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Transcriptome analysis of Eisenia fetida chronically exposed to benzo(a)pyrene
Accepted Manuscript Transcriptome analysis of Eisenia fetida chronically exposed to benzo(a)pyrene Srinithi Mayilswami, Kannan Krishnan, Ravi Naidu, Mallavarapu Megharaj PII: DOI: Reference:
S2352-1864(16)30174-2 http://dx.doi.org/10.1016/j.eti.2016.12.002 ETI 101
To appear in:
Environmental Technology & Innovation
Received date: 14 June 2016 Revised date: 7 December 2016 Accepted date: 13 December 2016 Please cite this article as: Mayilswami, S., Krishnan, K., Naidu, R., Megharaj, M., Transcriptome analysis of Eisenia fetida chronically exposed to benzo(a)pyrene. Environmental Technology & Innovation (2016), http://dx.doi.org/10.1016/j.eti.2016.12.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Highlights (for review)
Highlights Eisenia fetida chronically exposed in soil with 10 mg/kg of BaP. mRNA was isolated and sequenced from BaP exposed Eisenia fetida. The transcriptomes were in silico assembled from mRNA sequences Genes involved in calcium homeostasis, apoptosis and lipid metabolism are altered
Graphical Abstract
• Eisenia fetida exposed to 10 mg Kg-1 of BaP for 8 m
• mRNA isolation and sequencin
• Transcriptome assembly
• Identification of differential expressed transcripts
Apoptotic, reproduction related, lipid metabolism development related genes are altere
*Revised Manuscript with No Changes Marked
1 2 3
Transcriptome Analysis of Eisenia fetida Chronically Exposed to Benzo(a)Pyrene Srinithi Mayilswami2, 3, Kannan Krishnan1, 2*, Ravi Naidu1, 2, Mallavarapu Megharaj1, 2
4
1
5
Technology, The University of Newcastle, Callaghan NSW 2308, Australia
6
2
7
Environment (CRC-CARE), Mawson Lakes, Adelaide SA 5095, Australia
8
3
9
Mawson Lakes, Adelaide SA 5095, Australia
Global Centre for Environmental Research, Faculty of Science and Information
Cooperative Research Centre for Contamination Assessment and Remediation of the
Centre for Environmental Risk Assessment and Remediation, University of South Australia,
10 11
*e-mail: [email protected],
12
Phone: +61 2 4913 8732.
13
Key words: differential gene expression, Transcriptome assesmbly, Molecular markers,
14
Polycyclic aromaric hydrocarbons
15
Abstract
16
Benzo(a)pyrene is a high molecular weight polycyclic aromatic hydrocarbon which is
17
carcinogenic and widespread pollutant in the environment. It is essential to identify the
18
presence of this chemical in soil as it is toxic to biota including humans. Eisenia fetida is a
19
sentinal organism in soil which can be used to diagnose the health of the soil. In order to
20
identify potential molecular markers from Eisenia fetida to diagnose the presence of benzo(a)
21
pyrene in soil, we exposed the organism to sub-lethal (10 mg Kg-1) concentrations for a
22
period of eight months and carried out transcriptome anaysis. From the transcriptome, we
23
have identified differentially expressed genes. Results showed that benzo(a)pyrene has
24
altered the expression of calcium binding and calcium homeostasis, apoptotic process,
25
cytoskeletal proteins, protein transport, nucleotide binding, lipid metabolism, peripheral 1
26
neuronal development, cell division, wound healing and nucleotide binding and processing
27
genes at transcription level. Several of the genes we reported here were not reported earlier.
28
The highly up regulated and down regulated genes could be used as a molecular marker to
29
diagnose the presence of benzo(a)pyrene in the soil.
30 31
Key words: Transcriptome assembly, differential gene expression, toxicogenomics,
32
molecular markers, polycyclic aromaric hydrocarbons
2
33
1 Introduction
34
Benzo(a)pyrene (BaP) is one of the polycyclic aromatic hydrocarbons (PAHs) which is
35
an established mutagen and carcinogen. PAHs including BaP are formed during incomplete
36
combustion of fossil fuels and organic material (Yunker et al., 2002). Several processes
37
including automobile exhaust, industrial emission, agriculture activities and domestic
38
emission and waste contribute to the release ofBaP in to the environment (Ravindra et al.,
39
2008). BaP induces several enzymes with mixed function in the cell (Gelboin, 1980) and as a
40
result, different type of cancer progression occurs in human and animals. BaP has been
41
shown to accumulate in several organisms such as mussels (Canova et al., 1998) microalgae
42
(Subashchandrabose et al., 2014), and mouse (Uno et al., 2006). Benzo(a)pyrene has also
43
been established as a mutagen in somatic cells. By using specific locus test for visible
44
markers, it could not be concluded that point mutation by BaP could be inherited (Russell et
45
al., 1981), however, the dominant lethal mutations induced by BaP in post-meiotic germ cells
46
were found to be inherited (Generoso et al., 1982). Moreover, from environmentally exposed
47
animals, there are evidences that PAHs are also mutagenic in male germ cells which leads to
48
potential health risks in the offspring (Somers et al., 2004; Somers et al., 2002; Yauk et al.,
49
2000; Yauk and Quinn, 1996).
50 51
Benzo(a)pyrene binds to aryl hydrocarbon receptor (AhR), the transcription regulator
52
and the ligand bound AhR binds to xenobiotic responsive elements (XRE) thereby causing
53
biological effects (Nebert et al., 2000). BaP is enzymatically converted by CYP1A1,
54
CYP1B1 and epoxy hydrolase into BaP-diol epoxide that forms Nucleotide-BaP-adducts that
55
is incorporated into DNA (Kim et al., 2007; Shi et al., 2009). These adducts prevents the
56
DNA polymerase from moving along the DNA during replication (Hsu et al., 2005) that
57
causes immature termination of DNA synthesis. As a result, the regulation of genes will be 3
58
defective and cellular metabolism will be abnormal, which leads to cell proliferation defects
59
and apoptosis (Solhaug et al., 2004).
60 61
To diagnose the presences of sub lethal concentrations of BaP in aquatic system and
62
in soil there are several biochemical based markers (Gastaldi et al., 2007), and specific genes
63
as molecular markers (Zheng et al., 2008). However, due to the limitations with the
64
traditional techniques, it is possible that some potential toxic effects might be neglected.
65
Global transcriptome analysis of the effect of a chemical might reveal both mechanism of
66
toxicity as well as general toxic effects (Huang et al., 2014). It is appropriate to use a soil
67
organism such as Eisenia fetida for developing a molecular marker to monitor BaP presence
68
in the soil.
69 70
Soil is considered to be one of the largest sinks for BaP and hence, it is essential to
71
use soil organism such as Eisenia fetida to develop molecular biomarkers by studying the
72
transcriptome profile change and differential gene expression in the presence and absence of
73
BaP. The alteration in transcript level would be a powerful indicator to diagnose the effects
74
of a toxic pollutant such as BaP at sub-lethal levels. Advancement in mRNA sequencing
75
techniques such as Next generation mRNA sequencing is one of the tools that can be used to
76
analyse such changes in transcriptome expression profiles. In this paper we report the change
77
in transcriptome expression in E. fetida chronically exposed to sub-lethal levels of BaP for
78
about eight months.
79
80
2 Materials and Methods
81
2.1
Chemicals and earthworm treatment
82 4
83
Benzo(a)pyrene was purchased from Sigma-Aldrich (Australia). Eisenia fetida was
84
maintained in natural soil added with fruits and vegetable waste, at 25 ± 1 °C, 60 to 80%
85
humidity and a 16:8 h L: D (Light: dark) cycle in our laboratory. E. fetida with wet weight of
86
0.5 g and well-developed clitellum were used for the chronic toxicity studies.
87
88
2.2
Experimental Design
89 90
E. fetida was introduced into soil (pH ~ 6.5) for the transcriptome analysis followed by
91
differential gene expression studies. The soil sample was collected from Adelaide region, air
92
dried for 24 hours and sieved using a 2 mm sieve. Earthworm assay was carried out using 10
93
mg Kg-1 of BaP; controls were maintained simultaneously. The concentration of BaP in 9
94
manufactured gas plant (MGP) site soils ranged between 58 and 738 mg Kg-1 soil [21].
95
Hence, the environmentally relevant concentration for chronic exposure study was selected as
96
10 mg Kg-1. BaP was dissolved in acetone and mixed with the soil using an end-to-end shaker
97
overnight to artificially contaminate the soil for the study and subsequently the earthworms
98
were introduced into the soil. 50 g of soil was mixed with required amount of BaP dissolved
99
in acetone and later they were mixed with the remaining 1950 g of soil (in total 2000 g)
100
which ensures homogenisation of spiking. The solvent was evaporated from the soil in a
101
fume hood. The soils were moistened with water so as to obtain 50 to 60% by mass of water
102
holding capacity and the earthworms were released into it. Ten worms per group in triplicates
103
were released in the containers. The containers were supplemented with powdered pulses and
104
about 40 g of fruits and vegetable every week. This procedure was followed for eight months
105
and simultaneously control group was also maintained under the same condition.
106
5
107
2.3
RNA Isolation and next generation sequencing
108 109
E. fetida treated as well as control were collected, placed into a 15 ml tube with
110
suspension buffer QIAGEN® Mini Kit (Cat No: 74104) and homogenized using Polytron
111
homogenizer. From the homogenized E. fetida total RNA was isolated using QIAGEN® Mini
112
Kit using manufacturer’s protocol. From the Qiagen column, total RNA was eluted using
113
elusion buffer and stored in -80 °C and transported to sequencing facility on dry ice. Using
114
Illumina HiSeq 2000, the paired-end RNA sequencing was carried out at The Ramaciotti
115
Centre for Gene Function Analysis. The sequencing data is submitted to Sequence Read
116
Archive (SRA), National Center for Biotechnology Information (NCBI) (Accession:
117
SRS1828175, SRS1827215).
118
119
2.4
Sequence analysis
120 121
The forward and reverse mRNA sequence reads were joined together and transcriptome
122
assembly was carried out using Trinity software (Grabherr et al., 2011). Trinity software was
123
installed in bigmem-1024 server, eRSA, Adelaide. The transcriptome assembly was carried
124
with parameters, k-mers was set at 2 and glue length was set at 4. The transcripts with longer
125
than 130 amino acid length were selected and annotated using BLAST (NCBI-BLAST-
126
2.2.28+) with UniProt database. The transcripts were compared using NPKF- normalized
127
counts and the transcripts that are more than fourfold altered (p = 0.001) in expression were
128
selected.
6
129
3 Results and Discussion
130
3.1
Transcript assembly and annotation
131 132
The mRNA from E. fetida was sequenced by next generation sequencing and de novo
133
assembled using Trinity software installed in Bigmem-1024 server, at eRSA, Adelaide. The
134
transcripts obtained from de novo assembly were translated in silico and the best possible
135
ORFs were carefully chosen. The total number of peptides that were translated (ORFs) from
136
the de novo assembled transcripts were 616110. These translated peptides contain all the
137
possible trans-spliced isoforms. These peptides were subject to homology search using
138
BLAST where 106161 peptides retrieved hits. Apart from the peptides that retrieved
139
homologous results, 3830 peptides retrieved homologues signal peptides and 8525 TmHMM
140
topology results and 33064 did not retrieve any result. The transcripts that were longer that
141
130 amino acids obtained from the database (UniProt) were analysed further (Mayilswami et
142
al., 2014).
143
3.2
Differential gene expression
144 145
About 2000 genes were recognized as differentially expressed with more than four-fold
146
difference compared to control. The differentially expressed genes with their fold difference
147
was subjected to gene cluster analysis (Fig. 1). The gene expression fold difference were
148
plotted in MA-plot (Fig 2). Genes that are differentially expressed more than 12-fold (p <
149
0.001) were selected for further analysis. About 223 differentially expressed transcripts were
150
retrieved. The genes with more than one isoforms and genes with no known functions were
151
removed from analysis. Among these differentially expressed genes, 63 were up-regulated
152
and 56 down-regulated. Based on the functional information obtained from UniProt, the
153
toxicity was interpreted and grouped according to functions. 7
154
155
3.3
Calcium binding and homeostasis
156 157
The transcripts that are altered in the presence of BaP are given in Tables 1 to 5. BaP
158
has been shown to cause Ca2+ elevation in human mammary epithelial cells at 2 hours and
159
sustained alterations in Ca2+ homeostasis at 18 hours (Tannheimer et al., 1997). MATN3 has
160
been shown to be down regulated at transcriptome level in HepG2 cells exposed to BaP
161
(Magkoufopoulou et al., 2011) which is the case in Eisenia fetida as well. However, a
162
contradictory result has also been reported (Lizarraga et al., 2012). Altered expression of
163
collagen (Hussain et al., 1979) and basement membrane perforation has been observed
164
(Kopf-Maier and Flug, 1996). In mice liver, BaP induces PRDX6 expression at mRNA level
165
(Halappanavar et al., 2011), however, it has been shown to be down regulated in Solea
166
senegalensis live at protein level (Costa et al., 2010). So far the toxicological studies have
167
suggested that BaP affects calcium homeostasis which is confirmed by transcriptome study
168
and we have identified possible genes that could be involved in this process.
169
8
170 171 172
Figure 1. Hierarchical clustering analysis of gene expression profiles control (C) and
173
benzo(a)pyrene (BaP) treated Eisenia fetida. The fold difference was set at 2 and the
174
significance (p) was set as 0.001.
175
9
176 177 178
Figure 2. Differential expression of chronic benzo(a)pyrene exposed (10 mg Kg-1 in soil)
179
versus control Eisenia fetida. MA-plot with log counts on x-axis, log fold-change on y-axis
180
showing differentially expressed genes (P value < 0.001) as red dots.
181 182 183 184 185 186 187 188
10
189
Table 1. The transcripts of calcium ion binding protein that are altered by BaP exposure in
190
Eisenia fetida Normalized UniProt_ID
gene
Function and cellular process
expression BMP1_MOUSE
-60.97
Calcium ion binding, cell differentiation Sarcoplasmic reticulum, AMP binding, glycogen
PYGM_MOUSE
-30.32 phosphorylase activity, response to hypoxia
NCS2_CAEEL
-27.21
Calcium ion binding extracellular matrix structural constituent, calcium ion
MATN3_HUMAN -16.99 binding, development of skeletal system NAD(P)H oxidase activity, Calcium ion binding, cytokineDUOX2_PIG
-10.56
mediated signalling pathway, peroxidase activity, cuticle development
CO4A1_DROME
-4.2
Collagen type IV, oviduct morphogenesis
CAHD1_HUMAN
3.08
Calcium ion transport
CAD23_MOUSE
5.4
auditory receptor cell stereocilium organization, calcium ion binding, calcium-dependent cell-cell adhesion CROCC_HUMAN 7.47
Actin cytoskeleton, ciliary rootlet, cell cycle metal ion binding, collagen, extracellular matrix structural
CRA1B_DANRE
9.07 constituent,
SQH_DROME
16.31
ZAN_PIG
18.04
wound healing, calcium ion binding, cytokinesis Integral to membrane, binding of sperm to zona pellucid, cell adhesion
SSPO_CHICK
18.96
Extracellular space, cell adhesion 11
phospholipase response to reactive oxygen species, PRDX6_BOVIN
21.67
Cytoplasmic membrane-bounded vesicle, glutathione peroxidase activity, regulation of kinase activity, cytokinesis, positive regulation
CALM_DICDI
30.21
of cyclic-nucleotide phosphodiesterase activity, positive regulation of ATPase activity
MLR_LUMTE
348.37
Myosin complex, calcium ion binding
191 192
193
3.4
Apoptotic process related genes
194 195
BaP is known to cause apoptosis (Das et al., 2014). There are five transcripts that are
196
found to be differentially expressed and among them four genes are down regulated (Table
197
2). Among the four genes, FHL2 and ITA6 are negative regulators of apoptosis and this could
198
be the response of the animal to prevent apoptotic process. FHL2 mRNA in mice has been
199
shown to be down regulated in response to BaP (Kerley-Hamilton et al., 2012), whereas in
200
HepG2 model, it has been shown to be up regulated (Lizarraga et al., 2012). ITA6 has been
201
shown to be downregulated both in mice and human (Halappanavar et al., 2011; Sparfel et
202
al., 2010). Contrary to Eisenia fetida results, BTG1 has also been shown to be up regulated in
203
HepG2 cells (Magkoufopoulou et al., 2011) as well as in mice (Kerley-Hamilton et al., 2012).
204
Jennen et al (2010) [35] have obtained a similar result to that of Eisenia fetida expression in
205
HepG2 cells. Similarly, Kerley-Hamilton et al. (2012) [33] has obtained APLP1 expression to
206
be increased in mouse. The apoptotic process related gene transcripts that are altered by BaP
207
are more or less what has been obtained in animal and cell line models.
208 12
209 210
Table 2. Gene transcripts that are altered which are involved in apoptotic processes. UniProt_ID
Normalized Function and cellular process gene expression DNA-dependent transcription, negative regulation of
FHL2_RAT
-23.65 apoptotic process cell adhesion, negative/positive regulation of
ITA6_HUMAN
-11.4 apoptotic process regulation of apoptotic process, spermatogenesis,
BTG1_MOUSE
-6.64
response to oxidative stress, response to peptide hormone stimulus, Ras protein signal transduction, positive regulation of
NF1_RAT
-5.3
neuron, apoptotic process, wound healing, response to hypoxia, apoptotic process, cellular response to norepinephrine
APLP1_HUMAN
9.39 stimulus, endocytosis, cell adhesion
211
212
3.5
Genes that are involved in cell projection and cytoskeleton
213 214
The genes that are altered by BaP exposure that are involved in cytoskeletal structures
215
and cell projection are given in Table 3. Among these genes ABCF1 mRNA has been
216
reported to be decreased upon BaP exposure in mice (Kerley-Hamilton et al., 2012). MYO6
217
has been shown to be up regulated in HepG2 cells up on BaP treatment (Magkoufopoulou et
218
al., 2011). TTLL3, RADI, MSH5, ITA6 and NRX1A are not reported to be affected by BaP 13
219
treatment. Cell protrusion and motility are carcinogenic properties of a cell and are expected
220
to be affected by BaP. Cytoskeletal proteins are primary responsible molecules for cell
221
protrusion and motility, BaP is altering the expression of these molecules and as a
222
consequence, the cells are becoming malignant.
223 224
Table 3. Gene that are involved in cell projection and cytoskeleton UniProt_ID
Normalized gene
Localization, Function and cellular
expression
processes Cilium axoneme, cytoplasm, cytoskeleton,
TTLL3_HUMAN
-22.95
protein-glycine ligase activity, initiating, tubulin-tyrosine ligase activity Cytoskeleton, filopodium, actin filament
RADI_HUMAN
-14.48 capping, microvillus assembly Synaptonemal complex, ATP binding, DNA-
MSH5_HUMAN
-13.18 dependent ATPase meiotic prophase II positive regulation of transcription from RNA polymerase II promoter, Metal ion binding,
ITA6_HUMAN
-11.4 cell adhesion, negative/positive regulation of apoptotic process, Cell junction, synapse, cell adhesion molecule
NRX1A_HUMAN
-3.26 binding, axon guidance, cell adhesion Nuclear envelope, ribosome binding, ATPase
ABCF1_HUMAN
9.66
activity, inflammatory response, translation activator activity
FLNA_DROME
10.68
Actin cytoskeleton, positive regulation of 14
cytoskeleton organization, olfactory learning microtubule cytoskeleton organization, cilium TEKT3_BOVIN
16.62 axoneme Axon, cell cortex, DNA-directed RNA
MYO6_HUMAN
20.41
polymerase II, endocytic vesicle, endocytosis, almodulin binding, synaptic transmission
225
226
3.6
Protein localization and transport related genes
227 228
The genes that are involved in protein transport and localization are listed in Table 4.
229
The primary mouse hepatocytes exposed to BaP as analysed by Affimetrix microarray and
230
CAHD1 mRNA has been shown to be up regulated (Mathijs et al., 2009). CROCC is up
231
regulated in Eiseina fetida, whereas it has been shown to be down regulated in mice BaP
232
exposure.
233 234 235
Table 4. Genes that are involved in protein localization and transport Normalized UniProt id
gene
Function and cellular process
expression RSPH9_DANRE
-23.62
Cilium axoneme
COR1B_PONAB
-12.72
Cytoskeleton Cytoplasm, protein transporter activity, nuclear
XPO6_HUMAN
-10.43
pore, nucleolus, protein export from nucleus, plasma membrane
COPB2_MOUSE
-5.5
Actin cytoskeleton, intracellular protein 15
transport, structural molecule activity intra-Golgi vesicle-mediated transport endoplasmic reticulum membrane, cytosol, zinc ion binding antigen processing and presentation SC24B_HUMAN
4.14 of exogenous peptide antigen via MHC class I and II Actin cytoskeleton, structural molecule activity,
CROCC_HUMAN 7.47
cell cycle, cell projection organization, protein localization, centrosome organization,
IMA3_MOUSE
8.44
Protein transporter activity
VTA1_BOVIN
9.8
Protein transport
236
237
3.7
Nucleotide binding proteins
238 239
The nucleotide binding genes that are differentially expressed when Eisenia fetida is
240
exposed to BaP are listed in Table 5. DHX36 is down regulated and ABCF1 is up regulated
241
in Eisenia fetida, however, in mice expression of these genes has been reported to be
242
reversed. (Kerley-Hamilton et al., 2012). MSH5, RN213, HS12A and MYO6 are not reported
243
to be altered.
244
3.8
Other genes and functions
245 246
Eisenia fetida exposed to low levels of BaP showed up- and down regulation of genes
247
that are involved in apoptosis, calcium homeostasis, protein transport, cytoskeletal structures,
248
nucleic acid binding and several other genes (Table 5) which are reported earlier in other 16
249
organisms but we noticedsome of those have not been reported before. These genes are
250
involved in transcription regulation, lipid metabolism, cell cycle regulation, metal ion binding
251
and membrane proteins. The highly differentially expressed genes that are not induced by
252
other related chemicals or generic chemicals may be used as biomarkers for the presence of
253
low levels of BaP.
254 255
Table 5. Ungrouped genes that are altered by BaP exposure. Normalized UniProt_ID
gene
Localization and Function
expression ATF4_DANRE
-90.79
Sequence-specific DNA binding
CHCH2_MOUSE
-44.73
Mitochondrion
HMG2_DROME
-36.72
Polytene chromosome, single-stranded DNA binding C560_CRIGR
-31.78
INO1B_XENLA
-28.59
Metal ion binding, tricarboxylic acid cycle Nucleotide binding inositol biosynthetic process, phospholipid biosynthetic process
FACR1_XENLA
-24.98
KNG2_BOVIN
-23.84
Lipid metabolic process Blood coagulation inflammatory response, vasodilation
UBCP1_DANRE
-23.83
Phosphoprotein phosphatase activity
NUCL_XENLA
-23.41
Nucleolus, DNA binding
PRS6A_RAT
-23.13
ATP binding, nucleoside-triphosphatase activity
SAP_CHICK
-22.8
Lysosome, sphingolipid metabolic process
PI16_MOUSE
-21.36
Extracellular region, integral to membrane, 17
peptidase inhibitor activity Nucleus, Z disc, 14-3-3 protein binding, actin SYNP2_MOUSE
-19.55 binding, muscle alpha-actinin binding Mitochondrion, isomerase activity, metal ion
ENOF1_XENLA
-18.33 binding Mitochondrial matrix, dihydrolipoyl
DLDH_PIG
-17.92 dehydrogenase activity, cell redox homeostasis
PTBP3_HUMAN
-16.5
Nucleus, mRNA processing Mitochondrion blastocyst hatching, embryo
GRN_CAVPO
-15.95 implantation ATPase activity, phosphorylative mechanism,
AT8A1_MOUSE
-14.57 cation transport Cell division microtubule-based movement,
KIF2A_MOUSE
-13.65 nervous system development Integral to membrane, oxidoreductase activity,
FRRS1_XENLA
-13.64 electron transport chain Purine ribonucleoside salvage type B pancreatic
ADK_RAT
-13.37 cell proliferation
GBB_PINFU
-13.15
Signal transducer activity Condensed chromosome, cell division
TEX14_MOUSE
-12.82 intercellular bridge organization Lysosome, cysteine-type peptidase activity,
CATL_DROME
-11.15 digestion, autophagic cell death Glycogen (starch) synthase activity, glycogen
GYS_DROME
-9.5 biosynthetic process 18
IF4G3_MOUSE
-7.35
OTU5A_DANRE
-7.13
DNA binding, spermatogenesis Protein K48-linked deubiquitination proteolysis, response to lipopolysaccharide Integral to membrane, structural molecule
CC108_HUMAN
-7.04 activity Extracellular region, serine-type endopeptidase
ITIH2_PIG
-6.96 inhibitor activity Cysteine-type peptidase activity, ubiquitin-
UBP48_RAT
-6.96 dependent protein catabolic process Mitochondrial matrix, cellular amino acid
ALAT2_HUMAN
-6.01 biosynthetic process DNA binding histone demethylase activity (H3-
KDM5B_CHICK
-5.36
trimethyl-K4 specific) metal ion binding, 2oxoglutarate as one donor
DIP2C_HUMAN
-5.36
Catalytic activity Cysteine-type endopeptidase activity, ubiquitin
USP9X_HUMAN
-4.97 hiolesterase activity, BMP signalling pathway
PLB1_RAT
-3.86
phospholipase A2 activity,
PCM1_CHICK
4.25
Centriolar satellite, cilium assembly
RHG44_MOUSE
4.77
GTPase activator activity
TM131_HUMAN
5.73
Integral to membrane
Y1281_ARCFU
7.54
Hydrolase activity, acting on glycosyl bonds
SDC_DROME
7.81
Neuromuscular junction energy homeostasis
PDIA1_PONAB
7.87
Endoplasmic
19
reticulum
lumen,
cell
redox
homeostasis, glycerol ether metabolic process Mitochondrion, SPTC2_MOUSE
8.1
complex,
serine
C-palmitoyltransferase
pyridoxal
phosphate
binding
sphinganine biosynthetic process CAND1_PONAB
8.3
DNA-dependent Cytoplasmic mRNA processing body, mitotic
DDX6_DROME
9.31
cell cycle G2/M transition DNA damage checkpoint
PLCL_MYTGA
9.97
Carbohydrate binding Cellular aromatic compound metabolic process
AMPE_RAT
10.75
regulation of systemic arterial blood pressure by renin-angiotensin
RAB18_RAT
11.46
Brain development, eye development ATP-dependent
IF4A2_RAT
helicase
activity
translation
11.78 initiation factor activity
CP1A5_CHICK
12.34
heme binding, electron carrier activity, 3'-5' exonuclease activity, DNA-directed DNA
DPO1_KLULA
12.5 polymerase activity Protein binding involved in heterotypic cell-cell
NFASC_MOUSE
12.51
adhesion,
peripheral
nervous
development TEX14_BOVIN
12.64
Condensed chromosome kinetochore
PI16_HUMAN
14.63
Peptidase inhibitor activity
MPU1_MOUSE
16.08
Transport
20
system
GTPase RHO1_DROME
activity,
establishment
of
protein
16.23 localization, wound healing
TMED4_MOUSE
18.94
Positive regulation of I-kB kinase/NF-kB cascade
LAMP1_CRIGR
19.17
Endosome and lysosomal membrane
AT1B1_ARTSF
19.35
Sodium:potassium-exchanging ATPase activity
PRS6A_RAT
20.56
Perinuclear
region
of
cytoplasm,
protein
catabolic process MET24_XENTR
20.63
EF1A2_RABIT
21.04
Methyltransferase activity GTP binding, translation elongation factor activity, Regulation of systemic arterial blood pressure by
AMPE_BOVIN
23.94
renin-angiotensin, zinc ion binding and cell proliferation related proteolysis dUTP diphosphatase activity metal ion binding
DUT_COXBU
25.4 dUMP biosynthetic process
NF70_DORPE
26.35
structural molecule activity Hydrogen-exporting
VA0D1_DROME
28.31
phosphorylative
ATPase
activity,
mechanism,
vacuolar
acidification Endoplasmic MAMC2_HUMAN 36.7
reticulum,
glycosaminoglycan
cross-linking
binding
peptide
chondroitin 4-sulfate glycosaminoglycan RL27_DANRE
42.86
structural constituent of ribosome translation
LEG9_BOVIN
47.79
carbohydrate binding
21
of by
Adult 14332_CAEEL
lifespan
determination,
embryo
56.81 development ending in egg hatching or birth
IF27A_MOUSE
66.43
Aging, response to virus
PPIA_BLAGE
105.29
Peptidyl-prolyl cis-trans isomerase activity
ALDOA_RAT
111.63
Response to hypoxia response to lipopolysaccharide TXL_EISFO
150.89
Defence response to bacterium, ion transport
256
257
3.9
Potential Molecular Markers for PFOA
258 259
From the current study, we should be able to pick set of molecular markers that are
260
specific to BaP. If we pick a single gene and follow the expression level to use as a molecular
261
marker, it is highly likely that other polycyclic aromatic hydrocarbons/ homologues of the
262
intended molecule also may induce those molecules. Hence, from this study, we are
263
proposing to pick five up regulated and five down regulated genes that are specific to BaP. In
264
that case, it is highly unlikely that other polycyclic aromatic hydrocarbons/ homologues will
265
induce same set of genes to same levels. Therefore, the potential molecular markers are
266
IF27A, PPIA, ALDOA, TXL, MLR, BMP1, ATF4, CHCH2, HMG2, C560. However, these
267
molecules need to be tested and validated and compared with expression profiles of other
268
polycyclic aromatic hydrocarbons/ homologues induction.
269
270
4 Conclusion
271
Benzo(a)pyrene exposure has resulted in changes in the expression of genes involved in
272
calcium homeostasis, cell cycle regulation and inflammatory response in E. fetida. The other
22
273
altered genes due to BaP exposure include those that are involved in nucleotide binding,
274
protein transport, vasodilation, cytoskeletal structure and cell division, microtubule-based
275
movement, establishment of protein localization, lipid metabolic process, spermatogenesis,
276
embryo development ending in birth or egg hatching, wound healing, nervous system
277
development and eye development. Many genes identified in E. fetida in this study have not
278
been reported to be changed by Benzo(a)Pyrene in other organisms. This gene expression
279
data will assist in understanding the toxicological effects at molecular level. Furthermore, this
280
data will be helpful in developing molecular markers for detecting benzo(a)pyrene
281
contamination in soil.
282 283
Acknowledgements:
284
The authors acknowledge the Ramaciotti Centre for Genomics, The University of New South
285
Wales, Sydney, form RNA sequencing and eResearch SA for computing facility. Dr. Srinithi
286
Mayilswami is recipient of IPRS and CRCCARE top-up scholarships.
287 288
Conflict of Interest:
289
The authors declare that they have no conflict of interest
290 291 292
References:
293 294 295 296 297 298 299 300 301 302
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S2352-1864(16)30174-2 http://dx.doi.org/10.1016/j.eti.2016.12.002 ETI 101
To appear in:
Environmental Technology & Innovation
Received date: 14 June 2016 Revised date: 7 December 2016 Accepted date: 13 December 2016 Please cite this article as: Mayilswami, S., Krishnan, K., Naidu, R., Megharaj, M., Transcriptome analysis of Eisenia fetida chronically exposed to benzo(a)pyrene. Environmental Technology & Innovation (2016), http://dx.doi.org/10.1016/j.eti.2016.12.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Highlights (for review)
Highlights Eisenia fetida chronically exposed in soil with 10 mg/kg of BaP. mRNA was isolated and sequenced from BaP exposed Eisenia fetida. The transcriptomes were in silico assembled from mRNA sequences Genes involved in calcium homeostasis, apoptosis and lipid metabolism are altered
Graphical Abstract
• Eisenia fetida exposed to 10 mg Kg-1 of BaP for 8 m
• mRNA isolation and sequencin
• Transcriptome assembly
• Identification of differential expressed transcripts
Apoptotic, reproduction related, lipid metabolism development related genes are altere
*Revised Manuscript with No Changes Marked
1 2 3
Transcriptome Analysis of Eisenia fetida Chronically Exposed to Benzo(a)Pyrene Srinithi Mayilswami2, 3, Kannan Krishnan1, 2*, Ravi Naidu1, 2, Mallavarapu Megharaj1, 2
4
1
5
Technology, The University of Newcastle, Callaghan NSW 2308, Australia
6
2
7
Environment (CRC-CARE), Mawson Lakes, Adelaide SA 5095, Australia
8
3
9
Mawson Lakes, Adelaide SA 5095, Australia
Global Centre for Environmental Research, Faculty of Science and Information
Cooperative Research Centre for Contamination Assessment and Remediation of the
Centre for Environmental Risk Assessment and Remediation, University of South Australia,
10 11
*e-mail: [email protected],
12
Phone: +61 2 4913 8732.
13
Key words: differential gene expression, Transcriptome assesmbly, Molecular markers,
14
Polycyclic aromaric hydrocarbons
15
Abstract
16
Benzo(a)pyrene is a high molecular weight polycyclic aromatic hydrocarbon which is
17
carcinogenic and widespread pollutant in the environment. It is essential to identify the
18
presence of this chemical in soil as it is toxic to biota including humans. Eisenia fetida is a
19
sentinal organism in soil which can be used to diagnose the health of the soil. In order to
20
identify potential molecular markers from Eisenia fetida to diagnose the presence of benzo(a)
21
pyrene in soil, we exposed the organism to sub-lethal (10 mg Kg-1) concentrations for a
22
period of eight months and carried out transcriptome anaysis. From the transcriptome, we
23
have identified differentially expressed genes. Results showed that benzo(a)pyrene has
24
altered the expression of calcium binding and calcium homeostasis, apoptotic process,
25
cytoskeletal proteins, protein transport, nucleotide binding, lipid metabolism, peripheral 1
26
neuronal development, cell division, wound healing and nucleotide binding and processing
27
genes at transcription level. Several of the genes we reported here were not reported earlier.
28
The highly up regulated and down regulated genes could be used as a molecular marker to
29
diagnose the presence of benzo(a)pyrene in the soil.
30 31
Key words: Transcriptome assembly, differential gene expression, toxicogenomics,
32
molecular markers, polycyclic aromaric hydrocarbons
2
33
1 Introduction
34
Benzo(a)pyrene (BaP) is one of the polycyclic aromatic hydrocarbons (PAHs) which is
35
an established mutagen and carcinogen. PAHs including BaP are formed during incomplete
36
combustion of fossil fuels and organic material (Yunker et al., 2002). Several processes
37
including automobile exhaust, industrial emission, agriculture activities and domestic
38
emission and waste contribute to the release ofBaP in to the environment (Ravindra et al.,
39
2008). BaP induces several enzymes with mixed function in the cell (Gelboin, 1980) and as a
40
result, different type of cancer progression occurs in human and animals. BaP has been
41
shown to accumulate in several organisms such as mussels (Canova et al., 1998) microalgae
42
(Subashchandrabose et al., 2014), and mouse (Uno et al., 2006). Benzo(a)pyrene has also
43
been established as a mutagen in somatic cells. By using specific locus test for visible
44
markers, it could not be concluded that point mutation by BaP could be inherited (Russell et
45
al., 1981), however, the dominant lethal mutations induced by BaP in post-meiotic germ cells
46
were found to be inherited (Generoso et al., 1982). Moreover, from environmentally exposed
47
animals, there are evidences that PAHs are also mutagenic in male germ cells which leads to
48
potential health risks in the offspring (Somers et al., 2004; Somers et al., 2002; Yauk et al.,
49
2000; Yauk and Quinn, 1996).
50 51
Benzo(a)pyrene binds to aryl hydrocarbon receptor (AhR), the transcription regulator
52
and the ligand bound AhR binds to xenobiotic responsive elements (XRE) thereby causing
53
biological effects (Nebert et al., 2000). BaP is enzymatically converted by CYP1A1,
54
CYP1B1 and epoxy hydrolase into BaP-diol epoxide that forms Nucleotide-BaP-adducts that
55
is incorporated into DNA (Kim et al., 2007; Shi et al., 2009). These adducts prevents the
56
DNA polymerase from moving along the DNA during replication (Hsu et al., 2005) that
57
causes immature termination of DNA synthesis. As a result, the regulation of genes will be 3
58
defective and cellular metabolism will be abnormal, which leads to cell proliferation defects
59
and apoptosis (Solhaug et al., 2004).
60 61
To diagnose the presences of sub lethal concentrations of BaP in aquatic system and
62
in soil there are several biochemical based markers (Gastaldi et al., 2007), and specific genes
63
as molecular markers (Zheng et al., 2008). However, due to the limitations with the
64
traditional techniques, it is possible that some potential toxic effects might be neglected.
65
Global transcriptome analysis of the effect of a chemical might reveal both mechanism of
66
toxicity as well as general toxic effects (Huang et al., 2014). It is appropriate to use a soil
67
organism such as Eisenia fetida for developing a molecular marker to monitor BaP presence
68
in the soil.
69 70
Soil is considered to be one of the largest sinks for BaP and hence, it is essential to
71
use soil organism such as Eisenia fetida to develop molecular biomarkers by studying the
72
transcriptome profile change and differential gene expression in the presence and absence of
73
BaP. The alteration in transcript level would be a powerful indicator to diagnose the effects
74
of a toxic pollutant such as BaP at sub-lethal levels. Advancement in mRNA sequencing
75
techniques such as Next generation mRNA sequencing is one of the tools that can be used to
76
analyse such changes in transcriptome expression profiles. In this paper we report the change
77
in transcriptome expression in E. fetida chronically exposed to sub-lethal levels of BaP for
78
about eight months.
79
80
2 Materials and Methods
81
2.1
Chemicals and earthworm treatment
82 4
83
Benzo(a)pyrene was purchased from Sigma-Aldrich (Australia). Eisenia fetida was
84
maintained in natural soil added with fruits and vegetable waste, at 25 ± 1 °C, 60 to 80%
85
humidity and a 16:8 h L: D (Light: dark) cycle in our laboratory. E. fetida with wet weight of
86
0.5 g and well-developed clitellum were used for the chronic toxicity studies.
87
88
2.2
Experimental Design
89 90
E. fetida was introduced into soil (pH ~ 6.5) for the transcriptome analysis followed by
91
differential gene expression studies. The soil sample was collected from Adelaide region, air
92
dried for 24 hours and sieved using a 2 mm sieve. Earthworm assay was carried out using 10
93
mg Kg-1 of BaP; controls were maintained simultaneously. The concentration of BaP in 9
94
manufactured gas plant (MGP) site soils ranged between 58 and 738 mg Kg-1 soil [21].
95
Hence, the environmentally relevant concentration for chronic exposure study was selected as
96
10 mg Kg-1. BaP was dissolved in acetone and mixed with the soil using an end-to-end shaker
97
overnight to artificially contaminate the soil for the study and subsequently the earthworms
98
were introduced into the soil. 50 g of soil was mixed with required amount of BaP dissolved
99
in acetone and later they were mixed with the remaining 1950 g of soil (in total 2000 g)
100
which ensures homogenisation of spiking. The solvent was evaporated from the soil in a
101
fume hood. The soils were moistened with water so as to obtain 50 to 60% by mass of water
102
holding capacity and the earthworms were released into it. Ten worms per group in triplicates
103
were released in the containers. The containers were supplemented with powdered pulses and
104
about 40 g of fruits and vegetable every week. This procedure was followed for eight months
105
and simultaneously control group was also maintained under the same condition.
106
5
107
2.3
RNA Isolation and next generation sequencing
108 109
E. fetida treated as well as control were collected, placed into a 15 ml tube with
110
suspension buffer QIAGEN® Mini Kit (Cat No: 74104) and homogenized using Polytron
111
homogenizer. From the homogenized E. fetida total RNA was isolated using QIAGEN® Mini
112
Kit using manufacturer’s protocol. From the Qiagen column, total RNA was eluted using
113
elusion buffer and stored in -80 °C and transported to sequencing facility on dry ice. Using
114
Illumina HiSeq 2000, the paired-end RNA sequencing was carried out at The Ramaciotti
115
Centre for Gene Function Analysis. The sequencing data is submitted to Sequence Read
116
Archive (SRA), National Center for Biotechnology Information (NCBI) (Accession:
117
SRS1828175, SRS1827215).
118
119
2.4
Sequence analysis
120 121
The forward and reverse mRNA sequence reads were joined together and transcriptome
122
assembly was carried out using Trinity software (Grabherr et al., 2011). Trinity software was
123
installed in bigmem-1024 server, eRSA, Adelaide. The transcriptome assembly was carried
124
with parameters, k-mers was set at 2 and glue length was set at 4. The transcripts with longer
125
than 130 amino acid length were selected and annotated using BLAST (NCBI-BLAST-
126
2.2.28+) with UniProt database. The transcripts were compared using NPKF- normalized
127
counts and the transcripts that are more than fourfold altered (p = 0.001) in expression were
128
selected.
6
129
3 Results and Discussion
130
3.1
Transcript assembly and annotation
131 132
The mRNA from E. fetida was sequenced by next generation sequencing and de novo
133
assembled using Trinity software installed in Bigmem-1024 server, at eRSA, Adelaide. The
134
transcripts obtained from de novo assembly were translated in silico and the best possible
135
ORFs were carefully chosen. The total number of peptides that were translated (ORFs) from
136
the de novo assembled transcripts were 616110. These translated peptides contain all the
137
possible trans-spliced isoforms. These peptides were subject to homology search using
138
BLAST where 106161 peptides retrieved hits. Apart from the peptides that retrieved
139
homologous results, 3830 peptides retrieved homologues signal peptides and 8525 TmHMM
140
topology results and 33064 did not retrieve any result. The transcripts that were longer that
141
130 amino acids obtained from the database (UniProt) were analysed further (Mayilswami et
142
al., 2014).
143
3.2
Differential gene expression
144 145
About 2000 genes were recognized as differentially expressed with more than four-fold
146
difference compared to control. The differentially expressed genes with their fold difference
147
was subjected to gene cluster analysis (Fig. 1). The gene expression fold difference were
148
plotted in MA-plot (Fig 2). Genes that are differentially expressed more than 12-fold (p <
149
0.001) were selected for further analysis. About 223 differentially expressed transcripts were
150
retrieved. The genes with more than one isoforms and genes with no known functions were
151
removed from analysis. Among these differentially expressed genes, 63 were up-regulated
152
and 56 down-regulated. Based on the functional information obtained from UniProt, the
153
toxicity was interpreted and grouped according to functions. 7
154
155
3.3
Calcium binding and homeostasis
156 157
The transcripts that are altered in the presence of BaP are given in Tables 1 to 5. BaP
158
has been shown to cause Ca2+ elevation in human mammary epithelial cells at 2 hours and
159
sustained alterations in Ca2+ homeostasis at 18 hours (Tannheimer et al., 1997). MATN3 has
160
been shown to be down regulated at transcriptome level in HepG2 cells exposed to BaP
161
(Magkoufopoulou et al., 2011) which is the case in Eisenia fetida as well. However, a
162
contradictory result has also been reported (Lizarraga et al., 2012). Altered expression of
163
collagen (Hussain et al., 1979) and basement membrane perforation has been observed
164
(Kopf-Maier and Flug, 1996). In mice liver, BaP induces PRDX6 expression at mRNA level
165
(Halappanavar et al., 2011), however, it has been shown to be down regulated in Solea
166
senegalensis live at protein level (Costa et al., 2010). So far the toxicological studies have
167
suggested that BaP affects calcium homeostasis which is confirmed by transcriptome study
168
and we have identified possible genes that could be involved in this process.
169
8
170 171 172
Figure 1. Hierarchical clustering analysis of gene expression profiles control (C) and
173
benzo(a)pyrene (BaP) treated Eisenia fetida. The fold difference was set at 2 and the
174
significance (p) was set as 0.001.
175
9
176 177 178
Figure 2. Differential expression of chronic benzo(a)pyrene exposed (10 mg Kg-1 in soil)
179
versus control Eisenia fetida. MA-plot with log counts on x-axis, log fold-change on y-axis
180
showing differentially expressed genes (P value < 0.001) as red dots.
181 182 183 184 185 186 187 188
10
189
Table 1. The transcripts of calcium ion binding protein that are altered by BaP exposure in
190
Eisenia fetida Normalized UniProt_ID
gene
Function and cellular process
expression BMP1_MOUSE
-60.97
Calcium ion binding, cell differentiation Sarcoplasmic reticulum, AMP binding, glycogen
PYGM_MOUSE
-30.32 phosphorylase activity, response to hypoxia
NCS2_CAEEL
-27.21
Calcium ion binding extracellular matrix structural constituent, calcium ion
MATN3_HUMAN -16.99 binding, development of skeletal system NAD(P)H oxidase activity, Calcium ion binding, cytokineDUOX2_PIG
-10.56
mediated signalling pathway, peroxidase activity, cuticle development
CO4A1_DROME
-4.2
Collagen type IV, oviduct morphogenesis
CAHD1_HUMAN
3.08
Calcium ion transport
CAD23_MOUSE
5.4
auditory receptor cell stereocilium organization, calcium ion binding, calcium-dependent cell-cell adhesion CROCC_HUMAN 7.47
Actin cytoskeleton, ciliary rootlet, cell cycle metal ion binding, collagen, extracellular matrix structural
CRA1B_DANRE
9.07 constituent,
SQH_DROME
16.31
ZAN_PIG
18.04
wound healing, calcium ion binding, cytokinesis Integral to membrane, binding of sperm to zona pellucid, cell adhesion
SSPO_CHICK
18.96
Extracellular space, cell adhesion 11
phospholipase response to reactive oxygen species, PRDX6_BOVIN
21.67
Cytoplasmic membrane-bounded vesicle, glutathione peroxidase activity, regulation of kinase activity, cytokinesis, positive regulation
CALM_DICDI
30.21
of cyclic-nucleotide phosphodiesterase activity, positive regulation of ATPase activity
MLR_LUMTE
348.37
Myosin complex, calcium ion binding
191 192
193
3.4
Apoptotic process related genes
194 195
BaP is known to cause apoptosis (Das et al., 2014). There are five transcripts that are
196
found to be differentially expressed and among them four genes are down regulated (Table
197
2). Among the four genes, FHL2 and ITA6 are negative regulators of apoptosis and this could
198
be the response of the animal to prevent apoptotic process. FHL2 mRNA in mice has been
199
shown to be down regulated in response to BaP (Kerley-Hamilton et al., 2012), whereas in
200
HepG2 model, it has been shown to be up regulated (Lizarraga et al., 2012). ITA6 has been
201
shown to be downregulated both in mice and human (Halappanavar et al., 2011; Sparfel et
202
al., 2010). Contrary to Eisenia fetida results, BTG1 has also been shown to be up regulated in
203
HepG2 cells (Magkoufopoulou et al., 2011) as well as in mice (Kerley-Hamilton et al., 2012).
204
Jennen et al (2010) [35] have obtained a similar result to that of Eisenia fetida expression in
205
HepG2 cells. Similarly, Kerley-Hamilton et al. (2012) [33] has obtained APLP1 expression to
206
be increased in mouse. The apoptotic process related gene transcripts that are altered by BaP
207
are more or less what has been obtained in animal and cell line models.
208 12
209 210
Table 2. Gene transcripts that are altered which are involved in apoptotic processes. UniProt_ID
Normalized Function and cellular process gene expression DNA-dependent transcription, negative regulation of
FHL2_RAT
-23.65 apoptotic process cell adhesion, negative/positive regulation of
ITA6_HUMAN
-11.4 apoptotic process regulation of apoptotic process, spermatogenesis,
BTG1_MOUSE
-6.64
response to oxidative stress, response to peptide hormone stimulus, Ras protein signal transduction, positive regulation of
NF1_RAT
-5.3
neuron, apoptotic process, wound healing, response to hypoxia, apoptotic process, cellular response to norepinephrine
APLP1_HUMAN
9.39 stimulus, endocytosis, cell adhesion
211
212
3.5
Genes that are involved in cell projection and cytoskeleton
213 214
The genes that are altered by BaP exposure that are involved in cytoskeletal structures
215
and cell projection are given in Table 3. Among these genes ABCF1 mRNA has been
216
reported to be decreased upon BaP exposure in mice (Kerley-Hamilton et al., 2012). MYO6
217
has been shown to be up regulated in HepG2 cells up on BaP treatment (Magkoufopoulou et
218
al., 2011). TTLL3, RADI, MSH5, ITA6 and NRX1A are not reported to be affected by BaP 13
219
treatment. Cell protrusion and motility are carcinogenic properties of a cell and are expected
220
to be affected by BaP. Cytoskeletal proteins are primary responsible molecules for cell
221
protrusion and motility, BaP is altering the expression of these molecules and as a
222
consequence, the cells are becoming malignant.
223 224
Table 3. Gene that are involved in cell projection and cytoskeleton UniProt_ID
Normalized gene
Localization, Function and cellular
expression
processes Cilium axoneme, cytoplasm, cytoskeleton,
TTLL3_HUMAN
-22.95
protein-glycine ligase activity, initiating, tubulin-tyrosine ligase activity Cytoskeleton, filopodium, actin filament
RADI_HUMAN
-14.48 capping, microvillus assembly Synaptonemal complex, ATP binding, DNA-
MSH5_HUMAN
-13.18 dependent ATPase meiotic prophase II positive regulation of transcription from RNA polymerase II promoter, Metal ion binding,
ITA6_HUMAN
-11.4 cell adhesion, negative/positive regulation of apoptotic process, Cell junction, synapse, cell adhesion molecule
NRX1A_HUMAN
-3.26 binding, axon guidance, cell adhesion Nuclear envelope, ribosome binding, ATPase
ABCF1_HUMAN
9.66
activity, inflammatory response, translation activator activity
FLNA_DROME
10.68
Actin cytoskeleton, positive regulation of 14
cytoskeleton organization, olfactory learning microtubule cytoskeleton organization, cilium TEKT3_BOVIN
16.62 axoneme Axon, cell cortex, DNA-directed RNA
MYO6_HUMAN
20.41
polymerase II, endocytic vesicle, endocytosis, almodulin binding, synaptic transmission
225
226
3.6
Protein localization and transport related genes
227 228
The genes that are involved in protein transport and localization are listed in Table 4.
229
The primary mouse hepatocytes exposed to BaP as analysed by Affimetrix microarray and
230
CAHD1 mRNA has been shown to be up regulated (Mathijs et al., 2009). CROCC is up
231
regulated in Eiseina fetida, whereas it has been shown to be down regulated in mice BaP
232
exposure.
233 234 235
Table 4. Genes that are involved in protein localization and transport Normalized UniProt id
gene
Function and cellular process
expression RSPH9_DANRE
-23.62
Cilium axoneme
COR1B_PONAB
-12.72
Cytoskeleton Cytoplasm, protein transporter activity, nuclear
XPO6_HUMAN
-10.43
pore, nucleolus, protein export from nucleus, plasma membrane
COPB2_MOUSE
-5.5
Actin cytoskeleton, intracellular protein 15
transport, structural molecule activity intra-Golgi vesicle-mediated transport endoplasmic reticulum membrane, cytosol, zinc ion binding antigen processing and presentation SC24B_HUMAN
4.14 of exogenous peptide antigen via MHC class I and II Actin cytoskeleton, structural molecule activity,
CROCC_HUMAN 7.47
cell cycle, cell projection organization, protein localization, centrosome organization,
IMA3_MOUSE
8.44
Protein transporter activity
VTA1_BOVIN
9.8
Protein transport
236
237
3.7
Nucleotide binding proteins
238 239
The nucleotide binding genes that are differentially expressed when Eisenia fetida is
240
exposed to BaP are listed in Table 5. DHX36 is down regulated and ABCF1 is up regulated
241
in Eisenia fetida, however, in mice expression of these genes has been reported to be
242
reversed. (Kerley-Hamilton et al., 2012). MSH5, RN213, HS12A and MYO6 are not reported
243
to be altered.
244
3.8
Other genes and functions
245 246
Eisenia fetida exposed to low levels of BaP showed up- and down regulation of genes
247
that are involved in apoptosis, calcium homeostasis, protein transport, cytoskeletal structures,
248
nucleic acid binding and several other genes (Table 5) which are reported earlier in other 16
249
organisms but we noticedsome of those have not been reported before. These genes are
250
involved in transcription regulation, lipid metabolism, cell cycle regulation, metal ion binding
251
and membrane proteins. The highly differentially expressed genes that are not induced by
252
other related chemicals or generic chemicals may be used as biomarkers for the presence of
253
low levels of BaP.
254 255
Table 5. Ungrouped genes that are altered by BaP exposure. Normalized UniProt_ID
gene
Localization and Function
expression ATF4_DANRE
-90.79
Sequence-specific DNA binding
CHCH2_MOUSE
-44.73
Mitochondrion
HMG2_DROME
-36.72
Polytene chromosome, single-stranded DNA binding C560_CRIGR
-31.78
INO1B_XENLA
-28.59
Metal ion binding, tricarboxylic acid cycle Nucleotide binding inositol biosynthetic process, phospholipid biosynthetic process
FACR1_XENLA
-24.98
KNG2_BOVIN
-23.84
Lipid metabolic process Blood coagulation inflammatory response, vasodilation
UBCP1_DANRE
-23.83
Phosphoprotein phosphatase activity
NUCL_XENLA
-23.41
Nucleolus, DNA binding
PRS6A_RAT
-23.13
ATP binding, nucleoside-triphosphatase activity
SAP_CHICK
-22.8
Lysosome, sphingolipid metabolic process
PI16_MOUSE
-21.36
Extracellular region, integral to membrane, 17
peptidase inhibitor activity Nucleus, Z disc, 14-3-3 protein binding, actin SYNP2_MOUSE
-19.55 binding, muscle alpha-actinin binding Mitochondrion, isomerase activity, metal ion
ENOF1_XENLA
-18.33 binding Mitochondrial matrix, dihydrolipoyl
DLDH_PIG
-17.92 dehydrogenase activity, cell redox homeostasis
PTBP3_HUMAN
-16.5
Nucleus, mRNA processing Mitochondrion blastocyst hatching, embryo
GRN_CAVPO
-15.95 implantation ATPase activity, phosphorylative mechanism,
AT8A1_MOUSE
-14.57 cation transport Cell division microtubule-based movement,
KIF2A_MOUSE
-13.65 nervous system development Integral to membrane, oxidoreductase activity,
FRRS1_XENLA
-13.64 electron transport chain Purine ribonucleoside salvage type B pancreatic
ADK_RAT
-13.37 cell proliferation
GBB_PINFU
-13.15
Signal transducer activity Condensed chromosome, cell division
TEX14_MOUSE
-12.82 intercellular bridge organization Lysosome, cysteine-type peptidase activity,
CATL_DROME
-11.15 digestion, autophagic cell death Glycogen (starch) synthase activity, glycogen
GYS_DROME
-9.5 biosynthetic process 18
IF4G3_MOUSE
-7.35
OTU5A_DANRE
-7.13
DNA binding, spermatogenesis Protein K48-linked deubiquitination proteolysis, response to lipopolysaccharide Integral to membrane, structural molecule
CC108_HUMAN
-7.04 activity Extracellular region, serine-type endopeptidase
ITIH2_PIG
-6.96 inhibitor activity Cysteine-type peptidase activity, ubiquitin-
UBP48_RAT
-6.96 dependent protein catabolic process Mitochondrial matrix, cellular amino acid
ALAT2_HUMAN
-6.01 biosynthetic process DNA binding histone demethylase activity (H3-
KDM5B_CHICK
-5.36
trimethyl-K4 specific) metal ion binding, 2oxoglutarate as one donor
DIP2C_HUMAN
-5.36
Catalytic activity Cysteine-type endopeptidase activity, ubiquitin
USP9X_HUMAN
-4.97 hiolesterase activity, BMP signalling pathway
PLB1_RAT
-3.86
phospholipase A2 activity,
PCM1_CHICK
4.25
Centriolar satellite, cilium assembly
RHG44_MOUSE
4.77
GTPase activator activity
TM131_HUMAN
5.73
Integral to membrane
Y1281_ARCFU
7.54
Hydrolase activity, acting on glycosyl bonds
SDC_DROME
7.81
Neuromuscular junction energy homeostasis
PDIA1_PONAB
7.87
Endoplasmic
19
reticulum
lumen,
cell
redox
homeostasis, glycerol ether metabolic process Mitochondrion, SPTC2_MOUSE
8.1
complex,
serine
C-palmitoyltransferase
pyridoxal
phosphate
binding
sphinganine biosynthetic process CAND1_PONAB
8.3
DNA-dependent Cytoplasmic mRNA processing body, mitotic
DDX6_DROME
9.31
cell cycle G2/M transition DNA damage checkpoint
PLCL_MYTGA
9.97
Carbohydrate binding Cellular aromatic compound metabolic process
AMPE_RAT
10.75
regulation of systemic arterial blood pressure by renin-angiotensin
RAB18_RAT
11.46
Brain development, eye development ATP-dependent
IF4A2_RAT
helicase
activity
translation
11.78 initiation factor activity
CP1A5_CHICK
12.34
heme binding, electron carrier activity, 3'-5' exonuclease activity, DNA-directed DNA
DPO1_KLULA
12.5 polymerase activity Protein binding involved in heterotypic cell-cell
NFASC_MOUSE
12.51
adhesion,
peripheral
nervous
development TEX14_BOVIN
12.64
Condensed chromosome kinetochore
PI16_HUMAN
14.63
Peptidase inhibitor activity
MPU1_MOUSE
16.08
Transport
20
system
GTPase RHO1_DROME
activity,
establishment
of
protein
16.23 localization, wound healing
TMED4_MOUSE
18.94
Positive regulation of I-kB kinase/NF-kB cascade
LAMP1_CRIGR
19.17
Endosome and lysosomal membrane
AT1B1_ARTSF
19.35
Sodium:potassium-exchanging ATPase activity
PRS6A_RAT
20.56
Perinuclear
region
of
cytoplasm,
protein
catabolic process MET24_XENTR
20.63
EF1A2_RABIT
21.04
Methyltransferase activity GTP binding, translation elongation factor activity, Regulation of systemic arterial blood pressure by
AMPE_BOVIN
23.94
renin-angiotensin, zinc ion binding and cell proliferation related proteolysis dUTP diphosphatase activity metal ion binding
DUT_COXBU
25.4 dUMP biosynthetic process
NF70_DORPE
26.35
structural molecule activity Hydrogen-exporting
VA0D1_DROME
28.31
phosphorylative
ATPase
activity,
mechanism,
vacuolar
acidification Endoplasmic MAMC2_HUMAN 36.7
reticulum,
glycosaminoglycan
cross-linking
binding
peptide
chondroitin 4-sulfate glycosaminoglycan RL27_DANRE
42.86
structural constituent of ribosome translation
LEG9_BOVIN
47.79
carbohydrate binding
21
of by
Adult 14332_CAEEL
lifespan
determination,
embryo
56.81 development ending in egg hatching or birth
IF27A_MOUSE
66.43
Aging, response to virus
PPIA_BLAGE
105.29
Peptidyl-prolyl cis-trans isomerase activity
ALDOA_RAT
111.63
Response to hypoxia response to lipopolysaccharide TXL_EISFO
150.89
Defence response to bacterium, ion transport
256
257
3.9
Potential Molecular Markers for PFOA
258 259
From the current study, we should be able to pick set of molecular markers that are
260
specific to BaP. If we pick a single gene and follow the expression level to use as a molecular
261
marker, it is highly likely that other polycyclic aromatic hydrocarbons/ homologues of the
262
intended molecule also may induce those molecules. Hence, from this study, we are
263
proposing to pick five up regulated and five down regulated genes that are specific to BaP. In
264
that case, it is highly unlikely that other polycyclic aromatic hydrocarbons/ homologues will
265
induce same set of genes to same levels. Therefore, the potential molecular markers are
266
IF27A, PPIA, ALDOA, TXL, MLR, BMP1, ATF4, CHCH2, HMG2, C560. However, these
267
molecules need to be tested and validated and compared with expression profiles of other
268
polycyclic aromatic hydrocarbons/ homologues induction.
269
270
4 Conclusion
271
Benzo(a)pyrene exposure has resulted in changes in the expression of genes involved in
272
calcium homeostasis, cell cycle regulation and inflammatory response in E. fetida. The other
22
273
altered genes due to BaP exposure include those that are involved in nucleotide binding,
274
protein transport, vasodilation, cytoskeletal structure and cell division, microtubule-based
275
movement, establishment of protein localization, lipid metabolic process, spermatogenesis,
276
embryo development ending in birth or egg hatching, wound healing, nervous system
277
development and eye development. Many genes identified in E. fetida in this study have not
278
been reported to be changed by Benzo(a)Pyrene in other organisms. This gene expression
279
data will assist in understanding the toxicological effects at molecular level. Furthermore, this
280
data will be helpful in developing molecular markers for detecting benzo(a)pyrene
281
contamination in soil.
282 283
Acknowledgements:
284
The authors acknowledge the Ramaciotti Centre for Genomics, The University of New South
285
Wales, Sydney, form RNA sequencing and eResearch SA for computing facility. Dr. Srinithi
286
Mayilswami is recipient of IPRS and CRCCARE top-up scholarships.
287 288
Conflict of Interest:
289
The authors declare that they have no conflict of interest
290 291 292
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