While previous studies look into preference behaviour to ethanol and resistant mutation to caffeine in C. elegans, cellular and neuronal developments are currently unknown. Our approach has been to understand the effects of ethanol and caffeine exposure at cellular and neuronal level, especially looking at the worms developments through their life-cycle. Our objective is to explore any regenerative effects of Vitamin D and Vitamin B on prolonged caffeine 1

and ethanol use.

[1][2]

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fect of common household neurotoxic and neuron generating substance on the development of

nervous system in C. elegans

Effect of common household neurotoxic and neuron generating substance on the development of nervous system in C. elegans

Abstract:

Caenorhabditis elegans (C. elegans) senses multiple chemical stimuli through sensory systems

and rapidly changes its physiology for survival. With a simple and well-characterized nervous

system, C. elegans is a suitable animal model for studying neurotoxicity on multicellular

eukaryotic organisms.

In this study, we show that C. elegans is a useful model organism for studying side effects of

ethanol, including the thiamine (vitamin B1) deficiency caused by prolonged alcohol

consumption. We designed a population studies assay for population count after prolonged

ethanol, caffeine, vitamin B1 and Vitamin D exposure. Despite no responses to caffeine, vitamin

B1 and Vitamin D, animal show reduction of population after 1 week of pre-exposure to

ethanol at high concentration. The effect can be reverse by giving the animal a supplement of

vitamin B1 which results in the population increase at the same amount as seen in the wild type

control.

Abstract:

Caenorhabditis elegans (C.

elegans) senses multiple

chemical stimuli through

sensory systems and rapidly

changes its physiology for

survival. With a simple and

well-characterized nervous

system, C. elegans is a

suitable animal model

for studying neurotoxicity on

multicellular eukaryotic

organisms.

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Abstract Methods

Results Discussion & Conclusion

References & Acknowledgments

Population Study: Examining adult and L4 worms showed a decrease in population when expose to high concentration of ethanol, vitamin B and D in comparison with control (P < 0.05, n = 3). No population change was observed in caffeine, or 1

its combinations (P > 0.05). However, animals exposed to both ethanol and vitamin B1 in high concentration developed a significant increase in population in comparison to ethanol or vitamin B alone (P < 0.05).1

(Fig.1a)

Fluorescence intensity assay: Fluormeter data also agreed to a significant reduction in population of C. elegans when expose to high and medium concentration of ethanol and vitamin B (P < 0.05, n = 3) . Animals exposed to 1

both ethanol and vitamin B shown a recovery in population close to that of the control.1

(Fig1b)

Fluorescence Microscopy: Fluorescence microscopy showed the majority of the neurons located in the head, where they are organised in a number of ganglia surrounding the pharynx, forming the brain. Moreover, chemosensory neurons making up a large fraction of the head ganglia sending their dendrites to the tip of the nose are visible.

Figure 1: a) Adult and L4 Population Counts. b) Fluorescence intensity assay.Worm samples being exposed to Ethonol (EtOH), caffeine (CAF), Vitamin B1 (B1), Vitamin D (D) and their combinations with three concentrations for 1 week, were tested for population and fluorescence intensity.

(b)

(a)

(f)

Figure 2: Nerves System Fluorescence Microscopy. a,b) 20x view of C.elegans showing its head and tail. c,d) Nervous system of C.elegans, showing the head ganglia (brain) and the tail ganglion. e) 40x view of head ganglia. The ganglia forming the brain and the chemosensory neuron with their extend dendrites to nosie. f) 40x view of pharnyx.

(a)

Head

Tail

(d)

Head Ganglia

Tail Ganglion

(c)Head Ganglia

· Worm samples expose to vitamin B and D suffer a significant 1

reduction in population and fluorescence , which can be related, to the effects that B and D have on the available food 1

(OP50 E.Coli). Addition of vitamin B and ddH O results in the 1 2

formation of non-soluble B residues that can restrict 1

acceptability to food. Moreover, vitamin D itself has antimicrobial property that allows the animal to produce an enzymes destroying E.coli . No significant changes in caffeine and its combinations suggest that caffeine have no significant effect on the worms' nervous system and proliferation.

· In all samples, we did observe all the main head neurons. However, no abnormal changes in the anatomy of nervous system could be detected. This observation can be related to strength of the controlling factors and genes affecting the nervous system. Therefore, we can conclude that neurotoxic and neuron generating substances used in this experiment do not influence the development of the C.elegans nerve system.

(Fig.1)

[4]

Population Study: Worm samples were prepared with NMG+OP50 buffer plate and 500 μl of the tested substances . These samples were incubated at 20 C for a week. Then, the worms were analysed using stereo microscopy at 8X zoom in three different viewpoints. One viewpoint

2corresponded to area of 362.6 mm in the plates.

(Table 1)

mg/dl mol/L mg/dl mol/L mg/dl mol/L mg/dl mol/L

2.76 0.6 5.83 0.3 11.54 0.3 10.12 0.3

1.38 0.3 2.91 0.15 5.76 0.15 5.06 0.15

0.7 0.15 1.46 0.075 2.89 0.075 2.52 0.075

Co

ncen

tration

s

Alcohol Caffeine Vitamin D Vitamin B1

Fluorescence intensity assay: DiO dye was added to each worm plate. After one hour, they were transferred to conical tubes for three times washing. Triplicate 100μl of each washed samples were transferred in to a 96 well plate and their fluorescence measured using fluorometer with 484nm excitation max. and 501 nm emission max. [3].

Fluorescence Microscopy: Three dyed sample warms were picked and immobilized with Agaros and microbids between microscope slides. The fluorescence microscopy was performed with inverted microscope at 20X and 60X zoom.

With a simple and well-characterized nervous system, C. elegans is a suitable model for studying neurotoxicity on multicellular organisms. Here, we examined the long term effects of ethanol and caffeine, on C.elegans and determined generative role of Vitamin D and B . Our results showed that 1

1 week exposure of ethanol can impede the proliferation and population growth of worms. And the same amount of exposure to of vitamin B can reverse the impeding effect of ethanol. 1

Introduction

Special thanks to all the people who helped us perform this experiment:

Prof. KilkennyProf. ShukalyukProf. William RyuChao LiuEdward SykesXi ChenAylia MohammadiGreg StegemanKonstantine Palanski Jarlath Byrne RodgersHang Li

[1] J. Lee, C. Jee and S. L. McIntire, "Ethanol preference in C. elegans," Genes, Brain and Behavior, vol. 8, no. 6 , p. 578–585, 2009.

[2] P. S. Hartman, "Caffeine-resistant mutants of Caenorhabditis elegans," Genetical Research, vol. 49, no. 2, pp. 105-110, 1987.

[3] "Fluorsecent dye spectra." UA Department of Molecular & Cellular Biology. N.p., n.d. Web. 31 Mar. 2012. <http://www.mcb.arizona.edu/IPC/spectra_page.htm>.

[4]Hewison , M.. "Antibacterial effects of vitamin D.." Nat Rev Endocrinol. vol.7, no.6, p.337-45,2011.

Kasra Tajdaran & Jirapat Likitlersuang

The Institute of Biomaterials and Biomedical Engineering

University of TorontoBME340H1

Table 1. Neurotoxic and neural generating substance added to worm samples in three different concentrations.

(e)

(b)