I fully understand that any violation of the above will result in disciplinary action by the institution and may also result in criminal action if not properly cited or necessary permission not granted where appropriate. Toxic effects of combined exposure to triclosan and glyphosate on zebrafish embryos submitted for the degree of Master of Technology. We studied the effects of combined exposure to Triclosan and Glyphosate on zebrafish embryos.
We observed that the combined exposure of TCS and glyphosate is more toxic compared to individual exposure of TCS or glyphosate. Combined exposure increased mortality, delayed hatching, increased morphological abnormalities and decreased larval reproduction. In addition, we observed that TCS exposure disrupted the integrity of the NMJ junction in a concentration-dependent manner as indicated by an increase in chevron angle and increased visible discontinuities when labeling the NMJ with Znp1 antibody, which labels motor neuron axons.
Overall, our results suggest that developmental toxicity is more severe in case of combined embryo exposure.
Introduction
- Triclosan
- Glyphosate
- Developmental stage
- Neuromuscular Development Morphology
TCS has been reported to be involved in eczema and osteoporosis in humans and has been found to be estrogenic and (Ma et al. 2013) has been found to have androgenic properties (Cai et al. The use of glyphosate in agricultural and non- agricultural applications have increased very rapidly in the last 45 years (Benbrook 2016; Myers et al. 2016.) Toxicology studies on zebrafish muscles are considered to be of very high relevance since 60% of body mass of adult zebrafish consists of skeletal muscles (Blagden et al. 1997 ).
Skeletal muscles arise from the paraxial mesoderm and undergo further segmentation to form somites (Devoto et al. 2006). However, skeletal muscles in the trunk region and pelvic fin are only present in the myotome region (Cole et al. Skeletal muscle cells (SMCs) originate from precursor muscle cells called adaxial cells (initially cuboidal in shape), which are formed from paraxial mesoderm (Du et al. al 1997).
The physical connection between the two types of tissue - nerve and muscle tissue is called the Neuromuscular Junction (NMJ) which provides the contractile activity of the muscles derived from the motor neurons (Panzer et al. 2005).
Review of Literature
Adverse effects of Triclosan
- In humans
- In animal models
- Cell lines
- Triclosan and neurotoxicity
- Triclosan and muscular dysfunction
- In humans
- In animal model
- In cell lines
In mice it has been shown to cause hypothermia and inclusive central nervous system (CNS) depression (Miller et al. 1983). It causes adverse effects on fetal development (Kumar et al. 2009) by inhibiting the action of estrogen sulfotransferase in the ovine placenta. Human induced pluripotent stem cell-derived cardiomyocytes undergo arrhythmic beating and downregulated homeostatic genes upon exposure to TCS (Weatherly et al. 2016).
TCS acts as a mitochondrial uncoupler by altering its ultrastructure, causing change in membrane potential and forcing fission mechanism (Weatherly et al. 2018). TCS also interferes with Ca 2+ signaling during skeletal muscle development in the early developmental stages of zebrafish (Heath et al. 2000). The chemical structure of glyphosate consists of a phosphono-methyl and an amino acid glycine (Li et al. 2013).
Glyphosate and its metabolites have been found to be present in human urine and other body fluids (Krüger et al. 2014; Niemann et al. 2015).
Scope of the study
Objectives
- To observe and compare toxicity of TCS and Glyphosate (Combined v/s Single exposure)
- To observe effect of TCS on neuromuscular properties of larva after 96 h exposure exposure
Materials and Methods
Zebrafish Housing
Zebrafish mating
Preparation of solution
- Preparation of E3 Medium
- Preparation of TCS Solution
To prepare the basic solution I, 14 mg of triclosan was dissolved in 1.4 ml of acetone solution and then mixed well. The table below shows the details of stock and working solution preparation.
- Preparation of Glyphosate solution
- Preparation of (acetone) solvent control solution
- Preparation of PBS (Phosphate Buffer Saline) solution
- Preparation of Depigmentation solution
- Preparation of blocking solution
- Preparation of PBST solution
- Preparation of formaldehyde solution
- Preparation of mounting media (PPD Glycerol)
- Experimental Design
- Exposure with Chemicals
- Abnormality rate
- Larval length
- Whole Mount Labelling
- Materials Required
- Procedure
- Imaging the embryos
- Deconvolution
- Definition
- Section 4.11.1 to 4.11.3 explains deconvolution process using WPL algorithm of PID plugin of ImageJ
- Requirements
- Creating a PSF image using Diffraction PSF 3D
- Longitudinal spherical aberration is an intricate feature of a microscope and cannot be estimated comprehensively. Keeping an approximation value of 0.00 is fair and generally
- CCD cell spacing is the most important factor among all PSF image measures. Each CCD camera has a unique pixel size (usually in micrometres) which can be found either online or
- Normalization – this field area can be set at default value of ‘1’
For NMJ analysis, larvae exposed for 96 h were fixed in 4% formaldehyde overnight at 4˚C prior to whole-mount labeling with znp1 primary antibody. Unfertilized/dead embryos were separated from healthy embryos manually with the help of a Pasteur pipette. Then to start the drug treatment, the embryos were first transferred to a 24-well plate with 10 embryos in each well and the exposure to the respective chemical was 96 hours with 1 ml of solution per well.
The solutions were replaced with fresh solution in a 24 h window and dead embryos were periodically removed throughout the experimental period. After every 24 h of exposure, zebrafish embryos were analyzed for mortality; dead larvae were removed and the solutions were replaced each time with fresh ones. At the end of the 72 h exposure period, the larvae were analyzed for the presence of any morphological abnormalities such as TND - tail non-detachment, YSE - yolk sac edema, SC - spine curvature, PCE - pericardial edema, TB - tail bending.
Fixation of zebrafish larvae – After 96 hours of TCS exposure, the zebrafish embryos were first washed twice with E3 medium (5 min each) and then fixed with paraformaldehyde solution (4%, pH 7.2) overnight at 4˚C. Permeabilization - Larvae were washed with PBS (twice for 5min each) to remove the depigmentation mixture, followed by 20 min heat treatment in PBS at 57°C. Antibody labeling – Larvae were incubated for 4 h at RT in mouse monoclonal antibody znp-1 at 1:100 dilutions in blocking solution.
For anomalies and length, bright-field images of larvae were taken using the 4x objective at a resolution of For both Chevron and NMJ angle irregularities, larvae were placed under the microscope and images were captured at 10x and 20x objective at a resolution of. Parallel Iterative Deconvolution – This plugin can be downloaded from the website https://www.softpedia.com/get/Multimedia/Graphic/Graphic-Plugins/Parallel-Iterative- Deconvolution.shtml in the form of a .zip file.
Now, when you open the ImageJ application, you will find this plugin already installed in the ImageJ plugin menu as shown in Figure 15. Diffraction_PSF_3D.class file from the website "http://fiji.sc/Diffraction_PSF_3D" and place it in the folder of appendices (figure 16). 3D PSF Diffraction”, a new window will open with 11 different slots, as you can see in Figure 20.
Slice spacing (z), same units – This value is important for 3D deconvolution purposes, but not at all useful in the case of 2D deconvolution.
Different steps of deconvolution STEP 1 – Selecting an image
STEP 2—creating a PSF image using Diffraction PSF 3D plugin
Saving each channel individually according to its respective colour STEP 5 – Deconvolute each channel separately using deconvolution plugin with
Merge all deconvoluted singled channel images
- STEP 1 – Selecting an image
- STEP 2— Creating a PSF image using Diffraction PSF 3D plugin
Refer to figure. 18-21)
STEP 4 -- Saving each channel individually according to its respective colour
Convert these split images to 8-bit if the images are in some other format and store all three images according to their color channels as shown in Figures 31-33. Note: If your channel images turn out to be 8-bit, you don't need to convert to 8-bit.
Keep everything else as shown in figure 37 except maximum number of iterations and show iteration checkbox. If you check this box, you can have an idea about which iteration can give you the best results and choose accordingly. You can save your image only after the allotted number of repetitions have already been completed.
STEP 6 – Merge all deconvoluted singled channel images
Results
- Combined exposure of TCS and Glyphosate is more toxic as compared to individual exposure of TCS and glyphosate
- Mortality rate
- Hatching rate
- Morphological abnormalities
- larval length
- ImageJ plugin PID (WPL algorithm) delivers higher quality deconvoluted images images
- TCS induced Change in chevron angle
The solutions were replaced with new ones every 24 hours and dead embryos were removed each time. To observe abnormalities in larvae, chemical treatment was initiated using 5 HPF embryos and continued up to 72 hours. At the end of the 72-h exposure, 15 larvae from each concentration were randomly selected and checked under the microscope for any abnormality, such as yolk sac edema, tail bowing, pericardial edema, and spinal curvature.
In our study, exposure to 0.3 µg/ml and 0.6 µg/ml did not cause any significant abnormalities. abnormality of larval zebrafish after 72-hour chemical exposure to TCS and glyphosate. a) 50G+0.6TCS showed a significant increase in %abnormality compared to 50G. TND - non-detachment of the tail, YSE - edema of the yolk sac, SC - curvature of the spine, PCE - pericardial edema, TB - flexion of the tail. To examine any change in larval length due to chemical exposure, chemical treatment was indicated using 5 hpf embryos and continued for 96 h.
At the end of 96 hours of exposure, 5 larvae from each concentration were randomly selected and their images were taken with 4x objective of Olympus X-73 microscope. Larval length decreases after 96 h of combined exposure with 50G and TCS (a) Representative larval images of decreasing length with concentration. Larval length decreases after 96 h of combined exposure with 100G and TCS (a) Representative larval images of decreasing length with concentration.
Then the total of 3 best algorithms from both plugins PID (WPL & MRNSD) and DLab2 (RL) were finally compared with a total of five example images. To analyze chevron angle, a particular area consisting of a total of 5 segments above the mid-trunk of larvae was selected for analytical purposes as shown in figure 10. To analyze NMJ discontinuities, a specific region consisting of a total of 11 segments above the mid trunk of larvae was selected for analytical purposes as shown in figure 10.
In our study, less than 20% of segments of control larvae showed NMJ discontinuities, while in the case of 0.3 µg/ml and 0.6 µg/ml TCS treated larvae both more than 30% and 60% segments respectively showed NMJ irregularities.
Discussion and Conclusion
NMJ refers to direct physical contact between nerve and muscle tissue and is responsible for the neuron-derived motor behavior of larvae.
Future directions
Association of urinary triclosan with bone density and osteoporosis in US adult women Journal of Clinical Endocrinology and Metabolism. Simultaneous determination of triclocarban and triclosan in municipal biosolids by liquid chromatography tandem mass spectrometry.” Journal of Chromatography. Triclosan: Current Status, Occurrence, Environmental Risks and Bioaccumulative Potential.” International Journal of Environmental Research and Public Health.
Positive and negative regulation of muscle cell identity by members of the Hedgehog and TGF-Beta gene families. Journal of Cell Biology. Estrogenic and androgenic activity of Triclosan in breast cancer cells. Journal of Applied Toxicology: JAT. Influence of soil composition on glyphosate and phosphate adsorption from Danish surface soils in contrast.
Inhibition of the Staphylococcus AureusNADPH-Dependent Enoyl-Acyl Carrier Protein Reductase by Triclosan and Hexachlorophene. Journal of Biological Chemistry. Characterization of Pseudomonas Aeruginosa Enoyl-Acyl Carrier Protein Reductase (FabI): A target for the antimicrobial triclosan and its role in acylated homoserine lactone synthesis. Journal of Bacteriology. The need for independent research into the health effects of glyphosate-based herbicides.” Environmental Health: A Global Access Science Source 17(1):51.
Effects of glyphosate and aminomethylphosphonic acid on an isogenic model of the human blood-brain barrier.” Toxicology Letters 304: 39–49. Acute Toxicity of Penta-, Hexa-, and Heptachlorohydroxydiphenyl Ethers in Mice.” Journal of Toxicology and Environmental Health. Concerns about Glyphosate-Based Herbicide Use and Risks Associated with Exposure: A Consensus Statement.” Environmental Health: A Globally Accessible Science Resource 15:19.
Differential effects of glyphosate and summation on human placental cells and aromatase." Environmental Health Perspectives. Uncoupling of mitochondria in living mouse and human mast cells and in primary human keratinocytes. Journal of Applied Toxicology: JAT.
