Elsevier

Toxicology

Volume 265, Issues 1–2, 9 November 2009, Pages 10-14
Toxicology

Correlations and co-localizations of Hsp70 with XPA, XPG in human bronchial epithelia cells exposed to benzo[a]pyrene

https://doi.org/10.1016/j.tox.2009.09.001Get rights and content

Abstract

Benzo[a]pyrene (BaP) is a ubiquitously distributed environmental pollutant known to cause DNA damage, which may be repaired through nucleotide excision repair (NER). The significantly negative correlation between Hsp70 levels and the level of DNA damage in workers exposed to coke oven emission had been found. However, little is known about how Hsp70 modulate the DNA repair process. In a series of experiments using the human bronchial epithelia cells (16HBE) exposed to different concentrations of BaP for 24 h, we measured expression of NER subunit xeroderma pigmentosum (XP) group A, C, F, G (XPA, XPC, XPF, XPG), excision repair cross-complementing 1 (ERCC1) and Hsp70, and analyzed their possible correlations. Co-localizations of Hsp70 with NER subunit were detected by confocal microscope. We found that in vitro exposure to BaP reduced cell viability in a dose-dependent manner ranging from 2 to 64 μM. Our results showed that levels of XPA, XPG and Hsp70 significantly increased at cells exposed to 1 or 2 μM BaP. In addition, curve estimation showed there was a significant correlation between relative ratios of Hsp70 and XPA, XPG in cells exposed to different concentrations of BaP. Moreover, confocal microscopy demonstrated increased co-localization of Hsp70 with XPA, XPG in nuclei of cells exposed to BaP. These results suggested that Hsp70 might play a role in nucleotide excision repair. However, the mechanisms underlying this observation need further investigation.

Introduction

Heat shock protein 70 (Hsp70) is highly conserved among both prokaryotes and eukaryotes, and acts as chaperones in diverse cellular processes (Georgopoulos and Welch, 1993, Hendrick and Hartl, 1993, Lindquist and Craig, 1988). Induced Hsp70 protects cells, tissues, and organisms against damage from a wide variety of stressful stimuli (Hightower, 1991, Niu et al., 2006, Wu et al., 1996, Wu et al., 2001). Hsp70 can translocate to the nucleus and accumulate there under heat shock or in other harmful conditions (Chughtai et al., 2001, Lepock et al., 2001, Szekely et al., 1995). However, the potential role of Hsp70 in the regulation of the response to some carcinogenic reagents, such as benzo[a]pyrene (BaP), remains unclear.

BaP can results in DNA lesion by electrophilic metabolites as (t)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE) and reactive oxygen species (ROS) (Kim et al., 2005, Xue and Warshawsky, 2005). BPDE strongly reacts with the N-2 position of guanine in DNA to form anti-BPDE adducts. BaP-DNA adducts can be removed by nucleotide excision repair (NER) (Pavanello and Clonfero, 2004). NER is a multistep process involving more than 25 polypeptides, including an initial damage recognition step, an incision to either side of the lesion, the removal of the damage-containing oligonucleotide, DNA repair synthesis and final reseal of the strand by a DNA ligase (Wood, 1996). XPA to G (xeroderma pigmentosum group A to G) and ERCC1 (excision repair cross complementation group 1) are the main proteins in this pathway. Some researches related to Escherichia coli supported the involvement of DnaK (Hsp70) in NER (Chow and Tung, 2000, Zou et al., 1998). However, what kind of role of Hsp70 may play in this process in eukaryote remains unknowed. Two previous studies in our lab found that Hsp70 inversely correlated to residual DNA damage, hinting that Hsp70 might be involved in BaP repair process (Gao et al., 2004, Xiao et al., 2002). So we supposed that there may be some NER subunits through which Hsp70 modulate the DNA repair process.

A large number of methods have been developed over the years to study protein–protein interactions. Co-localization is defined as the presence of two or more different molecules residing at the same physical location in a cell. If proteins interact in vivo they are expected to be co-localized or, at least, they will display overlapping distribution within the cell. The intracellular localization of two (or more) proteins can be studied by confocal microscopy. In this paper, we used immortalized human bronchial epithelial cells (16HBE) as experimental model to investigate the correlations between levels of Hsp70 and NER subunit and analysis the role of Hsp70 in NER process in cells exposed to BaP.

Section snippets

Chemicals

BaP (98% purity) was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco modified Eagle minimal essential medium (DMEM), antibiotic, trypsin and fetal calf serums (FCS) were purchased from GIBCO (Grand Island, NY, USA). Nitrocellulose membranes were obtained from Bio-Rad (Hercules, CA, USA). X-ray film for autoradiography was from Kodak (Rochester, NY, USA). Ethylenediaminetetra-acetic acid (EDTA), glycine, Tris base, sodium dodecyl sulfate (SDS), tetramethylethylenediamine (TEMED)

Cell viability of 16HBE cells exposed to BaP

After 24 h incubations, the viability of cells exposed to BaP decreased from 2 to 64 μM BaP and 18% at 64 μM BaP. Compared with the control group, the viability of the treated 16HBE cells decreased significantly at 8 μM (P < 0.05) and 16, 32 and 64 μM (P < 0.01) of BaP treatment (Table 1). There was a significant trend in decreased cell viability and increased dose of BaP treatment (Ptrend = 0.013), showing a clear dose-response relationship.

Changes in levels of XPA, XPC, XPF, XPG, ERCC1 and Hsp70 protein expression in 16HBE cells treated by BaP

Fig. 1 shows protein expression patterns of XPA, XPC, XPF, XPG,

Discussion

Various functions have been attributed to Hsp70 not only in protein folding, protein translocation through membranes, and protein degradation but also in the translation machinery in normal and stressed states (Nelson et al., 1992), which could be very important for the damage of cells caused by BaP. Hsp70 also shares ATPase activities that are thought to be necessary for the release of associated peptides (Mayer and Bukau, 2005, Shomura et al., 2005). Hsp70 can inhibit apoptosis by modulating

Conflict of interest

The authors declare they have no competing financial interests.

Acknowledgment

This work was supported by research funds from the National Nature Science Foundation of China (NNSFC 30525031).

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