Mechanisms of Toxicity of Triphenyltin Chloride (TPTC)Determined by a Live Cell Reporter Array

Mechanisms of Toxicity of Triphenyltin Chloride (TPTC)Determined by a Live Cell Reporter Array

Guanyong Su1,2,Xiaowei Zhang1,*, Jason C. Raine2,Liqun Xing1, Eric Higley2, Markus Hecker2,3, John P. Giesy1,2,,4,5, Hongxia Yu1,*

1 State Key Laboratory of Pollution Control and Resource Reuse & School of the Environment, Nanjing University, Nanjing, China

2 Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada

3 School of Environment and Sustainability, University of Saskatchewan, Saskatoon, SK, S7N 5B3Canada

4Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada

5Department of Biology and Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong, 83 Tat Chee Avenue,Kowloon, Hong Kong SAR, China

Corresponding author:

School of the Environment, Nanjing University, Nanjing, 210089, China

Tel: 86-25-89680623

Fax: 86-25-83707304

E-mail:

(Xiaowei Zhang)

(Hongxia Yu)

Supporting Table 1: Description of Gene Function Groups

Function Group / Description
Enzyme / Genes encoded enzymes, which played an important role in biochemical reaction.
Regulatory Protein / Genes produced protein after gene expression, which were transcriptional regulators, activators or inhibitors, especially for DNA process.
Structure Protein / Genes produced protein after gene expression, which were part of organism structure, for example, membrane protein,water MIP channel,
rRNA or tRNA / Genes in the “RNA” group didn't produce protein but RNA, which might be rRNA or tRNA.
Stress Responsive Pathway / Stress responsive genes.
Amino Acids Formation / Genes were related to the formation of amino acids.
Function Unclear / Genes' functions were still not very clear right now.

Supporting Table 2: Functions of 71 differentially expressed genes

Type / function / Classification
1 / pepE / Protein / (alpha)-aspartyldipeptidase / Enzyme
2 / aroH / Protein / 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase,tryptophan repressible / Enzyme
3 / serB / Protein / 3-phosphoserine phosphatase / Enzyme
4 / add / Protein / adenosine deaminase / Enzyme
5 / gcvT / Protein / aminomethyltransferase / Enzyme
6 / lacZ / Protein / beta-galactosidase, lac operon / Enzyme
7 / cysK / Protein / cysteine synthase A,O-acetylserinesulfhydrolase A subunit / Enzyme
8 / serA / Protein / D-3-phosphoglycerate dehydrogenase / Enzyme
9 / dcd / Protein / Deoxycytidine triphosphate deaminase / Enzyme
10 / dmsA / Protein / dimethyl sulfoxidereductase, chain A / Enzyme
11 / ligB / Protein / DNA ligase / Enzyme
12 / fpr / Protein / ferredoxin-NADP reductase / Enzyme
13 / galU / Protein / glucose-1-phosphate uridylyltransferase / Enzyme
14 / gadB / Protein / glutamate decarboxylase B subunit / Enzyme
15 / metA / Protein / homoserinetranssuccinylase / Enzyme
16 / icd / Protein / isocitrate dehydrogenase / Enzyme
17 / lipA / Protein / lipoate synthase / Enzyme
18 / cueO / Protein / multicopper oxidase with role in copper homeostasis / Enzyme
19 / aroK / Protein / shikimate kinase I / Enzyme
20 / ttdA / Protein / L-tartrate dehydratase / Enzyme
21 / cysB / Protein / Cysregulon transcriptional activator / Regulatory Protein
22 / cspD / Protein / DNA replication inhibitor / Regulatory Protein
23 / ftsK / Protein / DNA-binding membrane protein required for chromosome resolution and partitioning / Regulatory Protein
24 / gadX / Protein / DNA-binding transcriptional dual regulator / Regulatory Protein
25 / phoP / Protein / PhoP transcriptional regulator PhoP transcriptional dual regulator / Regulatory Protein
26 / mcrA / Protein / restriction of DNA at 5-methylcytosine residues / Regulatory Protein
27 / rmf / Protein / ribosome modulation factor / Regulatory Protein
28 / rsd / Protein / regulator of sigma D stationary phase protein, binds sigma 70 RNA polymerase subunit / Regulatory Protein
29 / wrbA / Protein / The purified WrbA protein has NAD(P)H:quinoneoxidoreductase activity / Regulatory Protein
30 / fdhD / Protein / affects formate dehydrogenase-N / Regulatory Protein
31 / flgM / Protein / Negative regulator of flagellin synthesis / Regulatory Protein
32 / manX / Protein / PTS system mannose-specific EIIAB component / Regulatory Protein
33 / cld / Protein / regulator of length of O-antigen component of lipopolysaccharide chains / Regulatory Protein
34 / rpsT / Protein / 30S ribosomal subunit protein S20 / Structure Protein
35 / rpsU / Protein / 30S ribosomal subunit protein S21 / Structure Protein
36 / mglB / Protein / D-galactose-binding periplasmic protein / Structure Protein
37 / ytfF / Protein / inner membrane protein / Structure Protein
38 / b1403 / Protein / IS21 protein 2 / Structure Protein
39 / ompC / Protein / outer membrane porin protein C / Structure Protein
40 / ompA / Protein / outer membrane protein 3a (II*Gd) / Structure Protein
41 / skp / Protein / periplasmic molecular chaperone for outer membrane proteins / Structure Protein
42 / osmC / Protein / resistance protein, osmotically inducible / Structure Protein
43 / aqpZ / Protein / water MIP channel / Structure Protein
44 / rrnB / rRNA / The 5S and 23S rRNAs are the RNA components of the large subunit (50S subunit) of the E. coliribosome.There are seven ribosomal RNA (rRNA) operons, called rrnA, rrnB, rrnC, rrnD, rrnE, rrnG, and rrnH / RNA
45 / rrnD / rRNA / The 5S and 23S rRNAs are the RNA components of the large subunit (50S subunit) of the E. coliribosome.There are seven ribosomal RNA (rRNA) operons, called rrnA, rrnB, rrnC, rrnD, rrnE, rrnG, and rrnH / RNA
46 / rrnC / rRNA / The 5S and 23S rRNAs are the RNA components of the large subunit (50S subunit) of the E. coliribosome.There are seven ribosomal RNA (rRNA) operons, called rrnA, rrnB, rrnC, rrnD, rrnE, rrnG, and rrnH / RNA
47 / argW / tRNA / tRNA(argW) is one of seven arginine tRNAs / RNA
48 / evgA / Protein / response regulator in two-component regulatory system with EvgS，involved in acid resistance, osmotic adaption, and drug resistance / Environmental Stress Response
49 / uspB / Protein / ethanol tolerance protein / Component of Organism
50 / uspA / Protein / universal stress global stress response regulator / Environmental Stress Response
51 / menG / Protein / S-adenosylmethionine: 2-demethylmenaquinone methyltransferaseproteinE. The interaction of RraA with the degradosome is facilitated by protein-RNA remodeling via the ATPase activity of RhlB / Amino Acids Formation
52 / yfbV / Protein / conserved inner membrane protein / Function Unclear
53 / mntR / Protein / conserved protein / Function Unclear
54 / yhgF / Protein / conserved protein (3rd module) / Function Unclear
55 / yjdI / Protein / conserved protein;Uncharacterized protein yjdI / Function Unclear
56 / yhcG / Protein / function unknow / Function Unclear
57 / yafK / Protein / hypothetical protein / Function Unclear
58 / insA_7 / Protein / hypothetical protein / Function Unclear
59 / yaiE / Protein / hypothetical protein，UPF0345 family，function unknown / Function Unclear
60 / b3007 / Protein / unknown CDS / Function Unclear
61 / yhhY / Protein / predicted acetyltransferase / Function Unclear
62 / yeaU / Protein / predicted dehydrogenase D-malate dehydrogenase (decarboxylating) / Function Unclear
63 / glgS / Protein / predicted glycogen synthesis protein / Function Unclear
64 / proQ / Protein / predicted structural transport element / Function Unclear
65 / yedW / Protein / putative 2-component transcriptional regulator（yedV） / Function Unclear
66 / yedP / Protein / Putative mannosyl-3-phosphoglycerate phosphatase / Function Unclear
67 / ymcC / Protein / putative synthetase / Function Unclear
68 / yfiE / Protein / putative transcriptional regulator (LysR family) / Function Unclear
69 / uspF / Protein / putative universal stress protein F / Function Unclear
70 / yifE / Protein / Similar to Yersinia pestis KIM, hypothetical protein y0333 / Function Unclear
71 / ybcW / Protein / Uncharacterized protein / Function Unclear
Species Scientific Name / Species Group / Endpoint / Duration (Days) / Concentration (ng/mL) / Calculation of NOEC (ng/mL)a / Mean of (ng/mL)
Ankistrodesmusfalcatus ssp. acicul(Wong et al. 1982) / Algae Moss or Fungi / EC50 / 8 / 2.0 / 4.0×10-1 / 4.0×10-1
Scenedesmusquadricauda(Fargasova 1997) / Algae Moss or Fungi / EC50 / NR / 9.1×10-1 / 1.8×10-1 / 1.0×102
Scenedesmusquadricauda(Fargasova 2002) / Algae Moss or Fungi / EC50 / 12 / 1.1×103 / 2.3×102
Scenedesmusquadricauda(Fargasova 2002) / Algae Moss or Fungi / EC50 / 12 / 3.5×102 / 7.0×101
Oryziaslatipes
(De Vries et al. 1991) / Fish / LC50 / 2 / 6.4×101 / 1.4 / 1.4
Oncorhynchusmykiss(Nagase et al. 1991) / Fish / LOEC / 110 / 2.3×10-1 / 9.3×10-2 / 9.3×10-2
Indoplanorbisexustus(Goel and Prasad 1978) / Molluscs / LC50 / 1 / 6.2×10-4 / 1.3×10-5 / 3.7
Indoplanorbisexustus(Goel and Srivastava 1981) / Molluscs / LC50 / 1 / 3.5×102 / 7.4
Lymnaeaacuminate (Goel and Srivastava 1981) / Molluscs / LC50 / 1 / 4.3×101 / 9.1×10-1 / 9.1×10-1
Tubifextubifex
(Fargasova 1997) / Worms / LC50 / 4 / 2.4 / 5.1×10-2 / 5.1×10-2

Supporting Table 3: References of NOEC for different species exposure to TPTC

aNOEC was calculated following “Guidelines for Deriving Numerical National Water Quality Criteria for the Protection of Aquatic Organisms and their Uses”, which was drafted by U.S. Environmental Protection Agency.

References:

De Vries H, Penninks AH, Snoeij NJ, Seinen W (1991) COMPARATIVE TOXICITY OF ORGANOTIN COMPOUNDS TO RAINBOW TROUT ONCORHYNCHUS-MYKISS YOLK SAC FRY. Science of the Total Environment 103 (2-3):229-244

Fargasova A (1997) Comparative study of ecotoxicological effect of triorganotin compounds on various biological subjects. Ecotoxicology and Environmental Safety 36 (1):38-42

Fargasova A (2002) Structure-affected algicidal activity of triorganotin compounds. Bulletin of Environmental Contamination and Toxicology 69 (5):756-762. doi:10.1007/s00128-002-0125-3

Goel HC, Prasad R (1978) Action of Molluscicides on Freshly Laid Eggs of Snail Indoplanorbis-Exustus (Deshayes). Indian Journal of Experimental Biology 16 (5):620-622

Goel HC, Srivastava CP (1981) Laboratory evaluation of some molluscicides against french water snails, Indoplanorbis and Lymnaea species. J Commun Dis 13 (2):121-127

Nagase H, Hamasaki T, Sato T, Kito H, Yoshioka Y, Ose Y (1991) Structure-Activity-Relationships for Organotin Compounds on the Red Killifish Oryzias-Latipes. Applied Organometallic Chemistry 5 (2):91-97

Wong PTS, Chau YK, Kramar O, Bengert GA (1982) Structure-Toxicity Relationship of Tin-Compounds on Algae. Canadian Journal of Fisheries and Aquatic Sciences 39 (3):483-488

SupportingTable 4: Classification of differentially expressed genes and proportion of each groups basing on the function of genes (see in Supplementary Table 1) and fold change.

Fold Change / Genes / Number
1.5 - 2.0 / cysK, dcd, dmsA, gadB,fpr,gcvT, pepE,metA,serA, ttdA,icd, ligB, lipA, cueO, aroK, fdhD, flgM, ftsK, gadX, manX,rsd,phoP,cysB, cspD, ompA, ompC, osmC, ytfF,rpsT, rpsU, b1403, rrnB, rrnD, argW, uspA, uspB, evgA,yaiE, yeaU, yedP,yedW, yfbV, proQ,yfiE, yhgF, ymcC, b3007, yafK,mntR / 49
2.0 - 3.0 / lacZ, serB,galU,aroH,add, wrbA,cld,rmf, mglB, skp, aqpZ, glgS, insA_7, uspF, yhcG, yhhY, yifE, yjdI / 18
3.0 - 4.0 / mcrA, menG, ybcW / 3
7.0 - 8.0 / rrnC / 1

Each color represents one toxic pathway induced by TPTC, and detailed description of each toxic pathway can be found in Supplementary Table 1. О“enzyme”; О “Regulatory Protein”;О“Component of Organism”;О“rRNA or tRNA”;О “Environmental Stress Response”;О “Amino Acids Formation”;О “Function Unclear”.

Supporting Figure 1: Gene expression profiles of three potential biomarkers.