Effects of Aluminium Chloride and Aluminium Chlorohydrate on DNA Repair in MCF10A Immortalised Non-Transformed Human Breast Epithelial Cells

JIB-09774; No of Pages 4 Journal of Inorganic Biochemistry xxx (2015) xxx–xxx

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Journal of Inorganic Biochemistry

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Effects of aluminium chloride and aluminium chlorohydrate on DNA repair in MCF10A immortalised non-transformed human breast epithelial cells

A. Farasani, P.D. Darbre ⁎

School of Biological Sciences, University of Reading, Reading RG6 6UB, UK article info abstract

Article history: Use of underarm aluminium (Al)-based antiperspirant salts may be a contributory factor in breast cancer Received 5 May 2015 development. At the 10th Keele meeting, Al was reported to cause anchorage-independent growth and double Received in revised form 30 June 2015 strand DNA breaks in MCF10A immortalised non-transformed human breast epithelial cells. We now report Accepted 4 August 2015 that exposure of MCF10A cells to Al chloride or Al chlorohydrate also compromised DNA repair systems. Long- Available online xxxx term (19–21 weeks) exposure to Al chloride or Al chlorohydrate at a 10−4 M concentration resulted in reduced

Keywords: levels of BRCA1 mRNA as determined by real-time RT-PCR and BRCA1 protein as determined by Western Aluminium chloride immunoblotting. Reduced levels of mRNA for other DNA repair genes (BRCA2, CHK1, CHK2, Rad51, ATR) were Aluminium chlorohydrate also observed using real-time RT-PCR. Loss of BRCA1 or BRCA2 gene function has long been associated with Antiperspirant inherited susceptibility to breast cancer but these results suggest that exposure to aluminium-based antiperspi- BRCA1 rant salts may also reduce levels of these key components of DNA repair in breast epithelial cells. If Al can not only BRCA2 damage DNA but also compromise DNA repair systems, then there is the potential for Al to impact on breast Breast cancer carcinogenesis. DNA repair © 2015 Elsevier Inc. All rights reserved. MCF10A cells

1. Introduction instability [21] and the hypothesis that antiperspirant use may contrib- ute to generating a region of genomic instability in the human breast Application of aluminium (Al)-based antiperspirant salts to the [1–4] is worthy of testing. underarm region has been implicated in the rising incidence of breast DNA damage and loss of DNA repair capability are fundamental cancer [1–4]. Breast cancers originate with a disproportionately high components of the mechanisms of carcinogenesis [22] leading to frequency (N50%) in the upper outer quadrant of the breast [5,6] for genomic instability which is a hallmark of cancer cells [23].Alis which an explanation remains lacking other than that there is more known to possess DNA damaging capability [3,24], and at the 10th target epithelial tissue in that region [7,8]. However, coincidentally, Keele meeting, Al was reported specifically to induce double strand this is also the region of the breast to which antiperspirant products breaks in the DNA of MCF10A immortalised, non-transformed human are applied. Al can be absorbed from application of antiperspirant to breast epithelial cells [25] at concentrations measured in nipple aspirate the skin of the underarm [9], and procedures equivalent to shaving fluids [18] and where control experiments showed no mutagenic activ- have been demonstrated to increase dermal absorption [10].Alhas ity in bacteria or similar DNA damage in keratinocytes or fibroblasts been measured in several compartments of the human breast including [25]. Loss of function of genes involved in repair of DNA double strand breast tissue [11–16], breast cyst fluid [17] and nipple aspirate fluid [18]. breaks, most notably BRCA1 and BRCA2, is known to be linked to Genomic instability has been reported in outer regions of human breast inherited susceptibility to breast cancer [26,27] but the increasing pen- tissue compared with inner regions [19,20] which is interesting in the etrance of these genes [28] indicates another environmental component light of the report of Al also being measured at greater levels in as well. In this context, it is of interest to investigate whether Al might human breast tissue (but not the fat) in outer than inner regions of influence expression of these or any other components of the DNA dam- the breast [13]. The cancer field theory, proposed many years ago, age sensing or repair protein complexes. In this paper we describe how suggests that cancer may arise stochastically in regions of genomic long-term exposure of MCF10A human breast epithelial cells to the Al- based antiperspirant salts Al chloride and Al chlorohydrate can result in downregulation of BRCA1 mRNA/protein. We also report downregulation of mRNAs for BRCA2, CHK1, CHK2, Rad51 and ATR which are ⁎ Corresponding author at: School of Biological Sciences, Hopkins Building, University of Reading, Whiteknights, Reading RG6 6UB, UK. other components of the DNA double strand break repair processes E-mail address: [email protected] (P.D. Darbre). associated with increased risk of breast cancer when lost [27,29].

2. Methods instructions. Membranes were blocked and immunoblotted as published [31] but using Tris-buffered saline (TBS) throughout. Primary 2.1. Stock culture of MCF10A human breast epithelial cells antibody to BRCA1 or β-actin (Cell Signaling) was diluted 1/1000 and HRP-linked secondary antibody (rabbit (Cell Signaling)) was diluted MCF10A human breast epithelial cells were purchased from 1/2000. Bands were detected using enhanced chemiluminescence the American Tissue Culture Collection at passage number 98. The (ECL) (GE Healthcare) according to manufacturer's instructions and cells were maintained as monolayer cultures in a medium containing quantitation was performed digitally using the GE ImageQuant 1:1 (v/v) Dulbecco's modified Eagle's medium and Ham's F12 LAS4000mini luminescent image analyser. Band signals were normal- (Invitrogen) supplemented with 5% (v/v) horse serum (Invitrogen), ised relative to digitally quantified total protein using the BioRad 500 ng/ml hydrocortisone (Sigma), 10 μg/ml insulin (Sigma) and stain-free system according to manufacturer instructions. All results 20 ng/ml epidermal growth factor (Sigma) in a humidified atmosphere show the average ± SE (n = 3) of biological replicates generated of 10% carbon dioxide in air at 37 °C. from three independent cell cultures and were analysed for statistical significance using ANOVA Dunnett in Minitab17. 2.2. Long-term growth with Al

Cells were maintained in stock culture medium as above with no 3. Results −4 further addition (control), 10 M Al chloride (AlCl3) (Sigma, UK) or 10−4 M Al chlorohydrate (AlChloro) (Alembic Ltd., UK). Al salts were 3.1. The experimental model made as stock solutions in distilled water and diluted 1 in 1000 (v/v) into culture medium. Controls contained the same volume of distilled MCF10A human breast epithelial cells are an immortalised but water. non-transformed cell line [32] and therefore offer a model system for investigating mechanisms of carcinogenesis [33]. Published re- 2.3. Real-time RT-PCR search has demonstrated that exposure to Al enables anchorage- independent growth of these cells [25], which is a property of epithelial Following long-term (18–20 weeks) growth without or with Al (see cells in vitro acknowledged to be closely correlated with transfor- above), cells were plated at a density of 12.8 × 105 cells/9 cm culture mation in vivo [34]. In the research reported here, Al chloride and Al dish (16 ml), and grown for a further 7 days in stock medium without chlorohydrate were chosen as relevant antiperspirant salts for study or with the respective concentration of Al salt. This enabled cells to be [35]. Since Al has been measured in many samples of human breast harvested (using a rubber policeman) at similar densities after a total tissue [11–18], Al is likely to be present long-term in the human breast exposure time of 19–21 weeks, and whole cell RNA was prepared and so effects were studied over the long-term on expression of DNA using the RNeasy mini kit with a Qiashredder column according to repair genes. A time period of 19–21 weeks was chosen in line with manufacturer instructions (Qiagen). First strand cDNA synthesis was previously published gene expression studies on Al in human breast performed using the QuantiTect reverse transcription kit (Qiagen) and cells [36] and mRNA/protein samples were harvested from indepen- polymerase chain reactions were carried out using the QuantiTect dent cell cultures after 19, 20 and 21 weeks to ensure biological repli- SYBER Green PCR kit (Qiagen) together with BRCA1 (QT00039305), cates. The concentration of 10−4 M Al was chosen for study as the BRCA2 (QT00008449), CHK1 (QT00006734), CHK2 (QT01016155), highest concentration of Al which had previously been shown to have Rad51 (QT00072688), ATR (QT00030779) or β-actin (QT01680476) no detrimental effect on proliferation of human breast cells in the QuantiTect primers (Qiagen) according to manufacturer protocols. long term [3].

Quantitative values were obtained from the threshold cycle (CT)number at which the exponential increase in ﬂuorescent signal from the PCR product was at the mid-point (50%). All reactions were performed 3.2. Effect of Al on levels of BRCA1 mRNA and protein in MCF10A cells in triplicate. The CT number for each gene target (BRCA1, BRCA2, CHK1, CHK2, Rad51, ATR) was normalised to the CT number for the Effects of long-term exposure to Al were investigated on levels corresponding β-actin reaction and values from cells grown with Al of BRCA1 mRNA and BRCA1 protein. Biological replicate cell lysates normalised to control values of cells grown without Al using the were prepared after 19, 20 and 21 weeks of exposure to 10−4 MAlchlo- −ΔΔC −4 2 T method of relative gene expression analysis [30].Averagevalues ride or 10 M Al chlorohydrate. Using real-time RT-PCR, reduced levels for the triplicate reactions were then calculated. This was then repeated of BRCA1 mRNA were observed after the long-term exposure to 10−4 M for each of the three biological replicates generated from independent Al chloride (P b 0.001) or 10−4 M Al chlorohydrate (P b 0.001) (Fig. 1). cell cultures after 19, 20 or 21 weeks of culture, and results presented Western immunoblotting showed that levels of BRCA1 protein were show the average ± SE (n = 3) of the three biological replicates. also reduced after long-term exposure to aluminium and an example −ΔΔC According to the 2 T method, results are presented relative to the of a Western immunoblot after 20 weeks is shown in Fig. 2A. Calculated control value of 1.0 for cells grown in the absence of Al. Statistically averages of biological replicates (19, 20, 21 weeks) normalised to total signiﬁcant differences were determined using ANOVA Dunnett in protein showed that levels of BRCA1 protein were reduced following Minitab17. long-term exposure to 10−4 M Al chloride (P = 0.043) or 10−4 MAl chlorohydrate (P b 0.008) (Fig. 2B). 2.4. Western immunoblotting

Cells were grown as for RT-PCR and lysates prepared as de- 3.3. Effect of Al on expression of mRNAs for other DNA repair proteins in scribed previously [31] but using lysis buffer [50 mM Tris–HCl pH 7.4, MCF10A cells 250 mM NaCl, 5 mM EDTA, 0.3% (v/v) Triton-X-100, 4-(2- aminoethyl)benzenesulfonyl ﬂuoride (AEBSF 0.3 mM), leupeptin Effects of long-term exposure to 10−4 M Al chloride or 10−4 MAl (10 μg/ml) and aprotonin (2 μg/ml)] (1 μl buffer/105 cells). Lysates chlorohydrate were then investigated on levels of mRNA coding for were incubated on ice 30 min, passed through needles 19G–25G, run other DNA repair proteins. Using real-time RT-PCR and the same collect- on 10% SDS-PAGE BioRad stain-free gels (25 μg protein per track) ed samples as above, levels of BRCA2 mRNA, CHK1 mRNA, CHK2 mRNA, and transferred onto BioRad PVDF membranes using the BioRad Rad51 mRNA and ATR mRNA were also all reduced following long-term Trans-Blot-Turbo semi-dry transfer system according to manufacturer's exposure to the Al salts (Fig. 3).

Fig. 1. Effects of long-term exposure to Al on levels of BRCA1 mRNA in MCF10A human Fig. 3. Effects of long-term exposure to Al on levels of mRNA for BRCA2, CHK1, CHK2, breast epithelial cells as determined using real-time RT-PCR. Cells were assayed after Rad51 and ATR in MCF10A human breast epithelial cells as determined using real- – −4 −4 19 21 weeks with no aluminium, with 10 MAlchloride(AlCl3), or with 10 MAl time RT-PCR. Cells were assayed after 19–21 weeks with no aluminium, with chlorohydrate (AlChloro). For each RT-PCR assay, BRCA1 values were normalised to −4 −4 10 MAlchloride(AlCl3), or with 10 M Al chlorohydrate (AlChloro). For each β-actin values, expressed as fold change from control cells grown without Al by the β −ΔΔ RT-PCR assay, values for each target gene were normalised to -actin values, C −ΔΔ 2 T method and the average of triplicate technical replicates calculated. Results C expressed as fold change from control cells grown without Al by the 2 T method shown are the average ± SE of biological replicate assays (n = 3) taken from cells and the average of triplicate technical replicates calculated. Results shown are the grown for 19, 20 or 21 weeks of culture. Fold change for cells grown with AlCl3 average ± SE of biological replicate assays (n = 3) taken from cells grown for 19, (dark grey bars) or Al Chlor (light grey bars) compared to control cells grown without 20 or 21 weeks of culture. Fold change for cells grown with AlCl (dark grey bars) b 3 Al (relative value 1.0). **P 0.001 from control without Al by ANOVA Dunnett. or Al Chlor (light grey bars) compared to control cells grown without Al (relative value 1.0). **P b 0.001, *P b 0.05 from control without Al by ANOVA Dunnett. 4. Discussion

These results demonstrate that long-term exposure to Al causes a re- risk factor for susceptibility to breast cancer development [26,27,29] duction in cellular levels of BRCA1 mRNA and BRCA1 protein in MCF10A but this is the first report that BRCA1 gene expression can also be immortalised non-transformed human breast epithelial cells. Inheri- reduced by long-term exposure to Al in cultured breast epithelial cells. tance of loss of function of the BRCA1 gene, which encodes a protein The finding also of downregulated mRNAs for BRCA2, CHK1, CHK2, involved in homology directed repair of DNA, is well established as a Rad51 and ATR indicates a more generalised reduction in DNA repair processes within the cells after long-term exposure to Al but this requires further validation at a protein level. Repair of damaged DNA is essential to the maintenance of genomic integrity, and impaired processes for sensing or repair of damaged DNA are associated with development of genomic instability which is an enabling characteristic of cancer cells [23]. Although the Al concentration of 10−4 M used in these experiments might be considered as high, it is not out of line with measurements of Al in human breast tissues which are also high. Levels of Al in human breast tissue have been reported as ranging from 4–437 nmol/g dry weight [13]: assuming tissue is about 80% water and 1 g of tissue has a volume of 1 ml, these concentrations equate to 0.80 × 10−6 M– 0.87 × 10−4 M. Levels in nipple aspirate fluids have been measured as ranging from 150–520 μg/l (mean 268.4 ± 28.1 μg/l) [18] which equate to concentrations of 0.56–1.93 × 10−5 M. Levels of Al have been reported in type I breast cyst fluids as ranging from 80–330 μg/l (median 150 μg/l) [17] which equate to concentrations of 0.30–1.22 × 10−5 M. This shows that the concentrations of Al used in this study could be reached in some human breast tissues and therefore that this study has biological relevance. However, there is clearly a need to now repeat these time courses using lower concentrations of Al in order to complete a fuller understanding of effects across the whole range of concentrations measured in human breast structures. Since the MCF10 immortalised non-transformed human breast epithelial cell lines have been shown to be a relevant model system for the study of breast carcinogenesis [32,33], validation of the findings

Fig. 2. Effects of long-term exposure to Al on levels of BRCA1 protein in MCF10A human in the human breast in vivo is a next challenge. A correlation in vivo is breast epithelial cells as determined using Western immunoblotting. Cells were assayed supported from previously published results documenting higher levels −4 −4 after 19–21 weeks with no aluminium, with 10 MAlchloride(AlCl3), or with 10 M of Al in outer than inner regions of the breast [13] where there are Al chlorohydrate (AlChloro). Protein was loaded at 25 μg/track. A. Example of a Western reported increases in genomic instability in breast tissue [19,20]. The β immunoblot for BRCA1 and -actin after 20 weeks. B. Quantitated BRCA1 protein was cancer ﬁeld theory [21] hypothesises that cancer arises stochastically normalised to total protein using BioRad stain-free technology. Results are the average ± SE of the biological triplicate experiments at 19, 20 and 21 weeks. *P b 0.05 in an area of genetically unstable cells and it has long been known from control without Al by ANOVA Dunnett. that breast cancer arises with a disproportionately high incidence in

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