Nanosized zinc oxide particles do not promote DHPN-induced lung carcinogenesis but cause reversible epithelial hyperplasia of terminal bronchioles

Abstract

Zinc oxide (ZnO) is known to induce lung toxicity, including terminal bronchiolar epithelial hyperplasia, which gives rise to concerns that nanosized ZnO (nZnO) might lead to lung carcinogenesis. We studied the tumor promoting activity of nZnO by an initiation-promotion protocol using human c-Ha-ras proto-oncogene transgenic rats (Hras128 rats). The rats were given 0.2 % N-nitrosobis(2-hydroxypropyl)amine (DHPN) in the drinking water for 2 weeks and then treated with 0.5 ml of 250 or 500 μg/ml nZnO suspension by intra-pulmonary spraying once every 2 weeks for a total of 7 times. Treatment with nZnO particles did not promote DHPN-induced lung carcinogenesis. However, nZnO dose-dependently caused epithelial hyperplasia of terminal bronchioles (EHTB) and fibrosis-associated interstitial pneumonitis (FAIP) that were independent of DHPN treatment. Tracing the fate of EHTB lesions in wild-type rats indicated that the hyperplastic lesions almost completely disappeared within 12 weeks after the last nZnO treatment. Since nZnO particles were not found in the lung and ZnCl2 solution induced similar lung lesions and gene expression profiles, the observed lesions were most likely caused by dissolved Zn(2+). In summary, nZnO did not promote carcinogenesis in the lung and induced EHTB and FAIP lesions that regressed rapidly, probably due to clearance of surplus Zn(2+) from the lung.

Fig. 1

Induction of EHTB by nZnO. a representative normal terminal bronchiolar epithelium (NTBE); b EHTB in H&E-stained slides; c images and localizations of DHPN-induced alveolar hyperplasia (arrow) and nZnO-induced EHTB (arrow heads); d images of PCNA immunostaining in NTBE; and e in EHTB

Fig. 2

Induction of FAIP and observation of nZnO particles. a representative image of FAIP in rats treated with nZnO; b image of Azan–Mallory staining in the lung of rats treated with nZnO, showing collagen fibers; c percentage of the fibrotic area in total lung tissue area. ***<0.001 by two-tailed Student’s t-test versus the vehicle group; and ###p < 0.001 by Spearman rank correlation test. d image showing alveolar macrophages with vacuous phagocytosis vesicles; e and f TEM images showing alveolar macrophages (arrow) and epithelium (arrow heads), no nZnO particles being observed

Fig. 3

nZnO-induced EHTB and FAIP are reversible. Wild-type rats were treated with 500 μg/ml nZnO by IPS 2 times/week for 4 weeks and killed at different time points of 1 day (1d) and 2, 4, 6, 8, and 12 weeks (wks) after the last IPS. a histological images of the lung tissues; b number of EHTB per cm² lung tissue and c Zn²⁺ content in the lung tissues at different time points. Bars = 50 μm

Fig. 4

Similar effects of ZnCl₂ solution and nZnO in induction of EHTB and FAIP in wild-type rats. a H&E-stained slides of the lungs of rats treated with vehicle; b with nZnO; and c with ZnCl₂ solution, showing EHTB and FAIP; d comparable number of EHTB per square centimeter of the lung tissues induced by treatment of ZnCl₂ and nZnO; e gene expression determined by RT-PCR of Orm1 and Tnfa, with Actb gene as an internal control; f real-time PCR analysis of gene expression of Orm1 and Tnfa, which was normalized with Actb expression; g induction of Orm1 expression in primary alveolar macrophages exposed to nZnO; and h effect of human alpha 1 acid glycoprotein (AGP) and bovine serum albumin (BSA) on dissolution of nZnO in vitro