Non-Mammalian In Vivo models: C. elegans As a Model System to Inform Hazard Identification

Piper Reid Hunt, PhDFDA/CFSAN/OARSA/DOTpiper.hunt@fda.hhs.gov

Conflict of Interest Statement

Disclaimer:The data, views, and opinions presented here are those of the presenter and do not represent FDA policies or positions.

The presenter has no competing interests.

Affiliation

Topic Overview

C. elegans biology

Comparison of C. elegans model to other models

Established methods for assessing toxicity to C. elegans

Utility of C. elegans model for hazard identification of mixtures

A novel method for assessing developmental toxicity in C. elegans

What is Caenorhabditis elegans?

Adult Length ~ 1mm

What is Caenorhabditis elegans?

myo-3::GFP

Caenorhabditis elegans

100μm

C. elegans adult and 1st stage larvae (L1) Oxidative Stress Response gst-4p::GFP transgene

C. elegans Transgenic Strains Can Rapidly Detect Changes in Toxicity Pathways

Conserved Pathways

Key Elements of the Apoptosis Pathway Are Conserved Between C. elegans and Mammals

(Rastogi et al., 2010)

Conserved Pathways

ATP Synthesis Pathways Are Conserved Between C. elegans and Mammals

Mitochondrial respiratory chain complex, high homology for:− Nuclear encoded genes− mtDNA encoded genes− Bioenergetics− Metabolism− Structure

(Tsang, Lemire, 2002)

Conserved Pathways

IGF-1 Signaling Is Conserved Between C. elegans and Mammals

(Christensen et al., 2006)(Klotz et al., 2015)

Conserved Pathways

Conserved Signal Transduction Pathways• Wnt pathway via β-catenin• Receptor serine/threonine kinase pathway• Receptor tyrosine kinase pathway• Notch-delta pathway• Apoptosis pathway• Receptor protein tyrosine phosphatase pathway• G-protein-coupled receptor pathway• Ligand-gated cation channel pathway• Gap junction pathway

(Leung et al., 2008)

C. elegans vs. Rats

Space: 1 rat vs. ~100,000 C. elegans nematodes

Experiment Duration: months vs. days

Both: intact digestive, reproductive, and neuronal systems

Validation studies needed to define domains of applicability(Hunt, 2017)

C. elegans vs. Zebrafish

Utility for Toxicity Testing C. elegans Zebrafish embryo

organ systems: reproductive, muscular, nervous YES YES

organ systems: visual, circulatory, skeletal NO YES

established assays can be used with non-water soluble, volatile and/or extreme pH compounds NO NO

neurobehavioral assays YES YES

mutant and transgenic strains can identify pathways of toxicity YES YES

life cycle 3 days 3 months

model Oral Absorptive

TSCAUpdate June 22, 2016

C. elegans Motility Detects Mammalian Neurotoxins

(Cole et al., 2004)

C. elegans and Mammalian Developmental Toxins

(Harlow et al., 2016)

• 72 Chemicals, 57 with Mammalian Reproductive Effects

• Egg Viability Assay• Cytochrome P450 expression• RNAi P450 Expression Knock Down

C. elegans and Mammalian Developmental Toxins

ToxCastTM Phase I and II Libraries (~900 chemicals)• Assessed C. elegans larval growth data for predicting developmental

outcomes in rabbits and rats

(Boyd et al., 2015)

- 58% concordance between rabbit and rat- 52% concordance between C. elegans and rabbit or rat

Growth Assessed by Flow/Laser Technology

COPASTM: Complex Object Parametric Analyzer and Sorter

(Hunt et al., 2013)

C. elegans LC50 Correlates to Rat LD50

(Hunt et al., 2012)

C. elegans LC50 Correlates to Rat LD50

(Li et al., 2013)

Cadmium Induces Intestinal Abnormalities in Mammals

control 7.8 ppm CdCl2

(Hunt et al., 2012)

C. elegans Developmental Stages

C. elegansLifecycle

(Altun, Hall, 2008)

C. elegans Developmental Stages

Motility Assessment with Infrared Beam Detection

WMicroTrackerTM measures small animal activity in beam interruptions

(Simonetta, Golombek, 2007)

Worm Development and Activity Test (wDAT)

6h, Stage: L1Length: 0.30mm

27h, Stage: L2Length: 0.48mm

48h, Stage: L4Length: 0.74mm

72h, Stage: AdultLength: 1.0mm

Low Level Mercury Exposure Associated with Hyperactivity in Children and Developing Rodents

(Cheuk, Wong, 2006; Gimenez-Llort et al., 2001; Rocha et al., 2001; Sagiv et al., 2012)

Mercury Exposure Associated with Reduced Weight Gain in Developing Rodents

L3

L3

L3

(Fredriksson et al., 1993)

Mercury Exposure Induces Hypokinesia in Mammals

Mercury Exposure Induces Hypokinesia in Mammals

(Bushana, Hunt, 2014)

C. elegans Larval Development with wDATvs. Larval Growth by Flow/Laser Technology

* * * *** * **

* p-value < 0.05** p-value < 0.005

Arsenic Exposure Associated with Hyperactivity in Children and Developing Rodents

(Roy et al., 2011; Rodriguez et al., 2010; Chandravanshi et al., 2014)

Arsenic Exposure in Developing Rodents: hyperactivity at low dose, hypoactivity at high dose

(Rodriguez et al., 2010; Bardullas et al., 2009; Itoh et al., 1990)

*

**

**

* p-value < 0.05* p-value < 0.005

Developmental Delay with Mixtures

** *

Developmental Delay with Mixtures

*

*

*

*

Summary

C. elegans assays are rapid and inexpensive, facilitating mixture assessment

Concordance has been demonstrated between:− C. elegans LC50 ranking and rat LD50 ranking− C. elegans motility and mammalian neurotoxicity− C. elegans larval growth and mammalian developmental toxicity− C. elegans gene expression and mammalian mechanisms of toxicity

Validation studies are urgently needed

References

Altun, Z. F. & Hall, D. H. 2008. Introduction to C. elegans Anatomy. Worm Atlas. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

Bardullas, U., Limon-Pacheco, J. H., Giordano, M., Carrizales, L., Mendoza-Trejo, M. S. & Rodriguez, V. M. 2009. Chronic low-level arsenic exposure causes gender-specific alterations in locomotor activity, dopaminergic systems, and thioredoxin expression in mice. Toxicol Appl Pharmacol, 239, 169-77.

Boyd, W. A., Smith, M. V., Co, C. A., Pirone, J. R., Rice, J. R., Shockley, K. R. & Freedman, J. H. 2015. Developmental Effects of the ToxCast Phase I and II Chemicals in C. elegans and Corresponding Responses in Zebrafish, Rats, and Rabbits. Environ Health Perspect, 124, 586-593.

Bushana, P. & Hunt, P. R. 2014. Novel C. elegans motility assay with high-throughput screening potential detects mammalian neurotoxins. OARSA Science Day. MOD I, 8301 Muirkirk Rd, Laurel, MD 20708: CFSAN.

Chandravanshi, L. P., Shukla, R. K., Sultana, S., Pant, A. B. & Khanna, V. K. 2014. Early life arsenic exposure and brain dopaminergic alterations in rats. Int J Dev Neurosci, 38, 91-104.

Cheuk, D. K. & Wong, V. 2006. Attention-deficit hyperactivity disorder and blood mercury level: a case-control study in Chinese children. Neuropediatrics, 37, 234-40.

Christensen, K., Johnson, T. E. & Vaupel, J. W. 2006. The quest for genetic determinants of human longevity: challenges and insights. Nat Rev Genet, 7, 436-48.

Cole, R. D., Anderson, G. L. & Williams, P. L. 2004. The nematode Caenorhabditis elegans as a model of organophosphate-induced mammalian neurotoxicity. Toxicol Appl Pharmacol, 194, 248-56.

References Fredriksson, A., Gardlund, A. T., Bergman, K., Oskarsson, A., Ohlin, B., Danielsson, B. & Archer, T. 1993. Effects of maternal dietary

supplementation with selenite on the postnatal development of rat offspring exposed to methyl mercury in utero. Pharmacol Toxicol,72, 377-82.

Gimenez-Llort, L., Ahlbom, E., Dare, E., Vahter, M., Ogren, S. & Ceccatelli, S. 2001. Prenatal exposure to methylmercury changes dopamine-modulated motor activity during early ontogeny: age and gender-dependent effects. Environ Toxicol Pharmacol, 9, 61-70.

Harlow PH, Perry SJ, Widdison S, Daniels S, Bondo E, Lamberth C, Currie RA, Flemming AJ. 2016. The nematode Caenorhabditis elegans as a tool to predict chemical activity on mammalian development and identify mechanisms influencing toxicological outcome. Sci Rep 6: 22965.

Hunt, P. R., Marquis, B. J., Tyner, K. M., Conklin, S., Olejnik, N., Nelson, B. C. & Sprando, R. L. 2013. Nanosilver suppresses growth and induces oxidative damage to DNA in Caenorhabditis elegans. J Appl Toxicol, 33, 1131-42.

Hunt, P. R., Olejnik, N. & Sprando, R. L. 2012. Toxicity ranking of heavy metals with screening method using adult Caenorhabditis elegans and propidium iodide replicates toxicity ranking in rat. Food Chem Toxicol, 50, 3280-3290.

Hunt PR. 2017. The C. elegans model in toxicity testing. J Appl Toxicol 37: 50-59. Itoh, T., Zhang, Y. F., Murai, S., Saito, H., Nagahama, H., Miyate, H., Saito, Y. & Abe, E. 1990. The effect of arsenic trioxide on brain

monoamine metabolism and locomotor activity of mice. Toxicol Lett, 54, 345-53. Klotz LO, Sanchez-Ramos C, Prieto-Arroyo I, Urbanek P, Steinbrenner H, Monsalve M. 2015. Redox regulation of FoxO transcription

factors. Redox Biol 6: 51-72. Leung MCK, Williams PL, Benedetto A, Au C, Helmcke KJ, Aschner M, Meyer JN. 2008. Caenorhabditis elegans: an emerging model

in biomedical and environmental toxicology. Toxicol Sci 106: 5-28.

References

Li Y, Gao S, Jing H, Qi L, Ning J, Tan Z, Yang K, Zhao C, Ma L, Li G. 2013. Correlation of chemical acute toxicity between the nematode and the rodent. Toxicology Research 2: 403-412.

Rastogi RP, Sinha R, Sinha RP. 2010. Apoptosis: molecular mechanisms and pathogenicity. EXCLI Journal 8: 155-181. Rocha JB, Rocha LK, Emanuelli T, Pereira ME. 2001. Effect of mercuric chloride and lead acetate treatment during the second stage

of rapid post-natal brain growth on the behavioral response to chlorpromazine and on delta-ALA-D activity in weaning rats. Toxicol Lett 125: 143-150.

Rodriguez, V. M., Limon-Pacheco, J. H., Carrizales, L., Mendoza-Trejo, M. S. & Giordano, M. 2010. Chronic exposure to low levels of inorganic arsenic causes alterations in locomotor activity and in the expression of dopaminergic and antioxidant systems in the albino rat. Neurotoxicol Teratol, 32, 640-7.

Roy, A., Kordas, K., Lopez, P., Rosado, J. L., Cebrian, M. E., Vargas, G. G., Ronquillo, D. & Stoltzfus, R. J. 2011. Association between arsenic exposure and behavior among first-graders from Torreon, Mexico. Environ Res, 111, 670-6.

Sagiv, S. K., Thurston, S. W., Bellinger, D. C., Amarasiriwardena, C. & Korrick, S. A. 2012. Prenatal exposure to mercury and fish consumption during pregnancy and attention-deficit/hyperactivity disorder-related behavior in children. Arch Pediatr Adolesc Med,166, 1123-31.

Simonetta SH, Golombek DA. 2007. An automated tracking system for Caenorhabditis elegans locomotor behavior and circadian studies application. J Neurosci Methods 161: 273-280.

Tsang, W. Y. & Lemire, B. D. 2002. Mitochondrial genome content is regulated during nematode development. Biochem Biophys Res Commun, 291, 8-16.

FDA Mission RelevanceThe Office of Applied Research and Safety Assessment (OARSA) at the FDA’s Center for Food Safety and Applied Nutrition (CFSAN) protects public health by providing science-based data to support the FDA’s regulatory food protection and cosmetic safety programs. Current CFSAN priorities include the development of improved methods for the detection of developmental toxicity and assessment of mixture toxicity. Our concordance study findings indicate a potential for the use of data from C. elegans assays to quickly provide human-relevant information for agency resource allocation and regulatory decision making. In this way, we seek to increase the number of chemicals and chemical mixtures assessed for safety, while at the same time reducing costs and laboratory mammal use.

Acknowledgements

Andrew Liem (transgenic gene expression) Priyanka Bushana (motility analyses) Cory Vaught (morphology analyses) Nicholas Olejnik (COPAS assessments) Tom Black (lab management)

Robert Sprando (DOT Director) Suzanne Fitzpatrick (CFSAN Senior Advisor for Toxicology)

Questions?

http://www.wormatlas.org/hermaphrodite/introduction/mainframe.htm?mobify=0