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fishENCODE: ADVERSE HEALTH EFFECTS TO FISH CAUSED BY ENDOCRINE DISRUPTING CHEMICALS (EDCs) - LITERATURE REVIEW AND PRELIMINARY RESULTS   ON PATTERNS OF NEURONAL ACTIVATION IN THE OLFACTORY BULBS OF WHITE CACHAMA Piaractus brachypomus AND HORMONAL CHANGES IN TILAPIA Oreochromis sp. CAUSED BY EXPOSURES TO COMMERCIAL PRESENTATIONS OF GLYPHOSATE  

Edwin Gómez-Ramirez1, Javier F. Borbón2, Jaime F. González2, Pedro R. Eslava Mocha3, Amutha Chinnaiah*4, P. Subramanian5, Leslie Guzmán Beltran3, Paula Esquinas1, Estefany L. García1, Gina P. Sierra1, Hernán Hurtado Giraldo3, Wilson Ramirez Duarte3, Dang Thi Hoang Oanh6, Truong Quoc Phu6, Miriam Alcivar7, Diego Alejandro7, Acacia Alcivar-Warren7-8
 
1Grupo de Ictiología, Laboratorios de Embriología y Fisiología Animal, Universidad Militar
Nueva Granada, Bogota, Colombia, edwin.gomez@unimilitar.edu.co;

We present the long-term goals and research activities by collaborators of the 'fishENCODE' project, part of the global "ONE HEALTH Epigenomics Educational Initiative" (1). The goals of fishENCODE are to study the intergenerational epigenetic changes and adverse health effects to fish caused by endocrine disrupting chemicals (EDCs) such as the metal cadmium (Cd) and the herbicide Glyphosate. In tilapia Oreochromis mossambicus, Cd is an EDC at 150 parts per trillion, ppt (2). The gathered data on length-weight relation, gonado-somatic index, hepato-somatic index, vitellogenins, egg numbers and reproductive hormones such as gonadotropins (GtH-I & GtH-II) and gonadal hormones such as progesterone, testosterone and estradiol levels all indicated that Cd is an endocrine disruptor in tilapia (2). Though there is evidence for endocrine disruption of Atrazine and Glyphosate to O. mossambicus at parts per billion (ppb) concentrations (3), no studies that take into account the interactions of very low-levels of Glyphosate with fish hormones and receptors have been published following new scientific criteria for EDCs (4). Glyphosate is used in GMO-based crops such as soybeans used in aquafeeds. It is also a chelator and an antibiotic, and there are concerns about antibiotic resistance and new zoonotic diseases (1).  
 
We summarize here the results from an in-depth review of the scientific literature published in PubMed and Web of Science on adverse health effects to animals and human cells caused by EDCs. The range of concentrations of Glyphosate and its degradation product, aminomethylphosphonic acid (AMPA) reported in water, soil/sediment, and tissues of native and farmed fish species worldwide will also be presented in a revised version of Table 1 from researchers of the USDA Pesticide Laboratory (5) who reported EDCs in catfish, salmon, and soybeans produced in the USA. Preliminary results will also be presented from Colombian researchers (6-8) on the patterns of neuronal activation in the olfactory bulbs of white cachama (Piaractus brachypomus), a native species for consumption that is cultured and extracted from natural water bodies; and male-specific hormonal changes in an introduced species, red tilapia Oreochromis sp. Olfactory bulbs are important because they are part of various chemical signals (amino acids, sex hormones and bile salts) captured from the environment through the nostrils and olfactory tract. Data from India also indicated a reduction of testosterone by Glyphosate in both adult and juveniles O. mossambicus (3).
 
Initial studies showed that chronic exposure to 0.1 mg/l of the mix Roundup® + Cosmoflux® 411F induced neuronal lesions in the telencephalon implying major energetic expense to white cachama juveniles (6). Researchers then exposed P. brachypomus to 3 treatments with 3 replicates: (T1: 0 ppm, T2: 0.1 ppm, T3: 1 ppm, 20 individuals/40L aquarium). Increased number of cells similar to mast cells of fish were reported in telencephalic hemispheres after exposure to sub-lethal concentrations of Glyphosate (Roundup® Activo) indicating possible necrotic and/or apoptotic reactions (6). Telencephalic hemispheres are the multi-integrative sensory centers that mediate important functions such as learning, reproductive behavior, orientation, migration, etc. Further research showed changes in c-fos gene activity in neuronal cells exposed to sub-lethal doses of Glyphosate (7). In another experiment of 4 treatments with 4 replicates (T1: 0 ppm, T2: 1 ppm, T3: 5ppm, T4: 10 ppm, 15 individuals/40: aquarium), researchers found that in all treatments, including T0, the cell layers of the olfactory bulbs with immunohistochemical expression (c-fos +) were: (CCM) layer of mitral cells (mitral cells and Ruffed) and (CIC) the inner layer of cells (inhibitory interneurons of mitral cells and Ruffed), which are involved in receiving synaptic stimulation of olfactory receptor neurons, and also with the projection of axons to the telencephalic hemispheres. The immunoreactivity of c-fos+ increased with increasing concentration of Glyphosate, especially to the CIC, suggesting that exposure of white cachama to sub-lethal concentrations of Glyphosate (Roundup®Activo) alters the expression levels of important signals in the integration of neuronal cells for the species (7).  
 
In Colombia (8), exposure of red tilapia, Oreochromis sp., juveniles (23 females, 25 males) to 7 ppm of Glyphosate (Roundup®Activo), 446 g glyphosate/L (volume/volume) for 11 and 21 days (7) showed that Glyphosate affected the levels of testosterone in male tilapia compared to controls. Hormone levels were analyzed using ELISA (Monobind - Accubind TM). Mean levels of testosterone in males exposed to Glyphosate for 11 days were lower than in males of the control group (9.4 and 13.2 pmoles/ml plasma, respectively; n=11 per treatment). However, when concentrations of testosterone were examined at 21 days, the mean levels increased from 6.7 to 15.2 pmoles/ml of plasma. There were no changes in testosterone of female tilapia juveniles used in the study (8).  
 
In India (3), exposure of adult and juvenile O. mossambicus to the commercial herbicides Atrazine (up to 120 part per billion or 1µg/L) and Glyphosate (Glyphosate 41% SL herbicides) up to 150 ppb for three months in laboratory conditions (70% survived in these concentrations), showed endocrine disruptive effects at ranges from 80 ppb to 120 ppb for Atrazine, and 100ppb-150ppb for Glyphosate. In juveniles, the testosterone level declined in both exposures at the end of three months (25 days interval in each), at the same time estradiol level was increased and also elevated the aromatase (CYP19) level gradually in response to dose, but their impacts were almost similar to both herbicides. In adult male the testosterone level was decreased when compared to the control at same time the estradiol and progesterone activity was raised up, simultaneously initiated the early breeding cycle. These lead to increasing level of plasma vitellogenin (VTG) in both sexes of adults and juveniles. Glyphosate induced aromatase activity, which lead to increased estradiol level in males. Induction of aromatase by Atrazine is responsible for disturbance of steroidogenesis. VTG protein was detected in male plasma and other tissues. Detection of VTG in plasma of adult male and juvenile fish is considered as a sensitive biomarker of endocrine disruption impact of Atrazine and Glyphosate (3).
 
More research is needed that take into consideration the interaction of low (ppt) and high (up to 15 ppm found in GM soybeans) concentrations of Glyphosate with the fish genome and epigenome. Glyphosate is also an antibiotic and may kill gut bacteria of fish and shrimp. It may induce subtle changes in epigenetic mechanisms important for development and reproduction, and those changes may not be seen until F-2 and F-3 generations are examined (9). Some epigenetic mechanisms (DNA methylation, histone methylation and acetylation, RNAi, DNA transposition, chromatin structure changes, etc.) are known to be altered after exposure of live animals, or human cells in vitro, to a range of concentrations of EDCs (9). DNA transposition appears to be involved in Acute Hepatopancreatic Necrosis Disease (AHPND), most commonly known as Early Mortality Syndrome (EMS) of shrimp (1). Concerns remain about the adverse effects of Glyphosate on the environment and on the central nervous system (10) and in reproductive, physiology and endocrine systems throughout early development and growth of both native and exotic farmed fish populations. Glyphosate is found in water bodies of the Orinoco and Magdalena River of Colombia where native white cachama live, and negative effects of Roundup®Activo were reported after exposure to 0.1 and 1 mg/L, concentrations that were thought to be safe to fish (6,7). The Glyphosate concentrations measured in those studies did not affect the growth and survival of white cachama, but the fish did not recover from the observed nervous system lesions after removing them from the 'environmentally safe' levels they were originally exposed to (6,7). The results from our collaborators in India clearly indicate the endocrine disruption phenomena. Future studies should include rigorous experimental designs that take into account the scientific criteria recently published for EDCs (3), to study changes in epigenetic mechanisms in F2-F3 generations induced by very low levels of Glyphosate. The results of fishENCODE project will contribute baseline information to address the serious need of protecting developing children brains against harmful chemicals (10).
 
 
References
(1)  Alcivar-Warren et al. 2015. ONE HEALTH Epigenomics - An educational initiative to conserve healthy ecosystems, to maintain healthy animals, to protect human health. Abstract submitted for presentation at Aquaculture America 2015, New Orleans Feb 19-22, 2015.
(2)  Amutha et al. 2013. The multi-faceted endocrine disrupting effects in Oreochromis mossambicus due to cadmium exposure. Proceedings of the AA13 meeting in Nashville, TN, abstract 1034:   https://www.was.org/meetingabstracts/ShowAbstract.aspx?Id=29402
(3)  Amutha C. and P. Subramanian. 2015. Endocrine disruption effect in even very low level use of commercial herbicides Atrazine and Glyphosate on the freshwater fish Oreochromis mossambicus. Unpublished.
(4)  Zoeller et al. 2014. A path forward in the debate over health impacts of endocrine disrupting chemicals. Environmental Health 3:118: http://www.healthandenvironment.org/news/announce
(5)  Holmes et al. 2014.  U.S. Department of Agriculture's pesticide data program result summary of pesticide residues in catfish, soybeans, and salmon. Proceedings of the Aquaculture America 2014 meeting in Seattle, WA, USA, abstract 1051:
  https://www.was.org/meetingabstracts/ShowAbstract.aspx?Id=32154
(6)  Eslava et al. 2014. Pathological effects of sublethal exposures to commercial presentations of Glyphosate (Roundup®) on native fish from the Colombian Orinoco. Proceedings of the AA15 meeting in Seattle, WA, abstract 545: https://www.was.org/meetingabstracts/ShowAbstract.aspx?Id=32017
(7)  Gomez-Ramirez et al. 2014.  Estudio ultraestructural del efecto de una presentación de glifosato (Roundup®Activo) en los hemisferios telencefálicos de cachama blanca (Piaractus brachypomus). Proceedings of LACQUA 2014 meeting, Guadalajara, Mexico.
(8)  Borbón, J.F. and Jaime F. González. 2015. Unpublished.
(9)  Oahn D.T.H., Alcivar-Warren A. and collaborators of the ONE HEALTH Epigenomics Educational Initiative. 2015. Epigenetic modifications induced by endocrine disrupting chemicals (EDCs): metals, PCB pesticides, PAHs, and antibiotics reported in sediment of mangroves and shrimp ponds, tissues of shrimp, molluscs and fish, and mammalian cells. Abstract submitted to Aquaculture America 2015, New Orleans Feb 19-22, 2015.
(10)  Philippe Grandjean. 2013. Only One Chance: How Environmental Pollution Impairs Brain development - and How to Protect the Brains of the Next Generation.  Oxford University Press. See also 'Chemical Brain Drain" at http://braindrain.dk/
 
Acknowledgements
We thank the following governmental and non-governmental organizations and educational institutions for their support: ONE HEALTH Epigenomics Foundation, Southborough, MA, USA;  FUCOBI Foundation of Quito, Ecuador; Universidad Militar Nueva Granada and Project CIAS 1461; Universidad de Llanos, Villavicencio; Universidad Nacional de Colombia.  
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