Abstract
The inhalation of humidifier disinfectants (HDs) is linked to HD-associated lung injury (HDLI). Polyhexamethylene guanidine (PHMG) is significantly involved in HDLI, but the correlation between chloromethylisothiazolinone (CMIT) and HDLI remains ambiguous. Additionally, the differences in the molecular responses to PHMG and CMIT are poorly understood. In this study, RNA sequencing (RNA-seq) data showed that the expression levels of metallothionein-1 (MT1) isoforms, including MT1B, MT1E, MT1F, MT1G, MT1H, MT1M, and MT1X, were increased in human pulmonary alveolar epithelial cells (HPAEpiCs) that were treated with PHMG but not in those treated with CMIT. Moreover, upregulation of MT1B, MT1F, MT1G, and MT1H was observed only in PHMG-treated HPAEpiCs. The protein expression level of metal regulatory transcription factor 1 (MTF1), which binds to the promoters of MT1 isoforms, was increased in PHMG-treated HPAEpiCs but not in CMIT-treated HPAEpiCs. However, the expression of early growth response 1 (EGR1) and nuclear receptor superfamily 3, group C, member 1 (NR3C1), other transcriptional regulators involved in MT1 isomers, were increased regardless of treatment with PHMG or CMIT. These results suggest that MTF1 is an essential transcription factor for the induction of MT1B, MT1F, MT1G, and MT1H by PHMG but not by CMIT.
Highlights
Our results revealed that the mRNA expression of MT1B, MT1F, MT1G, and MT1H was markedly upregulated in the HPAEpiCs treated with Polyhexamethylene guanidine (PHMG) but not in those treated with CMIT, suggesting that the suppression of MT1B, MT1F, MT1G, and MT1H is responsible for the discrepancy in the cellular responses between the PHMG and CMIT groups
early growth response 1 (EGR1) and NR3C1 are induced by CMIT exposure, it cannot lead to the upregulation of metal regulatory transcription factor 1 (MTF1), suggesting that MTF1 is a vital transcription factor, along with EGR1 and NR3C1, for the expression of MT1B, MT1F, MT1G, and MT1H
Compared to CMIT, PHMG causes two prime cellular responses. It induces the synthesis of specific MT1 isomers, including MT1B, MT1F, MT1G, and MT1H
Summary
Cell viability was measured using the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) in accordance with the manufacturer’s instructions. HPAEpiCs (1 × 104 cells) were seeded in a 96-well culture plate (SPL Life Science, Gyeonggi, Korea). After treatment with PHMG and CMIT for 12, 24, and 48 h, the HPAEpiCs were incubated with CCK-8 reagent for 2 h. Total RNA was extracted from HPAEpiCs using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA was synthesized using the amfiRivert cDNA Synthesis. Platinum Master Mix (GenDEPOT, Baker, TX, USA) in accordance with the manufacturer’s instructions. Amplification was performed on a thermal cycler (Eppendorf, Hamburg, Germany), and the DNA bands were visualized using a Gel Doc EZ imager (Bio-Rad. Laboratories Inc., Hercules, CA, USA). MT1 primers used in this study were designed according to the process described by Mao et al [21]: MT1B: sense 50 -GATCCCAACTGCTCCTGCACCACA-30 , antisense 50 -AAGAATGTAGCAAACCGGTCAGGGTAGTT-30 ; MT1E: sense 50 -CACTGGTGTGAGCTCCAGCATCC-30 , antisense 50 -AATGCAGCAAATGGCTCAGTGTTGTATTT-30 ; MT1F: sense 50 -GAATGTAGCAAATGGGTCAAGGTG-30 , antisense 50 -TCTCCTGCACCTGCGCTGGT-30 ; MT1G: sense 50 -CAACTGCTCCTGTGCCGCTGG-30 , antisense 50 -TGTAGCAAAGGGGTCAAGATTGTAGC-30 ; MT1H: sense 50 -ATCTGCAAAGGGGCGTCAG-30 , antisense 50 -GAATGTAGCAAATGAGTCGGAGTT-30 ; MT1M: sense 50 -TCCGGGTGGGCCTAGCAGTCG-30 , antisense 50 -AATGCAGCAAATGGCTCAGTATCGTATTG-30 ; MT1X: sense 50 -GACCCCAACTGCTCCTGCTCG-30 , antisense 50 -GATGTAGCAAACGGGTCAGGGTTGTAC-30
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