Gender-Specific Toxicological Effects of Chronic ... - ACS Publications

Subscriber access provided by UNIV OF CALIFORNIA SAN DIEGO LIBRARIES

Article

Gender-specific toxicological effects of chronic exposure to pure microcystin-LR or complex Microcystis aeruginosa extracts on adult medaka fish Séverine Le Manach, Nour Khenfech, Hélène Huet, Qin Qiao, Charlotte Duval, Arul Marie, Gérard Bolbach, Gilles Clodic, Chakib Djediat, Cécile Bernard, Marc Edery, and Benjamin Marie Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01903 • Publication Date (Web): 13 Jul 2016 Downloaded from http://pubs.acs.org on July 16, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

Environmental Science & Technology

1

Gender-specific toxicological effects of chronic exposure to pure

2

microcystin-LR or complex Microcystis aeruginosa extracts on adult

3

medaka fish

4 5

Séverine Le Manach1, Nour Khenfech1, Hélène Huet1,2, Qin Qiao1, Charlotte Duval1, Arul

6

Marie1, Gérard Bolbach3, Gilles Clodic3, Chakib Djediat1, Cécile Bernard1, Marc Edery1,

7

Benjamin Marie1*

8 9

1UMR

7245 MNHN/CNRS Molécules de Communication et Adaptation des Micro-

10

organismes, Sorbonne Universités, Muséum National d’Histoire Naturelle, CP 39, 12

11

Rue Buffon, 75005 Paris, France.

12

2Ecole

13

Maison-Alfort, France.

14

3Institut

15

Protéomique, Sorbonne Universités, Université Pierre et Marie Curie, 75005 Paris,

16

France.

Nationale Vétérinaire d’Alfort, Université Paris-Est, BioPôle Alfort, 94700

de Biologie Paris Seine/FR 3631, Plateforme Spectrométrie de masse et

17 18

* Corresponding author: [email protected]

19

ACS Paragon Plus Environment


20

Abstract

21

Cyanobacterial blooms often occur in freshwater lakes and constitute a potential health risk to

22

human populations, as well as to other organisms. However, their overall and specific

23

implications for the health of aquatic organisms that are chronically and environmentally

24

exposed to cyanobacteria producing hepatotoxins, such as microcystins (MCs), together with

25

other bioactive compounds have still not been clearly established and remain difficult to

26

assess. The medaka fish was chosen as the experimental aquatic model for studying the

27

cellular and molecular toxicological effects on the liver after chronic exposures (28 days) to

28

environmentally relevant concentrations of pure MC-LR, complex extracts of MC producing

29

or non-producing cyanobacterial biomasses, and of a Microcystis aeruginosa natural bloom.

30

Our results showed a higher susceptibility of females to the different treatments compared to

31

males at both the cellular and the molecular levels. Although hepatocyte lysis increased with

32

MC-containing treatments, lysis always appeared more severe in the liver of females compare

33

to males, and the glycogen cellular reserves also appeared to decrease more in the liver of

34

females compared to those in the males. Proteomic investigations reveal divergent responses

35

between males and females exposed to all treatments, especially for proteins involved in

36

metabolic and homeostasis processes. Our observations also highlighted the dysregulation of

37

proteins involved in oogenesis in female livers. These results suggest that fish populations

38

exposed to cyanobacteria blooms may potentially face several ecotoxicological issues.

39 40


Page 2 of 40

Page 3 of 40


41

Introduction

42

Cyanobacteria play important roles in aquatic ecosystems because they make a significant

43

contribution to primary production and, for some species, to the fixation of atmospheric

44

nitrogen1. However, local cyanobacteria proliferations can also disrupt the functioning of

45

aquatic ecosystems and constitute a major cause of concern for both public health and ecology

46

as a result of the ability of several species and genera to proliferate and to produce harmful

47

toxins, so-called cyanotoxins2. The most common and well-studied cyanotoxin-producing

48

cyanobacterium, Microcystis, is one of the most widespread and important toxin-producing

49

cyanobacteria genera in worldwide lakes, in terms of both abundance and distribution3-6.

50

The concerns about the toxicological potential of these freshwater cyanobacteria have mainly

51

focused on their production of microcystins (MCs), a diverse family of cyclic heptapeptide

52

hepatotoxins that are considered the most common toxins of cyanobacteria. Among MC

53

structural diversity variants, the MC-LR variant, the most frequently detected MC variant in

54

the environment, is also the variant that exhibits the highest concentrations in lakes7 and has

55

the greatest potential for toxicity to aquatic organisms8.

56 57

Cyanotoxins, such as MCs, mostly enter an organism using the food pathway, cross the

58

intestinal wall, and move through the portal venous system to reach the liver, in which they

59

accumulate: the liver exhibits very high tropism for various drugs and/or chemicals, such as

60

cyanotoxins, and notably, MCs9-10. MCs are known to enter a hepatocyte liver cell as a result

61

of their high content in transmembranal anionic biliary-acid transporters, which induce

62

molecular defects though a cascade of reactions following the inhibition of phosphatase PP1

63

and PP2 activity due to MC-specific fixation of those proteins7,8,11. The mechanisms of MC

64

toxicity and detoxification in fish are believed to be similar to those reported in mammals12-15.

65

However, for aquatic organisms such as fish, most investigations on MC toxicity remains



66

based on gavage experiments, one-time force-feeding experiments16, or short-term dietary

67

exposure bioassays17 that determined the acute effects at cellular and molecular levels18-21.

68

The main toxicological pathways that resulted in the acute effects of MC on hepatocytes are

69

the production of reactive oxygen species production, the occurrence of oxidative stress, and

70

cytoskeletal dysregulation, together with the induction of apoptosis18-22.

71 72

To date, only limited information is available on the fine chronic effects of an aqueous MC

73

exposure on fish under balneation20,23-26, which might potentially be the major and natural

74

route of MC toxicity to fish in their environment9,27-28. A small number of these studies were

75

dedicated to the investigation of chronic effects of MC exposure at the cellular or molecular

76

levels, and all of them were focused on pure MC-LR or -RR effects only4,20,26,29-32. Only a

77

restricted number of ecotoxicological studies investigated the potential chronic effects of

78

complex cyanobacterial cells and lysates that contain other compounds24,25,33-35, many of

79

which are only now being identified,36-40 and their potential toxicological effects are being

80

revealed33,41-45. In addition to the classically described cyanotoxins (microcystins,

81

cylindrospermopsins, anatoxins or saxitoxins), cyanobacteria can also produce numerous

82

other secondary metabolites such as microviridins46, microginins37, oscillapeptins47,

83

cyanopeptolins or aeruginosins40, through non-ribosomal peptide synthase/polyketide

84

synthase (NRPS/PKS) pathways37; these secondary metabolites may also have concrete

85

deleterious biological effects on fish.

86 87

Although various studies have already been performed to determine pure cyanotoxin effects,

88

it remains a key issue to elucidate the underlying molecular mechanisms of the toxicological

89

response of aquatic organisms, such as fish, that are chronically exposed to a range of

90

cyanobacterial metabolites for both environmental and toxicological purposes. To learn more


Page 4 of 40

Page 5 of 40


91

about the chronic effects of the Microcystis aeruginosa secondary metabolites, comprising

92

MCs, we investigated their cellular and molecular effects on a fish model, the medaka Oryzias

93

latipes18, exposed to various cyanobacterial extracts. To investigate the ecotoxicological

94

effect of various Microcystis biomass containing or not MCs, we performed complementary

95

pathological and molecular approaches on the liver of chronically exposed adult medaka fish.

96

The fish liver represents the most suitable organs for this study because it both constitutes the

97

primary target of hepatotoxins and is the principal detoxification organ, integrating the whole

98

organism responsiveness to xenobiotics11,17,32. Through the systematic analyses of the cellular

99

and molecular alterations induced in adult medaka after chronic exposures to various

100

cyanobacterial extracts, containing or not MCs, we contribute to generate new information on

101

the environmental hazard and risk assessment of cyanobacteria to aquatic organisms.

102 103

Experimental section

104

Preparation of exposure extracts

105

Microcystis strain cultures. The monoclonal Microcystis aeruginosa strains PCC 7820 and

106

PMC 570.08 high-producer MCs and non-producer MCs, respectively, along with other

107

secondary metabolites (supplementary figure S1A-B). The strains were maintained in the

108

Paris Museum Collection (PMC) of cyanobacteria and cultured in Z8 medium48 (25°C, 16 h:8

109

h light:dark photoperiod at 16 µmol of photon.m-2.s-1) under non-axenic conditions for large

110

biomass production, prior to methanol extraction.

111

Microcystis bloom sampling. The recreational lake located near the city of Champs-sur-

112

Marne (48°51′47′′ N, 02°35′53′′ E, France) has a surface area of 0.1 km2 with an average

113

depth of 2.5 m, and since 2006, it has experienced several episodes of cyanobacterial

114

blooms49. During the summer of 2011, raw water was sampled weekly, the chlorophyll a (Chl

115

a) concentration was measured and the cyanobacterial genera or species (>20 µm) were



116

determined as described previously50. The MC concentration was determined using AD4G2

117

ELISA tests (Abraxis). During one of the main Microcystis aeruginosa bloom (98% of the

118

total phytoplankton biomass) events with high-producing MCs that occurred on 09/19/2011, a

119

large cyanobacteria biomass was concentrated with a specific net for phytoplankton (200 µm)

120

and was collected in a 2 L-bottle for the secondary metabolite extraction (supplementary

121

figure S2).

122

Microcystis secondary metabolite extraction. The Microcystis aeruginosa biomasses from

123

the 2 cultured strains and from the bloom described above were filtered and freeze-dried. The

124

lyophilized cells were then sonicated in 80% methanol, centrifuged at 4°C (4,000 g; 10 min)

125

and filtered (GF/C 1.2 µm); then, the supernatant was evaporated as described previously51.

126

The dried extract was dissolved in 50% ethanol (Vol/Vol) and then partially evaporated to

127

remove the ethanol prior to the experimentation. The metabolite compositions of the 3

128

extracts were then investigated using LC-MS/MS performed on ESI-qTOF/TOF, and the MCs

129

were quantified using AD4G2 ELISA tests (Abraxis).

130

Metabolite analysis by mass spectrometry.

131

High performance liquid chromatography (HPLC) was performed on 5 µL of each of the

132

metabolite extracts using a capillary 1 mm-diameter C18 column (Discovery Bio wide pore

133

5 µm, Sigma) at a 50 µL.min-1 flow rate with a gradient of acetonitrile in 0.1% formic acid

134

(10 to 80% in 60 min). The metabolite contents were analyzed at least three times using an

135

electrospray ionization hybrid quadrupole time-of-flight (ESI-QqTOF) hybrid mass

136

spectrometer (QStar® Pulsar i, Applied Biosystems®, France) on positive mode with

137

information dependent acquisition (IDA), which allowed for switching between MS and

138

MS/MS experiments, as previously described44. The data were acquired and analyzed with the

139

Analyst QS software (Version1.1). Peak lists were generated from MS/MS spectra between

140

10 and 55 min, with a filtering noise threshold at 2% maximal intensity and combining


Page 6 of 40

Page 7 of 40


141

various charge states and related isotopic forms. Metabolite annotation was attempted

142

according to the precise mass of the molecules and their respective MS/MS fragmentation

143

patterns with regards to an in-house database of above 600 cyanobacteria metabolites that

144

were previously described in reference publications.

145 146

Medaka chronic exposure

147

Experimental design. Medaka fish (Oryzias latipes) belonging to the inbred Cab strain were

148

reared, and experiments were performed in accordance with European Union regulations

149

concerning the protection of experimental animals and the validation of experimental

150

procedures by the ethical committee of the “museum national d’histoire naturelle” – MNHN

151

(N°68-040 for 2013-18). One month prior to the chronic experiments, 5 month-old adult fish

152

were maintained at 25 ± 1°C with a 15 h:9 h light:dark cycle (reproductive cycle) to induce

153

their reproductive activity. Fish were randomly assigned to one of the 5 experimental groups,

154

namely “control” for no toxin balneation with control solvent conditions; “MC-LR” for the

155

exposure to pure MC-LR, “MicA+” for the Microcystis aeruginosa MC-producing strain;

156

“MicA-” for the Microcystis aeruginosa non- producing MC strain; and “Bloom+” for

157

Microcystis aeruginosa bloom producing MC. Each treatment comprised 15 males and 15

158

females maintained in a 30-L aquarium. The exposure dose of MCs was fixed to the

159

frequently observed environmental concentration of 5 µg equivalent MC-LR L-1 for MC-LR,

160

MicA+ and Bloom+ groups, whereas the quantity of MicA- extract was adjusted to be

161

equivalent to the biomass used for the MicA+ concentration, and no toxin or extract was

162

added to the control tank. The experiment was performed for 28 days, and the exposure

163

condition maintained by renewal of a third of the total aquarium volume (10 L) every 2 or 3

164

days. Fish were inspected three times daily, and no abnormal behavior, nor mortality was

165

observed throughout the experiment. After 28 days of exposure, the fish were briefly



166

anesthetized in buffered 0.1% MS-222 and sacrificed, and the liver samples were collected for

167

analysis. Circulating estradiol E2 levels were measured in 3 plasma pools of 3 males and 3

168

females using a commercial ELISA test (Biosense laboratories) following protein

169

quantification using a bicinchoninic acid (BCA) test, which was performed with a bovine

170

serum albumin (BSA) protein standard.

171 172

Liver sample analyses.

173

Histopathology. Liver samples were fixed in cold 10% buffered formalin (4°C, 48 h),

174

transferred into 70% ethanol, dehydrated in successive baths of ethanol (from 70 to 95%), and

175

then embedded in paraffin. Blocks were cut into 3- to 5-µm thick sections, and slides were

176

stained (with hematoxylin-eosin-saffron (HES) or periodic acid-Schiff (PAS)), according to

177

standard histological procedure. For each individual, the hepatocyte lysis surface was

178

determined from a blind assessment of 3-5 liver sections of the HES-stained slides, and the

179

glycolysis index (scores = 0-3) was visually determined on 3 sub-sampled areas from one

180

section of PAS-stained livers by two different researchers. Significant differences among the

181

various experimental groups (n=6-9 individual for each sex) were investigated with non-

182

parametric tests using Kruskal-Wallis or Mann and Whitney-Wilcoxon methods, which are

183

suitable for small data sets.

184

Quantitative proteomic analysis. Liver tissues from 3 fish per treatment were pooled, and

185

the content protein was extracted and quantified as previously described44. One hundred µg of

186

each liver protein pool, prepared as described above, was used for the digestion with 5 µg of

187

proteomic-grade trypsin (Sigma-Aldrich, USA) and the sample was labelled, following the

188

manufacturer’s protocol for the 8-plex iTRAQ kit (Applied Biosystems®, France).

189

Mass spectrometry analysis. iTRAQ-based quantitative proteomic analysis was performed

190

using nano-LC (Ultimate 3000, Dionex) coupled with an ESI-LTQ-Orbitrap (LTQ Orbitrap


Page 8 of 40

Page 9 of 40


191

XL, Thermo Scientific) mass spectrometer. Six µg of iTRAQ-tagged liver protein digests

192

solubilized in 10% ACN with 0.1% formic acid were injected in triplicate by the autosampler

193

and were concentrated on a trapping column (Pepmap, C18, 300 µm x 50 mm, 3 µm 100 Å,

194

Dionex) with water containing 10% ACN with 0.1% formic acid (solvent A). After 5 min, the

195

peptides were eluted onto a separation column (Pepmap, C18, 75 µm x 500 mm, 2 µm 100 Å,

196

Dionex) equilibrated with solvent A. The peptides were separated with a 2 h-linear gradient,

197

increasing from 10% to 80% ACN + 0.1% formic acid (solvent B) at a flow rate of 200

198

nl.min-1. Spectra were measured at a mass scan range of m/z 300-2000 at a resolution of

199

30,000 in the profile mode followed by data dependent CID and/or HCD fragmentation of the

200

ten most intense ions, with a dynamic exclusion window of 60 s.

201

Proteomic data treatment. All data were processed using Mascot 2.4.1 (Matrix Science,

202

UK) and using X!Tandem with Scaffold software (version 3.0; Proteome Software, USA)

203

compared against Ensembl databases for fishes (restricted to Oryzias latipes, Danio rerio and

204

Tetraodon nigroviridis sequences in the Ensembl database V68). The ion mass tolerance and

205

the parent ion tolerance were set to 0.50 Da. The methyl methane-thiosulfonate of cysteine

206

was specified as a fixed modification. The oxidation of methionine and the iTRAQ 8-plex of

207

tyrosine for iTRAQ-derivatized samples were specified as variable modifications. Scaffold

208

was used to probabilistically validate the protein identifications derived from the MS/MS

209

sequencing results using the X!Tandem algorithms.

210

Data analyses. Scaffold Q+ was used to quantify the isobaric tag peptide and protein

211

identifications as previously described44. Quantitative ratios were log2 normalized for final

212

quantitative testing, and the control value was used as the reference sample in both sexes. The

213

heatmap

214

(http://www.broadinstitute.org/cancer/software/GENE-E/) with Spearman correlation’s value

215

for hierarchical clustering analysis for both samples and proteins. The statistical significance

protein

quantification

was

represented


using

Gene-E

freeware


216

of the differential expression of the proteins was investigated using Kruskal-Wallis tests with

217

a 0.5 log2 fold change (FC) threshold. The molecular pathway was determined using the

218

Ingenuity Pathway Analysis software (V01-04; Qiagen) with the Human orthologous of

219

medaka proteins available from the Ensembl online platform (http://www.ensembl.org),

220

according to the specific Ingenuity Knowledge Database (using default parameters for all

221

tissues and cell lines, with relaxed filters), which constitutes a repository of biological

222

interactions and functional annotations.

223 224

Results and discussion

225

Metabolite compositions of Microcystis culture and bloom extracts.

226

The secondary metabolite compositions of the two monoclonal Microcystis strains and the

227

Microcystis dominated bloom were determined using liquid chromatography coupled with

228

mass spectrometry (ESI-MS/MS) as represented in figure 1 (A-C). Both extracts exhibited a

229

large diversity of molecules, and their molecular annotations were performed according to

230

their respective precise mass and, if available, with their corresponding MS/MS fragmentation

231

patterns, containing a global matching pattern or several signature ions that are specific to

232

some metabolite families37 (supplementary table S1). We observed 40, 25 and 28 metabolites

233

for the MicA+, MicA- and bloom+ extracts, respectively, and a global composition

234

comparison showed that only a very limited number of metabolites were common between

235

the different extracts (figure 1D). In MicA+ extract, 6 MCs variants could be detected and

236

annotated: MC-LR, (DAsp3)-MC-LR, (DMha3)-MC-LR, (DAsp3-DMha7)-MC-LR, MC-AR

237

and 1 other putative uncharacterized variant Along with these MC variants, together with

238

microginin FR5, 5 cyclamides, 4 cyanopeptolins, and other uncharacterized molecules were

239

also detected in the MicA+ extract. The Microcystis bloom+ extracts contained

240

cyanopeptides, comprising higher peak counts for MC-LR, MC-YR, MC-RR and (DAsp3)-


Page 10 of 40

Page 11 of 40


241

MC-LR (in decreasing order), along with 3 other putative uncharacterized MC variants, 6

242

potential cyanopeptolins, 2 potential aeruginosins, 1 aeruginosamide 560, and other

243

uncharacterized molecules. In contrast, the MicA- extract, which lack any MCs, contained

244

different microginin variants (FR3 and FR4, plus potentially Tyr-Tyr deleted fragments),

245

along with cyanopeptolin 974, anabaenopeptin F and various other components of unknown

246

structures.

247

We confirm here that Microcystis aeruginosa produced a wide diversity of secondary

248

metabolites, including microcystins, microginins, aeruginosins, cyanopeptolins, cyclamides or

249

anabaenopeptides as was suggested through recent genome mining approaches performed in

250

this species. These observations also highlight that blooms might produce a wider metabolite

251

diversity as blooms comprise a co-dominance of various clones, producing different

252

metabolite sets3,5,6. These combined observations illustrate the complexity, and the global

253

dissimilarities of the studied Microcystis extracts. Therefore, the chronic deleterious effects

254

were further investigated on adult medaka fish, with regard to similar environmental

255

concentrations of MCs.

256 257

Effect on liver hepatocyte lysis and glycogen contents

258

The liver is an important organ that plays various vital functions, which include the process

259

and the storage of nutrients, maintenance of serum composition, bile production, and

260

xenobiotic detoxification. Liver from both sexes of medaka exposed to control conditions

261

presented a typical architectural organization with polyhedral hepatocytes organized around

262

the capillary sinusoids and the bile canaliculi, appearing in typical cord-like parenchymal

263

structures. As shown in figure 2A-D, the liver of medaka fish exposed to control conditions

264

presented a noteworthy sexual dimorphism: this is illustrated at the cellular level from the

265

histological observations of the hepatocytes. Hepatocytes of female fish presented large



266

reserve vesicles (very likely containing glycoprotein and/or glycogen, stained in purple with

267

PAS44), which appears isolated from the rest of the cytoplasm contents, whereas hepatocytes

268

of male fish exhibited a more diffuse cytoplasm that contained small inclusions. Indeed, in

269

mature fish, as in other oviparous vertebrates, the liver of the female plays an important

270

function in the production of the oocyte envelope and vitellogenin reserves, whereas the male

271

liver hepatocytes do not exhibit such activity. The liver globally presents sexual morphologic,

272

molecular and functional dimorphisms52-56.

273

Here, we determined that significant cellular impacts were detected by histology observation

274

of liver sections in both hepatocyte lysis and glycogen content levels with exposures to MC-

275

LR, MicA+ and bloom+ treatments in both sexes (figure 2E-F and figure 3). Those increases

276

in the hepatocyte lysis area, concomitant with a clear decrease in intra-hepatocyte glycogen

277

reserves, represent genuine evidence of the cellular hepatotoxicity of the various treatments

278

that contain at least 5 µg.L-1 MC-LR or equivalent MC content. The increase in the

279

hepatocyte lysis area may be the result of diffuse cellular necrotic or apoptotic events induced

280

by hepatotoxic treatments57, and its association with the decrease of intracellular glycogen

281

contents might reveal the induction of a true chronic hepatic stress induction. Interestingly,

282

previous investigations performed on whitefish chronically exposed to MC-producing

283

Planktothrix rufescens34 have shown gastrointestinal histological alterations characterized by

284

“granulated cytosol, reduced glycogen stores, disintegration of the parenchymal liver

285

architecture, cell dissociation, …”, the severity of these effects being dependent on the

286

quantity of toxic cells. Taken together, these observations support the idea that noticeable

287

cellular liver damages are induced in a dose dependent manner by environmental relevant

288

amount cyanobacterial toxic compounds, such as the MCs.

289

We observed also that in all of the experiment treatments that the fish were exposed to in this

290

study, the female medaka fish livers exhibited higher lysis areas associated with lower


Page 12 of 40

Page 13 of 40


291

glycogen content than the male livers (p0.5 and P0.5 log2 FC) in male and female livers.

813

Proteinaceous quantification according to iTRAQ analyses was normalized according to male

814

(M) and female (F) control quantification values and is represented with a log2 fold change

815

scale. Down-regulated proteins are indicated in red and up-regulated proteins in green

816

(threshold of 0.5 log2 FC, darker colors indicate when dysregulation is greater than 1 log2

817

FC). * indicates the three proteins highly dysregulated in both sexes.


Page 34 of 40

Page 35 of 40


Fig. 1

B

C

Ion count

A D

Molecular mass (Da)



Page 36 of 40

Fig. 2

c

v

n

v

c

m

m n

20%

F ## ##

male female

% of cellular lysis

★★

15%

** *

3 Glycogen-‐reserve index

E

** **

10% 5%

Controle

MC-‐LR

MicA+

MicA-‐

Bloom+

male

** **

female

2

## ##

1 0

0%

** **

Controle


MC-‐LR

MicA+

MicA-‐

Bloom+

★★

Page 37 of 40


Fig. 3

MC-‐LR MicA-‐

Control

MicA+

Bloom+

MC-‐LR MicA-‐


Control

MicA+

Bloom+


Page 38 of 40

Fig. 4

M Control F Control M MicA-‐ M MC-‐LR M Bloom+ M MicA+ F MicA-‐ F MC-‐LR F Bloom+ F MicA+


Page 39 of 40


Fig. 5

15 3 34

M MC-‐LR M MicA+ M MicA-‐ M Bloom+ METABOLISM Glycosyltransferase Acyl-‐CoA binding protein Betaine-‐homocysteine methyltransferase 1* FaQy acid-‐binding protein Saccharopine dehydrogenase a Aldehyde dehydrogenase 1 Acyl-‐Coenzyme A oxidase 3 Acetyl-‐Coenzyme A acyltransferase 1 FaQy acid amide hydrolase Phytanoyl-‐CoA 2-‐hydroxylase* Phenylalanine hydroxylase HOMEOSTASIS PROCESS Alpha-‐type globin Alpha-‐type globin Beta-‐type globin Cytochrome P450 4B* TRANSLATION Ribosome binding protein 1 Nucleolin Ribosomal protein SA

0 -‐0.4 -‐0.7 -‐0.4 -‐0.4 0.1 0.5 0.6 0.7 0.7 0.6

-‐0.6 -‐1.1 0 -‐0.6 -‐1 0.1 0.1 0.3 0.7 0.5 0.7

-‐0.5 -‐0.4 0 2.5 -‐0.3 0.8 0.6 0.3 0.7 0.4 0.6

-‐0.1 -‐0.6 -‐0.8 -‐0.7 -‐0.4 0 0.3 0.5 0.4 0.6 1.1

0.1 0.2 0.3 0.6

0.5 0.5 0.5 0.9

0.6 0.7 0.8 0.4

0 0.3 0.2 0.6

-‐0.2 -‐0.2 0.2

-‐0.6 -‐0.6 0.7

-‐0.5 -‐0.1 0.2

-‐0.1 0 0.1

METABOLISM HisBdine ammonia-‐lyase ATP synthase subunit alpha Aldehyde dehydrogenase 4A1 Phosphoethanolamine methyltransferase UDP glucuronosyltransferase 1 FaQy acid binding protein 10b Phytanoyl-‐CoA 2-‐hydroxylase* FaQy acid binding protein 10a UDP-‐glucose pyrophosphorylase 2a Sulfurtransferase Betaine-‐homocysteine methyltransferase 1* Betaine-‐homocysteine methyltransferase 2 4-‐aminobutyrate aminotransferase Dihydropyrimidine dehydrogenase b Alanine-‐glyoxylate aminotransferase a Acetyl-‐CoA acetyltransferase 2 REPRODUCTION PROCESS Vitellogenin 1 Vitellogenin II precursor Choriogenin H precursor Choriogenin L HOMEOSTASIS PROCESS Glutaredoxin Transferrin-‐a Uricase Catalase Cytochrome P450 4B* Cytochrome P450 3A Cytochrome P450 8B Cytochrome P450 2A Glutathione S-‐transferase A-‐like Glutathione S-‐transferase zeta 1 Superoxide dismutase 1 OTHER FUNCTIONS CalreBculin like Ribosomal protein S17 Uncharacterized protein Cathepsin D Complement component C3-‐1 CyBdine deaminase


F MC-‐LR

F MicA+

F MicA-‐ F Bloom+

-‐0.9 -‐0.6 -‐0.3 -‐0.5 0.1 0.4 0.3 0.4 0.1 0.2 0.3 0.6 0.7 0 0.6 1.5

-‐1.7 -‐0.5 -‐1.3 -‐0.6 0.2 -‐0.7 0 -‐0.1 0.1 0.6 -‐0.1 -‐0.1 0 0.7 0 2.2

-‐1.7 -‐1.8 -‐0.7 -‐0.4 -‐1.7 0.1 0 0.4 0.2 0.3 0 0.2 0.4 0 0.2 1.7

-‐1.7 -‐1.8 -‐0.4 -‐0.4 -‐0.1 0.4 0.6 0.9 0.7 0.3 0.8 0.8 0.5 1.2 1 1.1

-‐0.6 -‐0.3 -‐0.6 -‐0.6

-‐0.5 -‐0.3 -‐0.2 -‐0.2

-‐0.5 -‐0.2 0.1 -‐0.2

-‐1 -‐0.7 -‐0.9 -‐0.9

0.4 0.2 0.4 0.3 0.6 0.7 0.6 0.7 0.2 0.2 0.5

-‐0.7 0.3 -‐0.1 -‐0.1 -‐0.3 0.1 0.1 0.1 -‐0.4 0.5 0.5

0.2 0.1 0.1 0 -‐0.3 0.1 -‐0.1 0.3 -‐0.1 0.1 0.5

0.2 0.6 0.7 0.7 0.6 0.7 0.9 0.4 1 0.8 0.6

-‐0.6 -‐0.7 0.4 0.2 0.4 0.6

0.1 -‐0.1 0.2 0.5 1.1 0.6

-‐0.2 -‐0.2 0.1 0.3 0.1 0.4

-‐0.2 -‐0.1 0.8 0.8 0.8 0.5


Page 40 of 40

1/ MC-‐LR

Microcys(s aeruginosa

2/ MicA-‐

250000 200000 150000

100000

50000

0 0

500

100000

1000

1500

2000

28-‐days chronic exposure to environmental concentraBons

50000

0 0

500

1000

1500

2000

4/ Bloom+

200000 150000 100000

Oryzias La(pes

50000 0 0

500

15% 10%

1000

Mass

1500

2000


## ##

male female

★★

** *

** **

5% 0%

3/ MicA+

150000

20% % of cellular lysis

Controle MC-‐LR MicA+

MicA-‐ Bloom+

Gender-Specific Toxicological Effects of Chronic ... - ACS Publications

Recommend Documents