In vivo epigenetic effects induced by engineered nanomaterials: A case
study of copper oxide and laser printer-emitted engineered nanoparticles
Xiaoyan Lu1,†, Isabelle R. Miousse2,†, Sandra V. Pirela1, Jodene K. Moore3, Stepan Melnyk4, Igor
Koturbash2*, Philip Demokritou1*
1
Center for Nanotechnology and Nanotoxicology, Department of Environmental Health, Harvard
T. H. Chan School of Public Health, Boston, MA 02115, USA
2
Department of Environmental and Occupational Health, College of Public Health, University of
Arkansas for Medical Sciences, Little Rock, AR 72205, USA
3
Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA
4
Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72205,
USA
†
Xiaoyan Lu and Isabelle R. Miousse contributed equally to this work.
*
Corresponding authors:
Philip Demokritou, Department of Environmental Health, Harvard T. H. Chan School of Public
Health, 665 Huntington Avenue, Boston, MA 02115, USA. Tel: +1 6174323481. E-mail:
pdemokri@hsph.harvard.edu
Igor Koturbash, Department of Environmental and Occupational Health, College of Public
Health, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. Tel: +1
5015266638. Email: IKoturbash@uams.edu
Supplementary Materials
Supplementary Methods
Analysis of 5-Methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) levels
RNaseA (Sigma, St. Louis, MO) was added to 1 μg of genomic DNA to a final concentration of
0.02 mg/mL and incubated at 37°C for 15 min. The purified DNA was digested into component
nucleotides using Nuclease P1, snake venom phosphodiesterase, and alkaline phosphatase as
previously described (James et al. 2010). Briefly, DNA was denatured by heating for 3 min at
100°C and rapidly chilled in an iced-water bath. One-tenth volume of 0.1 M ammonium acetate,
pH 5.3, was added to 2 units of nuclease P1 (Sigma) for every 0.5 A260 unit of DNA and the
mixture incubated at 45°C for 2 h. Subsequently, 1/10 volume of 1 M ammonium bicarbonate
and 0.002 units of venom phosphodiesterase I (Sigma) were added and the mixture incubated for
2 h at 37°C. To the mixture, 0.5 units of alkaline phosphatase (Sigma) were then added and the
incubation continued for an additional hour. The digested nucleotides were stored at −20°C until
liquid chromatography tandem mass spectrometry (LC/MS/MS) analysis. Base separation was
performed with a Dionex HPLC system (Thermo Scientific, Waltham, MA) coupled to an
electrospray ionization (ESI) tandem mass spectrometer (Thermo-Finnigan LCQ) using a
Phenomenex Gemini column (C18, 150 x 2.0 mm, 3 μm particle size) and established
methodology (Friso et al. 2002).
Analysis of methylation status of TEs
Methylation at the LINE1 and SINE B1 elements was assessed by methylation-sensitive
quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Genomic DNA was treated with
five methylation-sensitive restriction enzymes (New England Biolabs, Ipswitch, MA). Briefly,
500 ng of gDNA was treated with 0.5 U of SmaI enzyme in 1 × CutSmart buffer and incubated
at 25°C for 2 h. Then, 0.5 U of the enzymes HpaII, HhaI, and AciI in 1 × CutSmart buffer was
added to the mix and incubated at 37°C for 16 h. Finally, 0.5 U of BstUI enzyme in 1 ×
CutSmart buffer was added, and the mix was incubated at 60°C for 4 h. The resulting digested
DNA was measured by qRT-PCR using 2 ng digested DNA per reaction and SYBR Select
chemistry (Life Technologies, Carlsbad, CA). DNA samples 1) not digested with the restriction
enzyme mix and 2) RAW 264.7-derived DNA pre-treated with 5-azadeoxycytidine, a potent
demethylating agent, served as a positive control, while samples lacking the specific primers for
DNA amplification, and/or DNA template served as a negative control.
Supplementary Table 1. The primers for analysis of methylation status of DNA repetitive elements
Repetitive elements
Mus musculus
LINE-1 5’UTR
LINE-1 ORF1
LINE-1 ORF2
LINE-1 3’UTR
SINE B1
rDNA(for
normalization of
methylation)
Forward Primer
Reverse Primer
AGTGGATCACAGTGCCTGC
GAACACAAATGCCAGCCCAG
CTATCTACAAATCCAGCCCTACAG
GGCTTGTACAACCACTCTGGA
CGAGATGGCTCAGTGGGTAA
CCTGTGAATTCTCTGAACTC
GGGTAGCCTGCTTCCCTATG
TGTGAACTTGGTTTTGTCGTGG
CTTTTGATTTGCTGGTCTGGG
TCTGGATATATGCCCAGGAGAG
GTGTGTGTGTGTGTGTGCG
CCTAAACTGCTGACAGGGTG
Supplementary Table 2. Gene-specific primers used for qRT-PCR amplification
Gene Symbol
Dnmt1
Dnmt3a
Dnmt3b
Gapdh(control
gene)
Hmox1
Tet1
*GenBank mRNA
Assay Name
Mm01151063_m1
Mm00432881
Mm01240113_m1
Mm99999915_g1
RefSeq #
NM_001199431.1
NM_007872.4
AF068626.2*
NM_008084.2
Source
Life Technologies
Life Technologies
Life Technologies
Life Technologies
Mm.PT.56a.8600055
Mm01169087_m1
NM_010442
NM_001253857.1
Integrated DNA Technologies
Life Technologies
Supplementary Table 3. TEs expression primers used for qRT-PCR amplification
Gene Symbol
Mus musculus
LINE-1 5'UTR
SINE B1
Rps29(control gene)
Forward Primer
Reverse Primer
AGTGGATCACAGTGCCTGC
GTGGCGCACGCCTTTAATC
GGAGTCACCCACGGAAGT
GGGTAGCCTGCTTCCCTATG
GACAGGGTTTCTCTGTGTAG
TCCATTCAAGGTCGCTTAGTC
Supplementary Table 4. In vitro and in vivo dosimetric considerations
ENMs
CuO
PEPs
Intratracheal
instillation
dose
(mg/kg body
weight)
Intratracheal
instillation
mass per
alveolar
surface areaa
(μg/cm2)
Cell
delivered
in vitro
mass doseb
(µg)
2.5
2.5
0.76
0.76
59.7
59.7
Cell administered in
vitro mass dosec
(µg)
Cell administered in vitro
volumetric dosed
(µg/mL)
DMEM/10%FBS
(RAW264.7 cells)
SAGM
(SAEC
cells)
DMEM/10%FBS
(RAW264.7 cells)
SAGM
(SAEC
cells)
59.7
64.2
59.7
59.7
7.5
8.0
7.5
7.5
a
The average mouse weight used in this study was 25 g and the alveolar surface area of adult mouse is 82.2 cm 2
(Knust et al., 2009). Thus, a 2.5 mg/kg body weight instillation dose was equal to a total particle mass of 62.5 μg
instilled to each mouse, resulting to a 0.76 μg/cm2 intratracheal instillation mass per alveolar surface area.
b
Calculation of the corresponding cell delivered in vitro mass dose to the in vivo intratracheal instillation dose was
based on the intratracheal instillation mass per alveolar surface area (0.76 µg/cm2) and the surface area of 100 mm
diameter dish (78.5 cm2) according to previous studies (Kroll et al., 2011; Landsiedel et al., 2014). Thus, the
equivalent cell delivered in vitro mass dose was calculated to be 59.7 μg.
c
The conversion of the cell delivered to administered mass dose for a 24 h in vitro exposure was performed using the
Harvard dosimetric platform (Cohen et al., 2014). In summary, the fD was found to be 1 for CuO in both
DMEM/10%FBS and SAGM. For PEPs, the fD was 1 in SAGM and 0.93 in DMEM/10%FBS. Thus, cell
administered in vitro mass dose was calculated by the cell delivered in vitro mass dose divided by specific fD.
d
The cell administered in vitro volumetric dose was based on a 100 mm diameter dish and 8 mL of media volume.
References
Cohen JM, Teeguarden JG, Demokritou P. 2014. An integrated approach for the in vitro dosimetry of engineered
nanomaterials. Part Fibre Toxicol 11:20.
Friso S, Choi SW, Dolnikowski GG, Selhub J. 2002. A method to assess genomic DNA methylation using highperformance liquid chromatography/electrospray ionization mass spectrometry. Anal Chem 74:4526-4531.
James SJ, Melnyk S, Jernigan S, Pavliv O, Trusty T, Lehman S, et al. 2010. A functional polymorphism in the
reduced folate carrier gene and DNA hypomethylation in mothers of children with autism. Am J Med Genet B
Neuropsychiatr Genet 153B:1209-1220.
Knust J, Ochs M, Gundersen HJ, Nyengaard JR. 2009. Stereological estimates of alveolar number and size and
capillary length and surface area in mice lungs. Anat Rec (Hoboken) 292:113-122.
Kroll A, Dierker C, Rommel C, Hahn D, Wohlleben W, Schulze-Isfort C, et al. 2011. Cytotoxicity screening of 23
engineered nanomaterials using a test matrix of ten cell lines and three different assays. Part Fibre Toxicol 8:9.
Landsiedel R, Sauer UG, Ma-Hock L, Schnekenburger J, Wiemann M. 2014. Pulmonary toxicity of nanomaterials: a
critical comparison of published in vitro assays and in vivo inhalation or instillation studies. Nanomedicine
(Lond) 9:2557-2585.
Lu X, Miousse IR, Pirela SV, Melnyk S, Koturbash I, Demokritou P. 2015. Short-term exposure to engineered
nanomaterials affects cellular epigenome. Nanotoxicology May 4:1-11. [Epub ahead of print].doi:
10.3109/17435390.2015.1025115.