Accepted Manuscript Title: Effects of chronic, parental pharmaceutical exposure on zebrafish (Danio rerio) offspring Author: Michal Galus Sahaana Rangaranjan Anderson Lai Lana Shaya Sigal Balshine Joanna Y. Wilson PII: DOI: Reference:
S0166-445X(14)00026-5 http://dx.doi.org/doi:10.1016/j.aquatox.2014.01.016 AQTOX 3745
To appear in:
Aquatic Toxicology
Received date: Revised date: Accepted date:
11-12-2013 13-1-2014 18-1-2014
Please cite this article as: Galus, M., Rangaranjan, S., Lai, A., Shaya, L., Balshine, S., Wilson, J.Y.,Effects of chronic, parental pharmaceutical exposure on zebrafish (Danio rerio) offspring, Aquatic Toxicology (2014), http://dx.doi.org/10.1016/j.aquatox.2014.01.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effects of chronic, parental pharmaceutical exposure on zebrafish (Danio rerio) offspring Michal Galus1, Sahaana Rangaranjan1, Anderson Lai1, Lana Shaya1, Sigal Balshine2, and Joanna Y. Wilson*1
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Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Canada ON L8S 4K1 2 Psychology, Neurosciences, and Behaviour; McMaster University, 1280 Main Street West, Hamilton, Canada ON L8S 4K1 *Address correspondence to: Joanna Y. Wilson Department of Biology, McMaster University 1280 Main St. West, Hamilton, ON L8S 4K1 Canada Tel: (905) 525‐9140 extension 20075 Fax: (905) 522‐6066 EMAIL:
[email protected] Co‐author Email addresses: Michal Galus:
[email protected] Sahaana Rangaranjan:
[email protected] Anderson Lai:
[email protected] Lana Shaya:
[email protected] Sigal Balshine:
[email protected] 28
Keywords: carbamazepine, gemfibrozil, sperm, behaviour, breeding
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Abbreviations1
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1 CBZ – carbamazepine, GEM‐ gemfibrozil, CTL‐ control
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Abstract
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reproduction in offspring. Adult zebrafish (Danio rerio) were exposed for 6 weeks to 10
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μg L‐1 of carbamazepine (CBZ) and gemfibrozil (GEM), two commonly prescribed drugs.
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Embryos were collected, reared in clean water until sexual maturity and then assessed for
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reproductive output, courtship, sperm function and organ histology. While 34% of the control
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pairs produced clutches, only 11% of the fish with CBZ exposed parents or 17% of the fish with
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GEM exposed parents produced clutches. Reciprocal crosses indicated that exposure in males
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had more profound reproductive effects. When a control F1 male was crossed with either a F1
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female whose parents were CBZ or GEM exposed; no differences were observed in embryo
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production compared to controls. However, when a control F1 female was crossed with either a
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CBZ or GEM F1 male, 50% less embryos were produced. Male courtship was reduced in both
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CBZ and GEM F1 fish but the deficits in courtship displays were drug specific. Compared to
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control males, the sperm from GEM F1 males had shorter head lengths and midpieces whereas
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sperm from CBZ F1 males had longer midpeices. Although it remains unclear how specifically
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these morphological differences influenced sperm velocity, the sperm from GEM F1 males and
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from CBZ F1 males swam faster than the sperm of control F1 at 20s post activation. No
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significant differences were observed in the histology of the liver, kidney and gonads across
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treatment groups. These data are important as they show that chronic, low dose
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pharmaceutical exposure of parental fish is sufficient to cause significant reproductive effects in
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offspring.
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In this study we explored how parental exposure to pharmaceuticals influences
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Introduction The effects of parental contaminant exposure on offspring are a sorely understudied
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area of toxicology. Multi‐ or trans‐ generational effects are typically easier to study in
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laboratory using species with short life spans (Dietrich et al., 2010; Lürling et al., 2006; Péry et
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al., 2008) but can also be investigated with organisms that have lived for generations in
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polluted environments (Hontela et al., 1992; Nacci et al., 2002; Nye et al., 2007). In both cases,
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exposures are typically for the whole life cycle. Traditionally, life cycle assessments (LCA) utilize
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either a cradle‐to‐grave or cradle‐to‐gate approach (EPA, 1993). In both schemes, exposure is
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typically from the onset of fertilization until terminal sampling (full life cycle) or until 24 hr past
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sexual differentiation (partial life cycle, EPA, 1993). Rarely do studies extend beyond one
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generation. Yet, the effects documented in LCA may be caused during gamete production,
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embryogenesis, maternal deposition or uptake of contaminants into the egg, secondary sexual
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maturation or some combination of these important biological processes. The major challenge
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is that life cycle or multi‐generational studies are difficult and time consuming to conduct and
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thus the number of studies that use vertebrates are limited. In a fast reproducing species such
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as zebrafish, these types of experiments are clearly possible. Given that many fish have
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migratory behaviors or distinct breeding grounds, quantifying the risks of parental versus
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embryonic contaminant exposures may be quite important and provide distinct data from
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traditional LCA approaches.
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focused on effects caused by estrogens (Greytak et al., 2010), estrogen like compounds
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(Brennan et al., 2006) or polychlorinated biphenyls (PCBs) (Nacci et al., 2002). The number of
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Studies that have looked at trans‐generational impacts in aquatic species have generally
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studies that have explored the trans‐generational impacts with pharmaceuticals is extremely
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limited. To date, only one multi‐generational assessment involving fish (Parrott and Bennie,
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2009) has been published and the majority of studies that have been conducted use
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invertebrates (Dzialowski et al., 2006; Kim et al., 2012; Ortiz‐Rodríguez et al., 2012). In fish,
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maternal deposition of contaminants is one primary route of exposure for the developing
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embryo (Abrams and Mullins, 2009), however there are other mechanisms by which the
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progeny may be impacted by parental exposure. For example, fluctuations in circulating
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estrogen concentrations within the mother, during gametogenesis, have been linked to
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impaired growth rate and survival amongst offspring (Migliaccio et al., 1996). Furthermore,
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elevated cortisol levels, induced from behavioral stress during courtship in females, impacts the
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morphology and yolk‐sac size of F1 offspring in coral fishes (McCormick, 1998) and cichlid fish
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(Mileva et al., 2011). Male mediated effects are often associated to impairments with
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spermatozoa and DNA damage which has been linked to the incidence of skeletal abnormalities
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amongst F1 (Devaux et al., 2011). Such studies provide evidence suggesting that parental
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exposure to endocrine disrupting compounds may impair fish populations over several
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generations.
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Gemfibrozil (GEM) is a fibric acid derivative which is clinically prescribed to lower plasma
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triglycerides and total cholesterol (Spencer and Barradell, 1996). Although its exact mechanism
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of action is unclear, GEM is a peroxisome proliferator believed to elicit its effect by binding to
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the nuclear transcription factor peroxisome proliferator‐activated receptor‐alpha (PPARα) and
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promoting hepatic uptake and metabolism of free fatty acids (Martin et al., 1997). Peroxisome
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proliferators have been heavily studied in mammalian systems and chronic exposure to them
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has been linked to the onset of hepatic carcinoma (Gonzalez, 1997) and efficiency reduction in
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the anti‐oxidant defense system in rodents (O'Brien et al., 2001). Little work has been done to
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characterize the impact of GEM on fish, however goldfish (Carassius auratus) exposed to 1.5 μg
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L‐1 of GEM had decreased plasma testosterone (Mimeault et al., 2005) and adult zebrafish
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(Danio rerio) chronically exposed to 0.5 and 10 μg L‐1 of GEM show decreased reproductive
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output, atretic oocytes, and altered kidney histology (Galus et al., 2013b).
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and is frequently used in the treatment of epilepsy and bipolar disorder (Brodie et al., 1995).
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CBZ is believed to have two target sites within vertebrates, triggering unique cellular responses.
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Its anticonvulsive properties are believed to occur through modulation of voltage gated sodium
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channels, maintaining the channels in an inactive state and reducing neuronal excitability
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(Brodie et al., 1995). However, its anti‐depressive properties are believed to occur by lowering
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the turnover rate of arachidonic acid while keeping docosahexaeonic acid unaltered in brain
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(Rao et al., 2008). Japanese medaka (Oryzias latipes) acutely exposed to 6150 μg L‐1 of CBZ
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showed reduced feeding behaviour and swimming speed (Nassef et al., 2010). Sperm of the
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common carp (Cyprinus carpio L.), incubated for two hours with 2000 μg L‐1 of CBZ had reduced
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velocity (Li et al., 2010). Lastly, adult zebrafish chronically exposed to 0.5 and 10 μg L‐1 of CBZ
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showed decreased reproductive output, decreased plasma ketotestosterone in males, and
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altered kidney histology (Galus et al., 2013b).
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Carbamazepine (CBZ) is primarily prescribed as an anticonvulsant and mood stabilizer
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Of the small number of studies that examined the effects of GEM and CBZ in fishes,
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most involved concentrations well above what would be considered environmentally relevant
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and most exposure were acute. Both GEM and CBZ have been detected in waste water effluent
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and surface waters with average effluent concentrations ranging from 0.84 – 4.76 μg L‐1 and
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0.87 – 1.2 μg L‐1, respectively (Andreozzi et al., 2003; Kolpin et al., 2002; Metcalfe et al., 2003;
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Paxéus, 2004; Petrović et al., 2003; Zhang et al., 2008). Like other pharmaceuticals in the
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aquatic environment, CBZ and GEM enter receiving waters via waste water effluent discharge.
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Their release into surface waters is due to the low removal efficiency of these compounds in
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waste water treatment, which is usually between 40 – 50% for GEM and