Depressing Antidepressant: Fluoxetine Affects Serotonin Neurons

Apr 29, 2016 - Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressants. As endocrine disruptive contaminants in the environmen...
0 downloads 13 Views 2MB Size
Subscriber access provided by SUNY DOWNSTATE

Article

Depressing antidepressant: Fluoxetine affects serotonin neurons causing adverse reproductive responses in Daphnia magna. Bruno Campos, Claudia Rivetti, Timm Kress, Carlos Barata, and Heinrich Dircksen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00826 • Publication Date (Web): 29 Apr 2016 Downloaded from http://pubs.acs.org on May 3, 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 30

Environmental Science & Technology

1

Depressing antidepressant: Fluoxetine affects

2

serotonin neurons causing adverse reproductive

3

responses in Daphnia magna.

4

Bruno Camposa,b,†, Claudia Rivettia,b,†, Timm Kressb, Carlos Barataa, Heinrich Dircksen*b a

5

Dept. of Environmental Chemistry, Institute of Environmental Assessment and Water Research

6 7

(IDAEA-CSIC), Jordi Girona 18, E-08034, Barcelona, Spain b

Department of Zoology, Stockholm University, Svante Arrhenius väg 18A, S-106 91 Stockholm,

8

Sweden

9 10

*Corresponding Author:

11

Heinrich Dircksen

12

Department of Zoology, Stockholm University, Svante Arrhenius väg 18A, S-106 91 Stockholm,

13

Sweden

14

[email protected]

15

Phone: +46 8164076

16

Fax: +46 8167715

17 18

1 ACS Paragon Plus Environment

Environmental Science & Technology

Page 2 of 30

19

ABSTRACT

20

Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressants. As endocrine

21

disruptive contaminants in the environment, SSRIs affect reproduction in aquatic organisms. In the

22

water flea Daphnia magna, SSRIs increase offspring production in a food ration-dependent

23

manner. At limiting food conditions, females exposed to SSRIs produce more but smaller

24

offspring, which is a maladaptive life-history strategy. We asked whether increased serotonin

25

levels in newly identified serotonin-neurons in the Daphnia brain mediate these effects. We

26

provide strong evidence that exogenous SSRI fluoxetine selectively increases serotonin-

27

immunoreactivity in identified brain neurons under limiting food conditions thereby leading to

28

maladaptive offspring production. Fluoxetine increases serotonin-immunoreactivity at low food

29

conditions to similar maximal levels as observed under high food conditions and concomitantly

30

enhances offspring production. Sublethal amounts of the neurotoxin 5,7-dihydroxytryptamine

31

known to specifically ablate serotonin-neurons markedly decrease serotonin-immunoreactivity and

32

offspring production, strongly supporting the effect to be serotonin-specific by reversing the

33

reproductive phenotype attained under fluoxetine. Thus, SSRIs impair serotonin-regulation of

34

reproductive investment in a planktonic key organism causing inappropriately increased

35

reproduction with potentially severe ecological impact.

36 37

Keywords: Selective serotonin-reuptake inhibitor, serotonin neurons, reproduction, Daphnia

38

magna, freshwater ecotoxicology

39

2 ACS Paragon Plus Environment

Page 3 of 30

Environmental Science & Technology

40

INTRODUCTION

41

Assessing the risks of long-term exposure to low doses of pharmaceuticals is an identified

42

research need1, particularly for those that may act as neural and endocrine disruptors in vertebrate

43

and invertebrate species2,3. Selective serotonin reuptake inhibitors (SSRIs) are currently

44

antidepressants of choice widely used in psychiatric treatments of deregulated serotonin signaling

45

underlying depressive disorders4. In recent years, evidence of adverse endocrine disruptive effects

46

for these substances has accumulated5,6. Human populations treated with SSRIs show signs of

47

developmental toxicity characterized by an increased rate of poor neonatal adaptation, reversibly

48

impaired sexual function in both sexes, specifically impairing the orgasm and adverse effects on

49

sperm integrity (increased DNA damage)6-8. Deregulation of serotonin signaling pathways by

50

SSRIs also alters food intake and hence energy homeostasis, which may lead to obesity in

51

humans9. In fish, fluoxetine, the active ingredient of Prozac®, acts as a neuroendocrine disruptor

52

causing male infertility via abnormal sperm production, alters testosterone levels, feeding and

53

reproductive behavior5,10.

54

Daphnia magna, an important micro-crustacean in freshwater food webs3,11, is an established

55

model for testing the toxicology of substances for regulatory purposes12. Daphnia shares more

56

genes with humans than probably any other invertebrate, whose genome has been sequenced, and

57

is an accepted model organism for human health research13. By proximate approaches, SSRIs were

58

reported to change the perception of the food environment and to switch life-history responses

59

towards higher food levels: females reproduced earlier, producing more but smaller offspring at

60

limiting food conditions14,15. Producing more offspring of smaller size at low food conditions is

61

maladaptive since smaller offspring are less fit than larger ones16. Furthermore, Campos et al.14

62

have shown that the serotonin-receptor antagonist cyproheptadine reversed the effects of SSRIs at

63

limiting food conditions. SSRIs also deregulated genes related to serotonin signaling pathways in 3 ACS Paragon Plus Environment

Environmental Science & Technology

Page 4 of 30

64

D. magna17. This ultimately suggested that specific serotonergic neurons are involved in

65

reproductive regulation and are the causative elements of SSRI action. Here, we hypothesized that

66

increased levels of (synaptic) serotonin, via SSRIs action, change the perception of the food

67

environment and life history responses to increased offspring production.

68

In arthropods, serotonin neurons are well established18,19. They stimulate insect ecdysteroid and

69

juvenile hormone production, both responsible for controlling oogenesis and vitellogenesis, and, as

70

well known in decapod crustaceans, stimulate growth- and reproduction-controlling hormone

71

systems20 and are involved in control of behavior and metabolism21-24. However, in non-decapod

72

crustacean species, such as Daphnia, we know little about serotonin in nervous systems25 and its

73

possible functions, the latter being only inferred from effects of exogenous SSRIs14,26,27. Since the

74

Daphnia genome encodes serotonin biosynthesis enzymes28, serotonergic neurons involved in the

75

control of growth and reproduction likely exist in D. magna.

76

The neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) specifically disrupts serotonergic neurons

77

by oxidative damage after selective uptake. 5,7-DHT is rapidly taken up into serotonergic neurons,

78

where it is oxidatively converted into the two metabolites 5-hydroxytryptamine-4,7-dione and 6,6'-

79

bi(5-hydroxytryptamine-4,7-dione). The following redox reactions of these two metabolites result

80

in the depletion of intraneuronal oxygen levels and OH- increase. The deleterious neurotoxicity of

81

this substance is then provoked by hypoxia and OH--formation29.

82

We provide first evidence that the SSRI fluoxetine (Prozac®) increases serotonin-

83

immunoreactivity in newly identified optic-ganglia and brain neurons of D. magna and,

84

concomitantly, increases offspring production especially under limited food conditions. In order to

85

establish serotonin as a key regulator of reproduction, we manipulated this system in two

86

directions (i) by increasing serotonin levels in the system via high food rations and low food ration

4 ACS Paragon Plus Environment

Page 5 of 30

Environmental Science & Technology

87

plus fluoxetine and (ii) by reducing them via the specific neurotoxin 5,7-dihydroxytryptamine. The

88

latter provided additional evidence for the specificity of the treatment.

89 90

MATERIALS AND METHODS

91

Chemicals. Fluoxetine hydrochloride (CAS-No 56296-78-7; analytical standard, purity 100%)

92

Sigma-Aldrich, USA), 5,7-Dihydroxytryptamine creatinine sulphate (5,7-DHT; CAS No 39929-

93

27-6) 1,4-Diazabicyclo[2.2.2]octane hydrochloride (Fluka, Germany), and polyclonal rabbit anti-

94

serotonin serum were purchased from Sigma-Aldrich (USA, Germany). All other chemicals were

95

analytical grade and obtained from Merck (Germany).

96

Experimental animals. All experiments were performed using a well-characterized single clone

97

of D. magna (Clone F), maintained indefinitely as pure parthenogenetic cultures12. Individual or

98

bulk cultures of 10 animals/L were maintained in ASTM hard synthetic water as described

99

previously16. These cultures were maintained at high food levels, fed every other day with

100

Chlorella vulgaris Beijerinck (5 x 105 cell/mL), corresponding to 1.8 µg carbon/mL). C. vulgaris

101

was grown axenically in Jaworski/Euglena gracilis 1:1 medium (Culture Collection of Algae and

102

Protozoa, 1989; http://www.ccap.ac.uk). We changed the culture medium every other day and

103

removed neonates within 24 h. Photoperiod was set to 14 h light: 10 h dark cycle and temperature

104

at 20 ± 1 ºC.

105

Experimental design. According to the adverse outcome pathway (AOP) framework, effects of

106

pollutants on their molecular targets do not necessarily occur at the same time as they become

107

apparent in individuals30. We tested if this was the case for fluoxetine and 5,7-DHT in adult

108

females assayed for serotonin-immunoreactivity in the brain-optic ganglia complex and

109

reproduction to assess the effect of exposure time and food ration levels.

5 ACS Paragon Plus Environment

Environmental Science & Technology

Page 6 of 30

110

Under optimum conditions, the normal life cycle of D. magna can be summarized as follows: (i)

111

embryonic development takes approximately 3 days at 20 ºC and >12 h of daily light, (ii) neonates

112

are released as free swimming individuals, (iii) after a juvenile period of around 5 days, during

113

which period animals undergo four molts, they become reproductively active adults. At this stage,

114

they transfer the first eggs into the brood pouch and embryonic development starts. Thus, it takes

115

approximately 8 days from birth to the first reproduction, i.e. the release of neonates. The adult

116

then successively releases its broods about every 3 days followed by its ecdysis. In a

117

reproductively active female, three broods are developing simultaneously: one clutch of eggs

118

develops in the brood pouch, while the second is provisioned in the ovaries, and the third clutch

119

differentiates from nurse cells to oocytes31. This means that effects on egg provisioning (when the

120

SSRI effect is elicited) and, hence, on the total reproductive output require at least 6 days (two

121

consecutive broods) to be reliably measurable as offspring production. SSRIs increase the rates of

122

juvenile development and offspring production in D. magna in a food-dependent manner14,15. For

123

the AOP framework, the timing of events occurring at the molecular level on primary targets and

124

those measured at the individual level (i.e. reproduction) is crucial to link the initiating with the

125

key events30. In this regard, we designed four assays to study exposure to 40 µg/L of fluoxetine

126

under different ontogenetic periods (see design summary in Figure 1). We previously reported that

127

effects of fluoxetine and of other SSRIs enhancing offspring production also occurred at lower

128

concentrations (3-10 µg/L)14,15, which were closer to those reported in the field32,33. Here we used

129

the concentration affecting mostly the reproduction, facilitating the establishment of links between

130

molecular and reproductive responses.

131

In assay 1, we exposed juveniles from birth until the first release of neonates (~8 days). In assays

132

2, 3, and 4, reproductive females were exposed during one, two and three consecutive broods, i.e.,

133

approximately 3, 6 and 9 days, respectively (Figure 1). The first batch of neonates (hatching within 6 ACS Paragon Plus Environment

Page 7 of 30

Environmental Science & Technology

134

the first 48 - 72 h) was always discarded and not evaluated as these animals were not exposed to

135

the tested chemicals during their egg provisioning period12. Thus, we counted only neonates from

136

the second and third broods to assess effects on total offspring production. This means that in

137

females exposed during 6 and 9 days (in assays 3 and 4, respectively, but not in assay 2) we

138

counted the number of neonates released in one and two consecutive clutches, respectively. These

139

assays were performed at low (1*105 cells/mL) and high food levels (5*105 cells/mL) using

140

C. vulgaris as the sole food source14. The high food level equals ad libitum or surplus food supply,

141

while the low food level is limiting though sufficient to allow the animals to develop and

142

reproduce. The purpose of assays 2, 3, and 4 was to assess the temporal dynamics in the primary

143

target of SSRIs, serotonin immunoreactivity in the brain, across different food conditions (with or

144

without SSRIs). Specifically, when adult animals reared at high food levels were switched to low

145

food with and without fluoxetine, this treatment was key to the understanding of when serotonin

146

depletion occurred, if any, and was affected by fluoxetine exposure. Assays 1, 3, 4 allowed to

147

relate serotonin immunoreactivity with the reproductive effects. A negative control via specific

148

chemical ablation of serotonin-neurons by 5,7-DHT was used in order to provide the two-

149

directional manipulations (see above), which lent strong support towards the specificity of the

150

effects. We used 1 and 10 µg/L 5,7-DHT to affect serotonin-immunoreactivity and reproduction in

151

adult females exposed for a short time (6 h) or from birth to first reproduction. These tests were

152

performed at high food ration level only.

153

Exposure assays were initiated with neonates (