Multigenerational Effects of the Antibiotic ... - ACS Publications

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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

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Environmental Science & Technology

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Multigenerational effects of antibiotic tetracycline on transcriptional

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

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organizations

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

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Schamphelaereb, and Sang Don Kimc*

7 a

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Research and Development Division, Korea Institute of Nuclear Nonproliferation and

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

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

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University, Ghent, B-9000, Belgium c

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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

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Radiation Research Division for Industry and Environment, Advanced Radiation

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

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56212, Republic of Korea

17 18 19 20

*

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Tel: +82-62-970-2445

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Fax: +82-62-970-2434

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E-mail: [email protected]

Author to whom correspondence should be addressed:

1

ACS Paragon Plus Environment


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Abstract

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

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

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

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

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crustacean Daphnia magna to the antibiotic tetracycline across multiple generations and

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effect concentrations were investigated. The results demonstrated that tetracycline induced

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

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

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were

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

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

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

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

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

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response curve showed higher correlative connections among transcriptional, physiological,

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

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the connectivity between adjacent generations decreased with increasing generation number.

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The results clearly highlight that exposure concentration and time trigger different

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

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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

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2


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Introduction

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Pharmaceuticals in natural environments are of significant concern because their

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pharmacological actions have resulted in unexpected deleterious effects on exposed

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Potential toxic effects of pharmaceuticals have been increasingly studied, considering

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

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

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

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



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

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

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

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

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

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21

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

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

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

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

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

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

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ecologically relevant and realistic assessments are becoming increasingly important with

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

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effects of these emerging pollutants.1,

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

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

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

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

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

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

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

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

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

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

26, 27

However, studies conducted regarding

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39

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

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stressors, two studies demonstrated transcriptional responses using microarray technology

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

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

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

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

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found significant effects on serotine metabolism and neurological processes.51

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We previously identified that the multigenerational exposure of tetracycline has

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

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

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

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

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

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reported. Information is also scarce regarding the relationship between transcriptional

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

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

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

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

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

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

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

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

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

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



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

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

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Materials and Methods

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Maintenance of D. magna cultures and target compound exposure

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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kept at -80˚C to await further experiments.

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Tetracycline concentration and stability

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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Probe and array design

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

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

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

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

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

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

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

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

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

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probes.

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RNA extraction and microarray hybridization

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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National Center for Biotechnology Information Gene Expression Omnibus with the accession

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number GSE94439.

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Data analysis

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


Question 3: What is

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

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

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

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

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Correlation analysis was conducted for the relationship between genetic responses

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

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

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

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acceptable p-value of

Multigenerational Effects of the Antibiotic ... - ACS Publications

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