Study design and experimental set-up

The detailed methodology and experimental set-up including a graphical timeline was described recently [6]. Briefly, subjects were exposed four times for 4 h with 2 weeks intervals in an exposure unit at our institute [10] to each exposure scenario: filtered air (sham) and 3 different ZnO particle concentrations (0.5, 1.0 and 2.0 mg/m³). ZnO particle synthesis was based on pyrolysis of atomized aqueous zinc formate solutions with a hydrogen-oxygen flame. The particle size of the generated primary particles was determined with scanning electron microscopy (SEM, model JSM-7500F, JEOL Ltd., Tokyo, Japan) and was about 10 nm [11]. Depending on the ZnO concentration the primary particles formed aggregates and agglomerates in a range from 48 nm (0.5 mg/m³ ZnO) to 86 nm (2.0 mg/m³ ZnO), determined with a scanning mobility particle sizer (SMPS, model 3080, TSI Inc., Shoreview MN, USA, equipped with a long differential mobility analyzer and a butanol condensation particle counter, model 3776, TSI Inc.) [10]. Measurements on airborne ZnO particles with an electrometer (Modell 3068B, TSI Inc., Shoreview MN USA) could not detect any electric charge.

X-ray powder diffraction of ZnO particles, that were sampled via thermophoresis, was determined using a diffractometer from Stoe with a Bragg-Brentano geometry (XRD, model Stadi P with Co Anode and scintillation counter, Stoe & Cie GmbH, Darmstadt, Germany). Comparing this with standard data, it was observed that all the peaks were matched with the standard data of hexagonal phase of zinc oxide (JCPDS card no. 36–1451).

An elementary analysis of the ZnO particles (Mikroanalytisches Labor Pascher, Remagen, Germany) yielded a purity of 99.7%. The specific surface area as determined by a BET device (BET, model Gemini VII 2390a, Micromeritics GmbH, Aachen, Germany) was 20.2 g/m³.

A ceiling fan was used to homogenize the freshly generated ZnO nanoparticle atmospheres in the exposure unit [12]. Briefly, constant target concentrations with 0.5, 1.0 and 2.0 mg/m³ ZnO were planned. Sham exposures (0 mg/m³ ZnO) were also performed with the flame generator operated with purified water without zinc salt. The purity of the airborne ZnO was 99.71%. The air exchange rate was set at 12 per hour (360 m³/h) with a room temperature of 23.5 °C (+/− 0.3 °C) and a relative humidity of 47.0% (+/− 1.7%).

Potential participants were tested for their suitability to participate in the study in a baseline examination including a questionnaire, medical examination, lung function test and exercise testing. Smoker or participants with chronic diseases were excluded with the exception of sensitizations to seasonal environmental allergens. The recruitment of these volunteers was realized by advertising at universities and student residences. Sixteen healthy nonsmoking volunteers (8 women, 8 men) with a median age of 26 years (range 19–42) and a median BMI of 24 kg/m² (range 19–29) participated in the study [6]. The subjects had no previous exposure to airborne zinc compounds. Standard baseline laboratory parameters were within normal ranges.

The subjects were examined during the 4 h-periods at rest and during periods of moderate physical exercise on a cycle ergometer set to 15 L/[min∙m²] corresponding to an individual work load of 30–96 watt. Each 30-min rest was followed by a 30-min exercise period, for four times. Exposures were randomized and double blinded, with the exception of the exposures to 2.0 mg/m³ ZnO, which were not blinded according to instructions by the ethics committee. Examinations were performed before, during, directly after (after about 10 min at rest), and approximately 24 h after exposure. Additionally, examinations were performed at recruitment (baseline test) and about two weeks after the last exposure (final test).

For each subject a total number of 46 10-min-sections from all electrocardiographic records were analyzed. A 10-min-section was defined as the 2nd third of every 30-min-period, thus the beginning and the end of the period were removed to ensure steady-state conditions and comparability.