations: GMD: geometric mean diameter; GSD: geometric standard deviations; SD: standard deviation; ZnONPs: ZnO nanoparticles.

 H.-C. Chuang et al. HEPA filtration. The exposure chamber has been previously described (Maciejczyk et al. 2005).

To understand the inhalation exposure conditions under the two modes of ZnONP generation, a TSI scanning mobility particle sizer (SMPS) with nano-DMA (model 3936, TSI, Shoreview, MN, USA) was used to continuously monitor the size distribution of ZnONP in the exposure chamber throughout the entire exposure period. The flow rates of the SMPS aerosol stream and sheath air were fixed at 0.3 and 3 l/min, respectively. The SMPS was calibrated with NIST-traceable PSL standard 100 nm particles before the experiment. The total particle number concentrations, the number-based GMD and GSD were reported. The total surface area was estimated using the TSI software (Aerosol Instrument Manager, AIM 9.0).

with a single intraperitoneal injection of 2 ml sodium pentobarbitone (200 mg/ml). A single 2 ml volume of PBS was used to lavage the lungs. The authors retained this first BALF in a separate tube for analysis of biochemical markers. Subsequently, they lavaged the lungs with a further three 2 ml volumes of PBS. All samples were centrifuged at 1500 g for 5 min at 4 C, the supernatant was removed and the cell pellet from the first lavage was combined with the cells from the subsequent lavage before resuspension in 1 ml PBS. Cytospin preparations were made using 10,000 cells centrifuged at 500 g for 5 min onto glass slides. Two hundred cells per slide were counted and the results expressed as total number of cells and number of neutrophils in the lung lavage. A Spotchem autoanalyser (SP4410, Germany) was used to determine lactic dehydrogenase (LDH) levels. The total protein concentration was determined with a total protein assay kit (Bio-Rad, Hercules, CA, USA).

Scanning electron microscopy

Determination of systemic inflammation

To investigate the physical characteristics of ZnONPs, the commercial ZnONPs and the ZnONPs generated using the evaporation–condensation method were loaded onto 13-mm aluminium scanning electron microscopy (SEM) stubs (Moreno et al. 2003). The prepared samples were carbon coated to an average thickness of 10 nm using a sputter coater (Bio-Rad, UK). The morphologies of the ZnONPs were observed using an Inspect SEM (FEI, Hillsboro, OR, USA) at an accelerating voltage of 3 kV with a 2.5 spot size.

Animals were anaesthetised intraperitoneally with sodium pentobarbital, and then blood was drawn from the abdominal aorta. A total of 10 ml of whole blood was taken from the rat aorta, 2 ml of which was then transferred immediately into ethylenediamine tetraacetic acid (4% EDTA) tubes. The other blood samples were used for the determination of serum oxidative-inflammatory markers. Each blood sample was centrifuged at 1500 g for 10 min. Differential cell counts were performed using the Spotchem SP4410 autoanalyser (Germany). The counts included white blood cells, lymphocytes and granulocytes. These analyses have been described in a previous study (Ho et al. 2011).


Scanning mobility particle sizer

Inductively coupled plasma-mass spectrometry To examine the zinc leached by ZnONPs, test solutions of 10 mg/ml (in 5% BSA in PBS) were shaken on a rotary shaker operating at 200 rpm under room temperature (Wong et al. 2010). Samples were taken at 0, 10, 40, 90 and 120 min, followed by 0.45-mm PVDF (polyvinylidene difluoride) filtration (ChromTech, Apple Valley, MN, USA). ICP-MS (Agilent 7500c, Santa Clara, CA, USA) was used to determine zinc concentrations (Chuang et al. 2012). Furthermore, ICP-MS was used to determine zinc concentrations in the supernatant fractions in BALF samples (soluble fraction of ZnONP) after centrifugation (1000 rpm for 10 min). The supernatants were then filtered through 0.45-mm PVDF filters. To determine the zinc concentrations in tissue samples after IT instillation, the lyophilised and blood samples were digested using concentrated nitric acid (Fisher Scientific, Waltham, MA, USA) in a MARS 5 microwave system (CEM, Matthews, NC, USA) in CEM advanced Teflon-lined composite vessels (Jones et al. 2006). The digestion temperature was increased to 150 C for 30 min. Nitric acid and deionised (>18 MW) water were added to the samples to a final concentration of 5% nitric acid. Deionised water blanks were analysed to detect any contamination during the analysis process. A solution of a certified rock standard (BCR1) was used to determine the accuracy of the analyses. The relative percentage difference was