Multigenerational Effects of the Antibiotic Tetracycline on

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

Multigenerational effects of antibiotic tetracycline on transcriptional responses of D. magna and its relationship to higher levels of biological organizations Hyun Young Kim, Jana Asselman, Tae Yong Jeong, Seungho Yu, Karel A.C. De Schamphelaere, and Sang D Kim Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05050 • Publication Date (Web): 12 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017

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 34

Environmental Science & Technology

1

Multigenerational effects of antibiotic tetracycline on transcriptional

2

responses of D. magna and its relationship to higher levels of biological

3

organizations

4 5

Hyun Young Kima, Jana Asselmanb, Tae Yong Jeongc, Seungho Yud, Karel A. C. De

6

Schamphelaereb, and Sang Don Kimc*

7 a

8 9

Research and Development Division, Korea Institute of Nuclear Nonproliferation and

Control (KINAC), 1534 Yuseong-daero, Yuseong-gu, Daejeon, 34054 Republic of Korea b

10

Laboratory of Environmental Toxicology and Aquatic Ecology (GhEnToxLab), Ghent

11 12

University, Ghent, B-9000, Belgium c

13

School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology (GIST), 123 Cheomdan-gwagiro, Buk-gu, Gwangju 61005, Republic of Korea d

14

Radiation Research Division for Industry and Environment, Advanced Radiation

15

Technology Institute, Korea Atomic Energy Research Institute, Jeongeup-Si, Jeollabuk-Do,

16

56212, Republic of Korea

17 18 19 20

*

21

Tel: +82-62-970-2445

22

Fax: +82-62-970-2434

23

E-mail: [email protected]

Author to whom correspondence should be addressed:

1

ACS Paragon Plus Environment


Page 2 of 34

24

Abstract

25

Given the risk of environmental pollution by pharmaceutical compounds and the effects of

26

these compounds on exposed ecosystems, ecologically relevant and realistic assessments are

27

required. However, many studies have been mostly focused on individual responses in a

28

single generation exposed to one-effect concentration. Here, transcriptional responses of the

29

crustacean Daphnia magna to the antibiotic tetracycline across multiple generations and

30

effect concentrations were investigated. The results demonstrated that tetracycline induced

31

different transcriptional responses of daphnids that were dependent on dose and generation.

32

For example, reproduction-related expressed sequence tags (ESTs), including vitellogenin,

33

were

34

multigenerational exposure induced significant change of molting-related ESTs such as

35

cuticle protein. Sixty-five ESTs were shared in all contrasts, suggesting a conserved

36

mechanism of tetracycline toxicity regardless of exposure concentration or time. Most of

37

them were associated with general stress responses including translation, protein and

38

carbohydrate metabolism, and oxidative phosphorylation. In addition, effects across the dose-

39

response curve showed higher correlative connections among transcriptional, physiological,

40

and individual responses than multigenerational effects. In the multigenerational exposure,

41

the connectivity between adjacent generations decreased with increasing generation number.

42

The results clearly highlight that exposure concentration and time trigger different

43

mechanisms and functions, providing further evidence that multigenerational and dose-

44

response effects cannot be neglected in environmental risk assessment.

distinctly

related

to

the

dose-dependent

tetracycline

exposure,

whereas

45 46

Key words: Daphnia magna, microarray, multigenerational exposure, antibiotics, tetracycline

47

2


Page 3 of 34


48

Introduction

49

Pharmaceuticals in natural environments are of significant concern because their

50

pharmacological actions have resulted in unexpected deleterious effects on exposed

51

organisms and humans.1-4 Among the various pharmaceutical compounds, tetracycline is a

52

widely used antibiotic for human, veterinary, aquaculture, and agricultural purposes including

53

prevention and treatment of infectious diseases and promotion of animal growth as feed

54

supplements.5 The low cost and high antimicrobial activity led to the extensive use of

55

tetracycline; for example, 3,200 tons in 2001 and 2,294 tons in 1997 were used in USA and

56

Europe, respectively.6 Mechanistic research has shown that tetracycline is not absorbed or

57

metabolized completely after administration, but excreted in active form of parent compound

58

in the fraction of 80% to 90% into the environment.7, 8 Furthermore, residues of tetracycline

59

persistently exist in the environment due to their hydrophilic character. As a result,

60

tetracycline has been commonly detected in freshwater (3.6–110.0 ng L-1),9, 10 seawater (1.0–

61

122 ng L-1),11, 12 soil and sediment (86–199 µg kg-1),13, 14 costal aquatic organisms (1.7–1.9 µg

62

kg-1),12 and agricultural vegetables (1.0–5.6 µg kg-1).14 In addition, it has been estimated that

63

tetracycline concentration in the feces of treated livestock were in the low ppm range,

64

comparable to the concentrations tested here (e.g., 4.0 mg L-1 in liquid manure and 43.4-

65

198.7 µg kg-1).13 Consequently, the use of tetracycline should be monitored because of its

66

associated hazards and the potential to affect the environment.15, 16

67

Potential toxic effects of pharmaceuticals have been increasingly studied, considering

68

uptake through various routes, exposure pathways, and toxic behaviors ranging from genetic

69

to population effects.17 In severe cases, near-extinction of vultures exposed to diclofenac via a

70

food web18 or increased feminization of the fish population after exposure to synthetic

71

estrogen19 were reported. Even though antibiotics such as tetracycline are targeted to bacteria 3



Page 4 of 34

72

and have no clear mode of action in higher organisms, studies regarding unexpected

73

detrimental effects on nontarget organisms have been continuously reported. For example, the

74

effects of tetracycline on pancreas, liver, and reproductive function were observed, showing

75

increased free radical levels and decreased antioxidant enzymes (e.g. superoxide dismutase,

76

catalase, glucose-6-phosphate dehydrogenase, and glutathione-S-transferase) in mammals.20,

77

21

78

production22 and steroidogenesis in Oryzias latipes.23 Tetracycline also reduced intracellular

79

calcium levels in the model plant Arabidopsis thaliana, which resulted in growth reduction

80

and other toxic effects.24 In addition, tetracycline released into the environment can enhance

81

antimicrobial resistance in microorganisms, which can severely threaten human health.25

Estrogenic effects of tetracycline were also reported, e.g., increased vitellogenin

82

With the risk of environmental pollution by pharmaceuticals, the necessities of

83

ecologically relevant and realistic assessments are becoming increasingly important with

84

regard to chronic, life-cycle, and especially multigenerational effects to understand long-term

85

effects of these emerging pollutants.1,

86

multigenerational effects and/or maternal transfer on Daphnia have been largely focused on

87

heavy metals28-31 and pesticides.32-34 Recently, multigenerational research of pharmaceuticals

88

has been carried out using Daphnia species35-37 as well as other aquatic organisms.38,

89

However, most of these multigenerational studies have only focused on organismal outcomes.

90

Daphnia species is the most commonly studied freshwater crustacean in the ecotoxicology

91

field, and plays a significant ecological role as a primary consumer of phytoplankton and a

92

food source for carnivores. In addition, Daphnia species have been developed as a genomic

93

model because of its cyclical parthenogenesis characteristics, showing relatively low genetic

94

variability.40 With the progress in sequencing of Daphnia pulex genome,41 a number of

95

transcriptional results using Daphnia species have been published to elucidate the molecular

26, 27

However, studies conducted regarding

4


39

Page 5 of 34


96

responses to various environmental stressors.42-50 For pharmaceuticals as environmental

97

stressors, two studies demonstrated transcriptional responses using microarray technology

98

and identified modes of action and target mechanisms in a single exposure generation.45, 51

99

Heckmann et al. demonstrated that ibuprofen disrupted crustacean eicosanoid metabolism,

100

resulting in the disruption of juvenile hormone metabolism and oogenesis.45 Campos et al.

101

studied the transcriptomic responses of selective serotonin reuptake inhibitors in Daphnia and

102

found significant effects on serotine metabolism and neurological processes.51

103

We previously identified that the multigenerational exposure of tetracycline has

104

distinct effects on D. magna on an individual level.52, 53 It was revealed that tetracycline

105

induced changes in internal energy balance with increasing generation, which is significantly

106

correlated with individual responses, such as reproduction and somatic growth. To the best of

107

our knowledge, however, the multigenerational effects of pharmaceuticals on nontarget

108

aquatic organisms (D. magna) using differential transcriptional responses have not been

109

reported. Information is also scarce regarding the relationship between transcriptional

110

responses and higher levels in multigenerational pharmaceutical exposure. Therefore, the

111

objective of the current study was to investigate transcriptional responses in D. magna

112

exposed to tetracycline in multigenerations and at different doses. Our first hypothesis was

113

that tetracycline affects transcriptional responses of target organism (D. magna) and a dose

114

effect and exposure time effect through successive generations can be clearly distinguished.

115

To do so, we observed the transcriptional responses of Daphnia to different tetracycline

116

exposure concentrations (control, 0.1, 1.0, and 10.0 mg L-1) and for different exposure

117

generations (F1, F2, F3, and F4) to the same concentration. We aimed to identify the main

118

effect of tetracycline as well as specific dose and generation patterns in gene expression.

119

Furthermore, we hypothesized that the transcriptional responses are closely related to higher 5



120

level responses. Therefore, we also analyzed the relationship between transcriptional results

121

and physiological and individual results, which were conducted in our previous studies.

122 123

Materials and Methods

124

Maintenance of D. magna cultures and target compound exposure

125

Animals from a single isoclonal population of D. magna, obtained from a permanent

126

laboratory culture, were used throughout the experiment. The D. magna culture procedure

127

and preparation of culture media was performed according to the EPA manual.54 In particular,

128

cultures were fed daily with a suspension of yeast, trout chow, and Cerophyll® (YCT, v/v/v =

129

1:1:1, 1.7 mL-1) mixture as well as green algae (Pseudokirchneriella subcapitata, 5 × 104 cells

130

mL-1). The culture medium for D. magna was reconstituted with hard water (i.e., CaSO4 120

131

mg L-1, NaHCO3 192 mg L-1, MgSO4 120 mg L-1, and KCl 8.0 mg L-1) and renewed three

132

times each week. The culture was maintained at 22 ± 1°C in a temperature- controlled room,

133

with a 16-hour light:8-hour dark photo cycle.

134

The experimental design is depicted in Figure 1, with four tetracycline concentrations

135

(control, 0.1, 1.0, and 10.0 mg L-1) and four exposure generations (F1, F2, F3, and F4). The

136

exposure concentrations in the present study were selected as they did not cause any acute

137

effects on the target species, based on the LC50 value of 36.52 ± 5.71 mg/L obtained in a

138

previous study.53 Four successive generations of D. magna were continuously cultured in

139

medium tanks for the exposure tests. For each generation, a 21-day life-cycle toxicity test was

140

performed in a static renewal system. One hundred daphnids in each aquarium were

141

continuously exposed to the target compound for 21 days through four generations without

142

any recovery period. The media solution containing target compound was renewed three

143

times a week during the exposure test. For the second generation’s (F2) exposure, neonates 6


Page 6 of 34

Page 7 of 34


144

from the prior generation after the third brood were transferred and started to be exposed to

145

the same concentration of tetracycline as in F1. The third-brood neonates were selected

146

because an earlier cluster of neonates are known to be more unstable than subsequent broods.

147

The test continued until the fourth generation (F4), using the same test method as that used

148

for the parental generation. For each generation, the life-cycle toxicity test in static renewal

149

system was performed. Daphnids in each aquarium were fed daily with 5 mL of YCT and

150

green algae per each test chamber. Tests were conducted in 5 L aquaria containing 3 L media

151

at 22 ± 1˚C. The exposure tests of each generation were conducted in duplicate. After 21

152

days, 30 daphnids were randomly sampled and immediately frozen with liquid nitrogen and

153

kept at -80˚C to await further experiments.

154 155

Tetracycline concentration and stability

156

The change of tetracycline concentration during the exposure period was observed to

157

compare the nominal and actual exposure concentration and the stability of the target

158

compound. The target compound was analyzed using LC/ESI-MS/MS (Agilent 6410 Triple

159

Quadrupole mass spectrometer, USA). First, tetracycline concentration in water, Daphnia

160

culture media, and food contained water were observed during the 48 hours of exposure time

161

(Table S1). The nominal tetracycline concentrations were 0.1 and 1 mg L-1. As a result, the

162

measured concentration of tetracycline was mainly influenced by Daphnia culture media. The

163

concentration was rapidly reduced about 50% within 2 hours and reached equilibrium. There

164

was no change in water and food contained water. The tendency of tetracycline to complex

165

with metals (e.g. Ca2+ and Mg2+) in culture media can explain the results.55 The results

166

indicated that the actual exposure concentration of tetracycline was 50% lower than the 7



167

nominal concentration. In addition, the residual concentration of tetracycline in Daphnia

168

culture media during 48 hours of exposure time was also observed (Table S2). The internal

169

concentration was increased to 0.29 and 1.60 ng mg-1, while the media concentration was

170

reduced to 72.61 and 405.79 µg L-1 when exposed to the nominal concentration of 100 and

171

1000 µg L-1, respectively. In the experiment design, tetracycline containing media was

172

renewed every 2 days (three times a week), so tetracycline exposure trend is likely to be

173

repeated every 2 days even in the long-term period of exposure.

174 175

Probe and array design

176

The target sequence of Daphnia clones was obtained from expressed sequence tag

177

(EST) database of D. manga (Daphniabase, http://daphnia.nibb.ac.jp) representing the

178

entirety of available D. magna sequences at that time. 8 × 60 K-format arrays (62,976 spots

179

of features per block and 8 blocks per array) were designed using Agilent’s eArray

180

(http://earray.chem.agilent.com/earray/), a web-based program. We could design probes for

181

10,935 target sequences of a total of 10,979 available ESTs. Three probes per target sequence

182

were designed (excluding, 3 target sequences for which only 1 probe could be designed and 1

183

target sequence for which only 2 probes could be designed.). Finally, a total 32,798 probes

184

were spotted on a block and blank portions were filled by random probes from the total

185

probes.

186 187

RNA extraction and microarray hybridization

188

Total RNA of D. magna (21 days old, 30 individuals/replicate) was extracted using

189

the TRIzol method (Invitrogen, USA) according to the manufacturer’s protocol. The quality

190

of RNA was examined using NanoDrop 1000 spectrophotometer (Nanodrop Technologies, 8


Page 8 of 34

Page 9 of 34


191

USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). Quality criteria

192

consisted of a total RNA quantity of 24.6-49.3 µg, protein contamination (260/280 ratio) and

193

organic contamination (260/230 ratio) of approximately 1.8 or greater, and an RNA integrity

194

number (RIN) higher than 7. RIN is a measure of RNA degradation, ranging from 1 for poor

195

quality to 10 for the best quality. Because of the lack of RNA quantity obtained from one

196

individual, 30 daphnids were regarded as one sample, and in total two replicates were used

197

for microarray analysis. Each total RNA sample (200 ng) was labeled and amplified using

198

Low Input Quick Amp labeling kit (Agilent Technologies). The Cy3-labeled aRNAs were

199

resuspended in 50 µL of hybridization solution (Agilent Technologies). Afterward, labeled

200

aRNAs were loaded on Agilent SurePrint G3 Custom (D. magna) GE 8 × 60 K array (Agilent

201

Technologies) and covered by a Gasket 8-plex slide (Agilent Technologies). The slides were

202

hybridized for 17 hours at 65ºC. The hybridized slides were washed in 2 X SSC, 0.1 % SDS

203

for 2 minutes, 1 X SSC for 3 minutes, and then 0.2 X SSC for 2 minutes at room temperature.

204

The slides were centrifuged at 3000 rpm for 20 sec to dry. The arrays were scanned using an

205

Agilent scanner with associated software. Feature intensities and ratios were calculated with

206

Feature Extraction v10.7.3.1 (Agilent Technologies). All data have been deposited to the

207

National Center for Biotechnology Information Gene Expression Omnibus with the accession

208

number GSE94439.

209 210

Data analysis

211

The data analysis was conducted in R using the LIMMA package with specific

212

functions for one-channel Agilent array data. The data were background corrected using the

213

normexp method.56 Additionally, data were normalized between arrays using a quantile

214

normalization method and averaged first across probes and then across ESTs (expressed 9



Page 10 of 34

215

sequence tags), resulting in a single average expression value for each EST.57 Then a linear

216

model was fitted to all the arrays (LIMMA analysis using lmFit). The initial experimental

217

design was selected to maximize the number of exposed generations and the number of

218

concentrations rather than the number of replicates within a single treatment. However, a

219

small number of replicates may reduce the statistical power of the analysis. Therefore, we

220

have analyzed the data across time and generations to increase our statistical power as these

221

points were replicated 4 times (i.e. 4 generations (F1 – F2 – F3 – F4) and 4 concentrations (0

222

– 0.01 – 0.1 – 1 mg L-1). To this end, we fitted a linear model to all data and then tested

223

different statistical contrasts. In particular, we focused on four different research questions as

224

highlighted in Figure 1, Question 1: what is the main effect of tetracycline across generations?

225

Therefore, we compared all control (F10 to F40, i.e., 8 samples) and all exposure data (F11 to

226

F41, i.e., 8 samples). Question 2: What is the dose-response effect of tetracycline in the

227

parental generation? This question will identify the effect of increasing doses of tetracycline

228

in a linear model by focusing on the changes across four doses of tetracyline (F10, F101, F11,

229

and F10) rather than a direct comparison between different treatments.

230

the generation response effect of tetracycline exposure versus control? This question focuses

231

on identifying similar changes that occur in each generation when comparing control and

232

tetracycline samples; it includes four generations (generation 1: F10-F11, generation 2: F20-

233

F21, generation 3: F30- F31, generation 4: F40-F41). Question 4: What are the transcriptional

234

responses of tetracycline treated D. magna over generations (F11 vs. F21 vs. F31 vs. F41) in

235

which we focused on increasing effect that tetracycline may have across generations? Next,

236

adjusted p-values were calculated by using empirical Bayes statistics and corrected for

237

multiple testing using the Benjamini-Hochberg correction.58 Annotation of the ESTs was

238

downloaded from daphniabase (http://daphnia.nibb.ac.jp/cgi-bin/showBest). The annotation 10


Question 3: What is

Page 11 of 34


239

available through daphniabase (http://daphnia.nibb.ac.jp) provided annotation for 6,111 ESTs

240

(55%). Next, overrepresentation of annotation terms in the significant contrasts was tested

241

using a Fisher exact test corrected for multiple testing with the Benjamini-Hochberg method.

242

We defined gene families as a group of more than 4 genes with the same annotation term.

243

Correlation analysis was conducted for the relationship between genetic responses

244

and higher level effects induced by tetracycline stress. Pearson correlation coefficients

245

between the components within and across levels were calculated using SPSS 17 (Chicago,

246

IL, USA). The relationships were determined by correlation coefficient with a significantly

247

acceptable p-value of

Multigenerational Effects of the Antibiotic Tetracycline on

Recommend Documents