Gold nanoparticles were generated as described in previous reports 10 11 12 13 , and the rats exposed in a whole-body-type exposure chamber (1.3 m³, Dusturbo, Seoul). The generation system consisted of a small ceramic heater connected to an AC power supply and housed within a quartz tube case. The heater dimensions were 50 × 5 × 1.5 mm³, and a surface temperature of about 1500°C within a local heating area of 5 × 10 mm² was achieved in about 10 seconds 12 . For long-term testing, the source material (about 700 mg of 24 ct gold) was positioned at the highest temperature point. The quartz tube case was 70 mm in diameter and 200 mm long. Clean (dry and filtered) air was used as the carrier gas, and the gas flow maintained at 30.0 L/min (Re = 572, laminar flow regime) using a mass flow controller (MFC, AERA, FC-7810CD-4V, Japan) 10 11 . This generator has already been shown to generate nanoparticles from 1.8 to 6.1 nm (below 10 nm) in diameter that do not agglomerate in air. XRD analysis using an X-ray diffractometer with CuK2 radiation previously showed that the particles generated are metallic gold, not gold oxides 13 . In the current study, the system produced different concentrations of nanoparticles (high, middle, and low) in three separate chambers. For the high-concentration chamber, the nanoparticle generator was operated at 45 L/min (liters per minute) and mixed with 200 L/min of clean ambient air. A portion of the high nanoparticle concentration was then diverted to the middle-concentration chamber using an MFC for the first dilution (1.49 ± 0.06 L/min, mean ± S.E.), and a portion of the middle nanoparticle concentration then diverted to the low-concentration chamber using a second MFC (14.94 ± 0.02 L/min).

In each chamber, nanoparticle distribution in terms of size was measured directly in real-time using a differential mobility analyzing system (DMAS); combining a differential mobility analyzer (Short type-DMA, 4220, HCT Co., Ltd. Korea, range 2-150 nm) and condensation particle counter (CPC, 4312, HCT Co., Ltd. Korea, 0-10⁸/cm³ detection range). The nanoparticles were measured using sheath air at 5 L/min and polydispersed aerosol air at 1 L/min for the DMA and CPC, respectively.

The control chamber was supplied with HEPA-filtered fresh air. Particle concentration in the fresh-air control chamber was measured using a particle sensor (4123, HCT Co., Ltd. Korea) that consisted of two channels; 300-1000 nm and over 1000 nm, to verify the performance of the HEPA filters. Previous studies have shown that the control chamber contains negligible nanoparticles if the HEPA filter is functioning correctly 14 15 .

The filters used to sample the fume particles were coated with carbon, mounted on an electron microscope grid (200 mesh, Veco, Eerbeek, Holland), and visualized under a field emission-transmission electron microscope (FE-TEM, JEM2100F, JEOL, JAPAN). The diameters of 800 randomly selected particles collected using a nanoparticle collector (nanoparticle collector, 4650, HCT Co., Ltd. Korea) were measured at a magnification of 100,000, and the gold particles analyzed using an energy-dispersive x-ray analyzer (EDX, TM200, Oxford, UK) at an accelerating voltage of 200 kV.

Six-week-old male and female, specific-pathogen-free (SPF) Sprague-Dawley rats (Slc:SD) (originally derived from the Charles River SD in 1968) were purchased from SLC (Tokyo, Japan) and acclimated for 1 week before starting the experiments. During the acclimation and experimental periods, the rats were housed in polycarbonate cages (3 rats per cage) in a room with controlled temperature (23 ± 2°C), humidity (55 ± 7%), and a 12-h light/dark cycle. The rats were fed a rodent diet (Harlan Teklab, Plaster International Co., Seoul) and filtered water ad libitum. The 7-week-old rats, weighed approximately 200 g for the males and 145 g for the females at the beginning of exposure. The rats were divided into 4 groups (10 rats in each group/sex): fresh-air control, low-dose group (target dose, 2.5 × 10⁴ particles/cm³, 1.0 × 10⁶ nm²/cm³ surface area), middle-dose group (target dose, 2.5 × 10⁵ particles/cm³, 6.0 × 10⁶ nm²/cm³ surface area), and high-dose group (target dose, 1.2-2.8 × 10⁶ particles/cm³, 3.0 × 10⁸ nm²/cm³ surface area). The animals were exposed to the gold nanoparticles for 6 hours/day, 5 days/week, for 13-weeks, and housed in individual wire cages without food and water during the 6-hour exposure periods. The animals were examined daily on weekdays for any evidence of exposure-related effects, including respiratory, dermal, behavioral, nasal, or genitourinary changes suggestive of irritation. Body weights were evaluated at the time of purchase, at the time of grouping, after one day of exposure, once a week during exposure, and before necropsy. The experiment was approved by the KCL Institutional Animal Care and Use Committee.

At the conclusion of the 90-day experiment, the rats were 20 wks old. Before necropsy, food was withheld for 24 h and the rats were anesthetized with an overdose of sodium pentobarbital. Blood was then drawn from the abdominal aorta, collected in vacutainers, and analyzed for ALB (albumin), ALP (alkaline phosphatase), Ca (calcium), CHO (cholesterol), CRE (creatinine), gamma-GT (gamma-glutamyl transpeptidase), GLU (glucose), GOT (glutamic oxaloacetic transaminase), GPT (glutamic pyruvic transaminase), IP (inorganic phosphorus), LDH (lactate dehydrogenase), MG (magnesium), TP (total protein), UA (uric acid), BUN (blood urea nitrogen), TBIL (total bilirubin), CK (creatine phosphokinase), Na (sodium), K (potassium), Cl (chloride), TG (triglyceride), and A/G (ratio of albumin to globulin) using a biochemical blood analyzer (Hitachi 7180, Hitachi, Japan). The blood was also analyzed for the WBC (white blood cell count), RBC (red blood cell count), Hb (hemoglobin concentration), HTC (hematocrit), MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration), RDW (red cell distribution width), PLT (platelet count), MPV (mean platelet volume), NE# (number of neutrophils), NE% (percent of neutrophils), LY# (number of lymphocytes), LY% percent of lymphocytes), MO# (number of monocytes), MO% (percent of monocytes), EO# (number of eosinophils), EO% (percent of eosinophils), BA# (number of basophils), and BA% (percent of basophils) using a blood cell counter (Hemavet 0950, CDC Tech., USA).

To evaluate aggregation of red blood cells or blood coagulation attributable to the gold nanoparticles, 3.2% sodium citrate was used for anticoagulation, and the activated partial thromboplastin time (APPT) and prothrombin time (PT) measured using a blood coagulation analyzer (ACL 7000, Instrumentation Laborato Co., U.S.A.).

Since a gender difference in silver accumulation was previously noted in kidneys 14 15 , kidney function was measured based on the N-acetyl-beta-D-glucosaminidase (NAG) and protein in the urine using metabolic cages for 5 rats from each exposure group and the control.

After collecting blood samples, rats were killed by cervical dislocation, and all the organs carefully removed, including the adrenal glands, bladder, testes, ovaries, uterus, epididymis, seminal vesicle, heart, thymus, thyroid gland, trachea, esophagus, tongue, prostate, lungs, nasal cavity, kidneys, spleen, liver, pancreas, brain and olfactory bulb. The organs were then weighed, fixed in a 10% formalin solution containing neutral phosphate-buffered saline, embedded in paraffin, stained with hematoxylin and eosin, and examined under light microscopy.

The lung function of four rats from each dose group was evaluated every week during the 90-day exposure using a ventilated bias flow whole-body plethysmograph (WBP) (SFT3816, Buxco Electronics, Sharon, CT) that consisted of a reference chamber and animal chamber interconnected by a pressure transducer (MAX1320, Buxco Electronics, Sharon, CT; 15 ). Measurements taken during pulmonary function testing included the tidal volume (TV, ml), minute volume (MV, ml/min), respiratory frequency (BPM, breath/min), inspiration time (Ti, s), expiration time (Te, s), peak inspiration flow (PIF, ml/s), and peak expiration flow (PEF, ml/s). After the last 6-hour exposure, the rats were placed in an animal chamber, left for 40 min to stabilize, and measurements were taken in the whole-body plethysmograph for 5 minutes.

The same animals that were used for lung function testing were also subjected to a BAL at the end of the 90-day exposure period. The rats were deeply anesthetized with an overdose of sodium pentobarbital. Blood was collected from the abdominal aorta and the rats were then exsanguinated by severing the abdominal aorta. The lungs were lavaged 14 times with 3 ml aliquots of a warm calcium- and magnesium-free phosphate buffer solution (PBS), pH 7.4. The samples were also centrifuged for 7 min at 500 × g and the cell-free BAL fluid discarded. The cell pellets from all washes for each rat were then combined, washed, and resuspended in 1 ml of a phosphate buffered saline (PBS) buffer and evaluated 16 17 18 19 20 . Total cell numbers were determined using a hemacytometer. The cells were first smeared and then stained with the Wright Giemsa Sure Stain 21 to allow a count of the total number of cells, macrophages, polymorphonuclear cells (PMNs), and lymphocytes. BAL levels of total protein, albumin, and LDH were also measured using a blood biochemical analyzer (Hitachi 7180, Hitachi, Japan).

After necropsy, samples of the blood (0.5 ml), liver, lungs, brain, kidneys, and olfactory bulb were analyzed to determine organ distribution of gold. Care was taken to avoid contaminating tissues with gold from the fur and skin of each animal. After wet digestion using a flameless method, the tissue concentrations of gold were analyzed using an atomic absorption spectrophotometer equipped with a Zeeman graphite furnace (Perkin Elmer 5100ZL, Zeeman furnace module, USA) based on the NIOSH 7300 method 22 . The detection limit was 2 ppb and the limit of quantification was 7 ppb.

All results are expressed as means ± standard error (SE). An analysis of variance (ANOVA) test and Duncan's multiple range tests were used to compare the body weights, bronchoalveolar lavage cell distributions, lung function test parameters, and all other comparisons for the three dose groups with those for the control rats. Histopatholgic results were analyzed using a Chi-square analysis. The level of significance was set at p < 0.05 and p < 0.01.

Nanoparticle distributions in chambers are shown in Table 1. For the high-concentration chamber, the geometric mean diameter (GMD), concentration, and surface area of the gold nanoparticles measured by the DMAS were 5.06 nm, 1.85 × 10⁶ particles/cm³, 20.02 μg/m³, and 3.64 × 10⁸ nm²/cm³, respectively; for the middle-concentration chamber were 4.12 nm, 2.36 × 10⁵ particles/cm³, 0.38 μg/m³ and 1.68 × 10⁷ nm²/cm³, respectively, and for the low-concentration chamber were 4.3 nm, 2.36 × 10⁴ particles/cm³, 0.04 μg/m³, and 1.9 × 10⁶ nm²/cm³, respectively. The gold nanoparticles observed by TEM were spheroid in shape in both non-aggregated and non-agglomerated forms, with diameters under 6 nm (Figure 1,A-C). TEM-EDX analysis indicated that only elemental gold was present (Figure 1D). The diameters were log normally distributed between 1 and 6 nm, and the CMD (count median diameter) and GSD were 2.47 nm and 1.42, respectively (Figure 2). The distribution of gold nanoparticles was well maintained during the 90-day exposure period, as shown in Figure 3.

No significant gross effects were observed during the 90-day exposure period. There was a loss of hair on the front left leg of one male in the high dose group at 9 weeks and a loss of hair on both front legs of one female in the high dose group at 12 weeks of exposure. There was no apparent reason for the loss of hair in these two animals. In our experience, hair loss in isolated animals is not an unusual event. Examination of the eyes of control and high-dose animals did not reveal any effects. Food intake increased in the high-dose male group when compared to the control (p < 0.05), middle-dose (p < 0.05), and low-dose (p < 0.01) groups (Figure 4A). Food intake also increased in the low-dose female group when compared to the control (p < 0.01), middle-dose (p < 0.01), and high-dose (p < 0.05) groups (Figure 4B). While there were no significant changes in the body weights of the male rats (Figure 5A), there was a significant body weight gain in the low-dose female group (p < 0.05) when compared to the control, middle-dose, and high-dose groups after 4 weeks of exposure (Figure 5B). There was a 0.3 × 0.2 cm white mass in the right kidney of one rat from the high-dose group. No significant organ weight changes were observed in either the male or female rats at the conclusion of the study (Table 2 and 3).

No significant dose-related differences were observed for the hematology values (Table 4 and 5) and blood biochemical measurements (Table 6 and 7).

Increases in gold concentrations in the lung tissue from the high dose group as compared to controls were statistically significant (p < 0.01) and increased with dose in both the males and females (Table 8 and 9). Gold concentrations in kidneys also increased in a dose dependent manner with statistical significance (p < 0.01). Interestingly, there was gender-related difference in gold concentrations in the kidneys (Table 8 and 9), with female kidneys showing more gold accumulation than the male kidneys. Apart from the lungs and kidneys, no dose-dependent increase of gold nanoparticles was found in the blood, liver, or olfactory bulb from both the male and female rats. Gold levels in blood were less than the tissues measured so that any small amount of contamination of other tissues with blood or blood contained in tissue was unlikely to greatly influence tissue measurements of gold content. There was a significant increase in gold nanoparticle concentration only in brain tissue from high-dose females (Table 9).

Total number of cells, alveolar macrophages, polymorphonuclear cells (PMN), and lymphocytes in BAL fluid after 90-days of exposure are shown in Figure 6. When compared to the control group, there were no significant changes in total cell numbers, alveolar macrophages, PMN, or lymphocytes of any exposed males. There was a significant increase in lymphocytes in middle and high-dose females (Figure 6B). Albumin, LDH, and total protein in BAL did not increase significantly in either male or female rats (data not shown).

Among the pulmonary function test parameters, there were significant changes in tidal volume and minute volume during the 90-days of gold nanoparticle exposure (p < 0.01-0.05) (Figures 7,A and 7B and 8,A and 8B). Dose-dependent tidal volume decreases in male rats led to minute volume decreases in the high-dose animals. A tendency towards a dose-dependent decrease in the tidal volume appeared to be present in female rats, but was not statistically significant.

The only significant changes in histopathology occurred in lungs (Table 10 and 11), where there was minor focal inflammation with an inflammatory infiltrate of mixed cell type (lymphocyte/neutrophil/macrophage) were noted in both treated male and female rats. The increases were dose-dependent in female rats (Figure 9).

To evaluate changes in red blood cell aggregation or blood coagulation, erythrocyte aggregation, activated partial thromboplastin time (APPT), and prothrombin time (PT) were tested. No significant differences were found between the control and any treated animals in APPT or PT (Figure 10,A and 10B). There were no significant differences among dose groups in kidney function as measured based on the NAG and protein in the urine (Figure 11,A and 11B).