Hydra as a Dual-Utility Model for Cytotoxicity and Genotoxicity: Bridging
Environmental Toxicology and Therapeutic Applications
Jagadisha Tavarekere Venkataravanappa a*, Nayana Mitta Lakshminarayana Guptab,
Shreya Srivastavab, Yuvaraj Shapur Gopalkrishnashettyb , Parvathee Somanb , Saraswathi
Saraswathi b, Shambhavi Kumarib. Konakanchi Sureshc, Venkateswarlu raavid
a*
Department of Medical Genetics, JSS Medical College and Hospital, JSS-AHER, Mysuru570015, Karnataka, India.
b
Department of Life Sciences, Kristu Jayanti Deemed to be University, Bengaluru-560077,
Karnataka, India.
c
Department of Physiology, Sri Madhusudan Sai Institute of Medical Sciences and Research,
Satya Sai University for Human Excellence, Chikkaballapur, Karnataka, India.
d
Department of Cell Biology and Molecular Genetics, Sri Devaraj Urs Academy of Higher
Education and Research, Kolar - 563 103, Karnataka, India.
Corresponding Author*
Dr. Jagadisha T.V., M.Sc., PGDGT., PhD
Assistant Professor
Department of Medical Genetics
JSS Medical College and Hospital
JSS-AHER, Mysuru- 570015
Ph.-No: 8892698143/9449442521,
E-mail:jagadish.tv.1993@gmail.com
Running title: Hydra as a Model for Environmental and Therapeutic Toxicology
Declaration of Interest: The authors declare no conflict of interest
Author's Email IDs:
● Jagadisha Tavarekere Venkataravanappa:jagadish.tv.1993@gmail.com
●
Yuvaraj Shapur Gopalkrishnashetty: yuvarajsg2002@gmail.com
● Parvathi Soman : parvathysoman2018@gmail.com
● Nayana Mitta Lakshminarayana Gupta: nayanamitta16@gmail.com
● Shreya Srivastava : shreya@gmail.com
● Saraswathi Saraswathi: saraswathi571@gmail.com
● Shambhavi Kumari: shambhavik2504@gmail.com
● Suresh Konakanchi: suresh.c@smsimsr.org
● Venkateswarlu raavi; Venkyneuro@gmail.com
Hydra as a Dual-Utility Model for Cytotoxicity and Genotoxicity: Bridging
Environmental Toxicology and Therapeutic Applications
ABSTRACT
Background:
Hydra, a simple freshwater cnidarian, is increasingly recognised as a valuable alternative
model organism in toxicological research due to its ease of cultivation, remarkable regenerative
capacity, and well-characterised cell lineages.
Objective:
This review aims to consolidate current knowledge on the cytotoxic and genotoxic effects of
environmental pollutants, nanoparticles, heavy metals, and Hydra-derived toxins. It also
explores the applicability of Hydra in biomedical research, particularly in cancer therapeutics.
Methods:
A comprehensive review of literature was conducted focusing on the impact of substances such
as copper and zinc oxide nanoparticles, cobalt ions, and pharmaceuticals on Hydra vulgaris
and related species. Various assays, such as morphological assessments, regeneration
inhibition, comet assay, micronucleus assay, and gene expression analysis, were evaluated.
Results:
Zinc oxide nanoparticles exhibited size-dependent toxicity, with smaller particles causing more
pronounced morphological changes and inhibition of regeneration. Copper and cobalt exposure
led to oxidative stress, ROS generation, DNA damage, and apoptosis. Hydra-derived toxin
HALT-1 showed potent cytolytic activity against cancer cells, highlighting its therapeutic
potential. Genotoxic effects were confirmed through DNA strand break assays and
chromosomal damage indicators.
Conclusion:
Hydra is an effective and ethical model for assessing both cytotoxic and genotoxic effects of
environmental and therapeutic agents. Its regenerative biology and molecular tractability make
it a promising system for future research in environmental toxicology and drug development,
including cancer immunotoxins.
Keywords: Hydra, cytotoxicity, genotoxicity, nanoparticles, regeneration, ROS, HALT-1,
model organism, environmental toxicology, cancer therapeutics.
1. INTRODUCTION
One of the earliest model organisms used in biology is the hydra, it is a freshwater diploblast
with a basic well-defined body plan, a well-organized nervous system, along with stem cells.
Even as an adult, it displays a remarkable capacity for regeneration, lacks organismal aging,
and displays numerous embryonic traits. It features an oral-aboral axis and radial symmetry
similar to that of a starfish. Freshwater polyps of the class Hydrozoa and phylum Cnidaria are
called Hydras,that demonstrate extraordinary regenerating powers. A Hydra polyp may rebuild
its head and foot in a number of days after being bisected.H. oligactis, H. viridissima ,Hydra
oligactis, Hydra mangnipapillata ,H. braueri , Hydra viridissima, Hydra pseudoligactis are
different Hydra species but the most well-known is Hydra vulgaris. The capacity of hydra to
replenish and regenerate differentiated cells from pluripotent stem cells, including nerve cells,
makes it unique.Hydra polyps may completely repair missing structures after being chopped
into pieces (Bode, 2003). Following tissue breakdown and cell reassembly, regeneration may
take place. Hydra polyps can completely rebuild the missing structures when they are divided
into smaller parts. With the exception of the basal disc and tentacles, almost every portion of
the hydra's body has some degree of regeneration ability, However, regeneration can also
happen when tissues separate from cells that reagglomerate. (Noda, 1971; Gierer et al., 1972).
Toxicological research first used artificial embryos made from separated cells to quantify
regeneration (Johnson et al., 1982). subsequently, In order to study regeneration, (Wilby 1988)
proposed using mitotically active multipotent stem cells from the mouth and above the budding
zone in dissected stomach sections (located below the hypostome). Hydra can reproduce both
sexually and asexually. When the right conditions exist, hydra reproduce asexually by budding,
which generates animals that are genetically identical (Pollino and Holdway, 1999). The
availability of food and temperature are two factors that hydra might experience in their
surroundings that can affect their lifetime and asexual reproduction. Maintaining and
cultivating Hydra is not that difficult. Large colonies of lab animals develop asexually by
budding, which makes them an affordable option for toxicity testing. Mass cultures of hydra
are simple to maintain in the lab (Loomis and Lenhoff, 1956). A temperature of 18°C is
maintained in glass or plastic plates, and the animals are fed brine prawns (Artemia nauplii)
three to four times a week. For an infinite amount of time, hydra populations can reproduce
asexually and increase logarithmically (Loomis, 1953). Hydra growth can be attributed to a
number of variables, such as temperature, pH, and water utilized in environmental research.
three dissolved oxygen, food quantity, and ionic equilibrium. Hydra's sensitivity and
widespread distribution across numerous aquatic environments have made it a useful tool for
biological substance toxicity testing. Hydra is an aquatic invertebrate that can detect the
toxicological effects of medications that enter natural waters through sanitary channels and
sewage. Hydra has been demonstrated to be adversely affected by medications and drugs that
target the mammalian receptor. Hydra can be used to precisely assess the toxicological effects
of medications introduced into natural environments through sewage and other means. It has
also been shown that drugs and medical treatments that target the mammalian receptor are
harmful to Hydra. To determine the acute or sub lethal toxicity of a particular drug, a variety
of bioassays are availableMitotically active pluripotent stem cells found in the stomach's
gastrointestinal area have the ability to create a new organism in less than 72 hours. This
accounts for Hydra's remarkable regenerative capacity. Polyps exposed to various medical
treatments may experience alterations in: (1) physical traits and developmental programs; (2)
regeneration or pattern formation; and (3) change in the rates of population growth; bioassays
measuring the asexual reproduction-based population expansion of Hydra are quick, sensitive,
and precise. Hydra's small size and quick rate of reproduction allow them to be cultivated in
big quantities. Hydra's high rate of reproduction makes them useful for sub chronic toxicity
testing, which assesses the effects of toxins on reproduction over brief time periods. Compared
to several freshwater invertebrates, H. vulgaris (formerly known as H. attenuata) has been
found to be among the species most susceptible to the acute and long-term toxicity of the
endocrine disrupting chemical 4-nonylphenol.(Pachura-Bouchet et al., 2006). (Pascoe et al.,
2003) found that three of the ten drugs examined—diazepam, digoxin, and amlodipine—
inhibited Hydra regeneration at a level of 10 mg/L over a protracted exposure period of 17
days. When utilizing environmentally relevant quantities in a tier two toxicity evaluation
conducted in accordance with EU regulatory guidance. Biochemical biomarker reactions have
also been observed in Hydra following drug exposure. In a battery of aquatic biotests, the
toxicity of eleven nanomaterials was investigated (Blaise et al., 2008). For inorganic
nanomaterials (NMs) like as copper and zinc oxides, indium tin oxides, and holmium oxide
nanoparticles in the range of 0.1–1 mg/L, the most sensitive species, Hydra, had a 96-hr EC50.
Hydra has also been used in bioassay studies to assess how urban and industrial effluents affect
the environment. They are usually employed as a part of an organism battery for testing. Four
industrial effluents were determined to be lethal and eight to be sub-lethal in a study on H.
vulgaris that examined the acute toxicity of ten different sources; the 96-hr LC50 ranged from
18.8 to 100 percent effluent (Blaise and Kusui, 1997). A minimal effect concentration (MEC)
of 6- 31 percent was obtained for the effluent produced by H. vulgaris based on adult
morphology and artificial embryo toxicity (Fu et al., 1991). According to the authors, after 72
and 96 hours, the LC50 for industrial wastewaters varied from 2.7 to >100%. The broad
genotoxic and cytotoxic effects of hydra are examined in this review.
1.1 ANATOMY OF HYDRA
A structure called a hypostome, which encircles the mouth opening and is surrounded by
tentacles carrying stinging cells (nematocysts), allows the hydra to catch prey. The hydra's head
is shaped like tubes with tentacles at the tip. (Holdway 2005) refers to the mouth and tentacles
as the hydranth. The head and foot are separated by the body column, and the creatures have a
foot at their base called a basal disc. The term "column" refers to the four parts of the organism:
The area that develops and yields buds is called the developing region; the stomach portion is
between the tentacles and the first (apical) bud; the base of the disc resembles a foot; and the
peduncle is between the lowest bud and the basal disc. The extracellular matrix (ECM) known
as the mesoglea divides the two cell layers that make up hydra, the epidermis and the
gastrodermis. The gastrovascular cavity is lined with endoderm, a water-filled sac that serves
as the location of nutrition absorption and food digesting in addition to acting as a hydrostatic
skeleton. Hydra have a simple neurological system that includes a nerve net that runs
throughout the body Compared to vertebrates, which have specialised organs and a central
nervous system, this structural complexity is simpler, but it is still more complicated than cells
grown in culture. Because of its composition and basic morphology( Figure 1) , Hydra is a
useful and sensitive indicator of environmental stressors such as pollution.
Fig.1- Hydra vulgaris morphology. The entire body, from the foot to the tentacles, is made
up of a bilayer structure, or the ectoderm and endoderm layers split apart by the
mesoglea, as seen in the inset. (Monica Terracciano., 2016).
1.2. REGENERATION IN HYDRA
Abraham Trembley first noted the capacity of hydra to regeneration in 1740. In the pond water,
he unintentionally found a green-colored organism that resembled a polyp. Initially he was not
sure that the organism was a plant or an animal, therefore, he relied on the fact that if it would
be an animal it would die on cutting the body into two halves but it will survive if it would be
a plant. He cut the body of hydra into two halves and after a couple of weeks he observed that
the two halves regenerated their lost parts and looked alike. He also observed many animallike characteristics such as contraction upon touch and development of buds from the parent
body which later detached and grew as an independent organism. Therefore they further
classified the organism as an animal.The remarkable regenerative capabilities of hydra are
demonstrated by their capacity to restore their whole body, even when reduced to a single cell.
According to early studies during the first few hours of regeneration only the sorting of cells
takes place – the epidermal and the gastrodermal layer, reconstructing the body's initial two
layers. Three to five days later, complete polyps with hypostomes, tentacles, and basal discs
appear( Figure 2.).
Fig.2- Regenerating hydra heads and feet at the designated time intervals following midgastric bisection are displayed. A blue arrow points to the fully healed foot. Tentacle
rudiments are indicated by green arrows. A fully regenerated head is indicated by a red
arrow (Matthias C. Vogg et al., 2019).
1.3 REPRODUCTION IN HYDRA
Hydra is able to reproduce both sexually and asexually. To reproduce asexually, the animals
form a bud in their body wall. After three days, the polyp fully develops and splits from its
parent. During sexual reproduction, the ovaries or testes differentiate within the epidermis and
the body wall thickens during sexual reproduction. Depending on whether the animal in
concern is a gonochoristic or hermaphrodite species, the developing testes release sperm cells
that can fertilize the exposed oocytes of that animal or another. The fully developed oocyte
needs to be fertilized within two hours of breaking through the ectoderm and entering the water
surrounding the animal to complete meiosis. For proper embryogenesis to occur, this is
essential. After fertilisation, within 12 hours, gastritis occurs. To protect the embryo until
hatching, which can occur two to twenty-four weeks later, the next stage is the creation of a
thick cuticle. Dormancy occurs two days before to hatching, during which time the embryo
forms its gut and undergoes extensive neurogenesis.
2. CYTOXICITY OF HYDRA
Cytotoxicity can be defined as the adverse effects of different substances reacting with the cell
or disrupting crucial processes for cell survival such as proliferation, Reproduction, normal
physiology and biochemistry. This section mainly focuses on the cytotoxicity of zinc oxide
nanoparticles and copper oxide on hydra, later on we also talk about the toxins produced by
hydra that can pose cytotoxicity.
Due to concerns about ethics and lack of relevance, the use of animals and other vertebrates in
toxicity tests for environmental chemicals has been under scrutiny and criticism recently.
It was put forward that Hydra, a freshwater cnidarian having a simpler biology and body
architecture, would be a suitable substitute model to evaluate the toxicity of pollutants in the
environment. (Blaise et al., 2018, Murphy and Quinn, 2018, Yum et al., 2014, Zeeshan et al.,
2016, Zeeshan et al., 2017). Hydra has also been adopted for the test organism for
pharmaceutical drug testing (Pascoe et al., 2003) due to its simple body organisation and
maintenance.
Although nanotechnology has become a prominent field of study, the main cause for concern
is the potential of nanoparticles, Hence proper toxicology assay is required prior to any
application of these nanoparticles. Any nanomaterial's potential for damage depends on A
nanomaterial's physicochemical characteristics and the biological system it interacts with these
are major determinants of its toxicity. The environmentally toxicology of CuO nanorods was
investigated in Hydra magnipapillata on genetic, cellular, and organismal levels.
Zinc oxide nanoparticles are also known to have acute cytotoxic effects on human and aquatic
organisms, ZnO NPs are generally used for coatings and additives, medication administration,
cleaning supplies, electronics, and personal hygiene, therefore, these substances are quite likely
to make an appearance in the environmental systems. This study focused to assess the effects
of ZnO NPs on Hydra magnipapillata, which were very similar to the impact of ZnO
nanoparticles on mammalian cells. The effects included apoptosis of the cells and cell cycle
disruption and natural killer cell mediated cytotoxicity along with the genotoxic effects such
as DNA Damage and phosphorylation. (Valdiglesias et al., 2013).
Numerous investigations have demonstrated that ZnO NPs are harmful to both people and
aquatic life. ZnO NPs were applied to human nerve cells at doses ranging from 0.01 to 0.03
mg/L in an invitro conditions. leading to both genotoxicity (H2AX phosphorylation and DNA
damage) and cytotoxicity (apoptosis and cell-cycle abnormalities).
Because benthic aquatic species gather in liquid environments and settle in sediments, exposure
to zinc oxide nanoparticles (ZnO NPs) may have an impact on these organisms. On the other
hand, ZnO NPs' biological effects on Little research has been done on benthic species and their
harmful impacts. on biological entities have not been investigated in depth. An earlier study
demonstrated the ecological hazards and toxicity of ZnO NP exposure to creatures that live in
sediment, such the amphipod Hyalela Azteca.
2.1 MORPHOLOGICAL CHANGES ASSAY
In the previously indicated culture environment, A six-well cell culture plate with 10 polyps
per well and one of seven dosages of ZnO NPs added was used. The median lethal
concentration (LC50) for both kinds of ZnO NPs was then determined by incubating the plate
for 48, 72, or 96 hours. Zeta Oxide NP20 in seven concentrations combined with ZnO NP100
Tests were conducted on the culture solution. The polyps without ZnO NPs were utilized.
is the opposite of the positive control. For 48 hours, every hydra polyp that was used was
starved. prior to being placed on culture plates, and not a single animal was given food.
while the toxicity tests were being conducted. Apart from this,10 polyps were taken in a petri
plate for the morphological examinations as well Considering the 48-hour half-life of zinc
oxide (ZnO NP20), 50μg/mL of ZnO NP of concentration 20 or ZnO NP of concentration 100
was dissolved in each culture medium. The polyps that were not exposed served as a negative
control and for each size of the copper nanoparticle triple plates were exposed to different time
periods and checked after 3,6,12 and 24 hrs from a dissecting microscope.
2.2 REGENERATION CHANGES OR INHIBITION ASSAY
In a Petri dish containing Ten hydra polyps were put in a medium for growth with 10 μg/mL
of ZnO NP20 dissolved in it. The polyps that made it through the ZnO NP treatment were
moved right away to the previously indicated regular culture solution and given time to regrow.
The hydra shed their tentacles. The head-decapitated, unexposed polyps served as the
adverse
supervision.
The
experiment
was
run
in
duplicate.
The
polyps
were thereafter cultured as previously mentioned inside the cultural space. The polyps'
regenerative pattern following ZnO NP20 exposure in comparison to individuals who did not.
Polyps were examined under a dissecting microscope at 24, 48, 72, and 96 hours.
Following 48, 72, or 96 hours of exposure to ZnO NP conc.20 or ZnO NP conc.100, the values
of the ZnO NPs in Hydra magnipapillata were ascertained. According to these findings, at all
three treatment durations, ZnO NP conc. 20 was found to be more hazardous as compared to
ZnO NP conc. 100. As a result, the exposure duration and particle size had an impact on the
toxic behaviour of the ZnO Nanoparticles.
After three hours of exposure, there were no modifications in morphology in the hydras treated
to ZnO NP conc. 20. After six hours of treatment, the morphological alterations were
represented by a thin column like body structure. Clubbed-end tentacles were among the
morphological alterations seen in the polyps and buds during a 12-hour exposure period. The
tentacles and body column underwent substantial retraction during a 24-hour of exposition.
After three or six hours of exposure, the hydra polyps treated to ZnO NP100 did not exhibit any
morphological alterations. After exposure for 12 hours, there was a decrease during the growth
of the columnar body of hydra as well as the first seen buds and tentacles were with clubbed
ends. Following a 24-hr exposure, the body column thinned, the tentacles' length significantly
decreased, and their tips grew more
The gene expression experimentation findings indicate that following a 12-hour exposure,
more genes' expression was impacted than following a 24-hour exposition. From this we may
infer that Compared to the 24-hr exposure group, the polyps' physiology and/or metabolism
altered more dramatically in the 12-hr exposure group.
Conversely, in the hydra polyps expositioned to ZnO NP conc.100, the quantity of genes with
differential expression was nearly same between the 12- and 24-hour experimental groups.
ZnO NP20 exposure over 12 and 24 hours impacted the molecular function of genes linked to
"heterocyclic binding," "organic cyclic compound binding," "ion binding," in that order. Genes
linked to "protein binding," "hydrolase activity," "small molecule binding" were also impacted.
The effects of ZnO NP20 exposure on cellular components were seen in genes related to
"intracellular," "intracellular part," "intracellular organelle," and "membrane-bounded
organelle," accompanied by "protein complex," "non-membrane-bound," and "organelle
lumen." Also KEGG pathway analysis also revealed the upregulation and downregulation of
several genes.
2.3 TOXINS DERIVED FROM HYDRA
A toxin derived from Hydra magnipapillata known as Toxin similar to hydra actinoporin
[HALT-1],which is a low molecular weight around 18 KDa toxin which when in contact with
the cell membrane has high haemolytic and cytolytic activity.HALT-1 was further tested for
their cytotoxic activities with mutated strain m HALT-1 and wild type strain wtHALT-1. In
fact, In immunotoxin research initiatives, they were being studied as a possible toxin moiety,
ideally as a targeted cancer treatment. Immunotoxins are nothing but a hybrid protein molecule
in which a toxin molecule is linked with a cell specificity molecule to achieve cell specific
killing. The traditional toxins used the recombinant immunotoxic drugs are inefficient to be
produced at bulk scale possess complicated internalization processes and inadequate tissue
penetration. MTT assay which is a colorimetric analysis of cytotoxicity based on cell viability,
proliferation and toxicity was used for the cytotoxicity testing Assay results revealed that m
HALT-1 strain showed a significant decrease in the cytotoxic effects by changing or mutating
the recognition domain of the membrane whereas wt HALT-1 induced a apoptosis mediated
cell death across the cell lines. Among these cytotoxic assays one was MTT assay that is used
here for the cytotoxicity testing of the cancerous cell lines. The early 1980s saw the first reports
of this approach, which uses a tetrazolium dye (MTT) as an indirect indicator of cell number.
(Mosman, T.,1983). The MTT dye decrease was assessed by the National Cancer Institute as a
potential screening test outcome. The poison was introduced to the cells during their
exponential development phase. The greatest amount of damage that may be caused can be
used to determine the duration of exposure; however, this is heavily impacted by the toxin's
stability. After the poison is eliminated, the cells are allowed to divide twice or three times in
order to assess their viability and rate of proliferation. Some of the cells are found to be viable
and also proliferate but some of them are live but cannot divide or differentiate. An indirect
method of estimating the quantity of surviving cells is to employ MTT dye. Tetrazolium dye
MTT is a yellow dye that dissolves in water. It is reduced by live cells to a purple form product
that is not soluble in water. Once the MTT formazan has been dissolved in the proper solvent,
its quantity may be determined using spectrophotometer. This assay's inability to differentiate
between cytotoxic and cytostatic cells, as well as cells with a sluggish growth rate, is one of its
main shortcomings. This process is specifically tailored to work with specific cytotoxic
medications.
The test for immunofluorescence (IF) staining was used to see where wildHALT-1 and
mutatedHALT-1 binding were located in the cell. Using Alexa Fluor 488 goat anti-mouse IgG
(H+L) secondary antibody and primary mouse anti-His tag antibody, an indirect
immunofluorescence experiment was carried out. The cell lines HepG2, HeLa, MCF-7, and
SW-620 were treated for 24 hours with wtHALT-1, mHALT-1, and camptothecin prior to
collection. After treatment, cells were fixed with 4% paraformaldehyde and then given three
further PBS washes before being cleaned with PBST. For five minutes, the cells were incubated
with 0.1% triton X-100 to permeabilize them. After that, the cells were blocked with 2% BSA
for 45 minutes and given three PBS washes to reduce non-specific binding. The primary
antibody against the His tag was then incubated on the cells for one
To produce a cytotoxic impact, these endotoxins typically must be incorporated and migrate
into the cytosol; most of them accomplish this enzymatically by blocking the production of
proteins. (Tejuca. et. al., 2009). The majority of protein-based toxins may be created as toxin
moiety in medications based on immunotoxins. [Aruna, 2006; Becker & Benhar, 2012; Mazor
et al., 2016; Mazor, King & Oastan, 2018; Shan, Liu & Wang, 2013; Mathew & Verma, 2009]
.The major hurdle for these protein based toxins was their size therefore scientists discovered
HALT-1 as a low molecular weight toxin moiety which could facilitate proper tissue
penetration (Tejuca et al., 2009; Mariottini & Pane, 2014). These toxins basically target the
sphingomyelins present on the cell membrane to achieve perforation or lysis of the cell (Schön
et al., 2008).They lead to a disturbance in the gradient of the cell wall that causes osmotic
swelling.
Actinoporin poisons form pores in many stages. The phosphocholine binding site is crucial for
the toxin's attachment or binding to sphingomyelins in the cell membrane during the initial
stage. Upon reaching the lipid-water interface, the toxin's N terminus underwent
conformational changes that resulted in its separation from the protein and entry into the lipid
bilayer. After that, three to four actinoporin oligomerizations led to the chemical cross- linkage
of the N terminal alpha helix, which generated a functional pore. ( Liew et al., 2015) According
to a recent study, the cell lytic activity of Hydra actinoporin-like toxin-1 (HALT-1) is
significantly reduced upon the insertion of a negatively charged amino acid sequence at its Nterminal region. This is likely because the toxin's binding capabilities are disrupted. [Liew et
al., 2015]. Cytotoxic assays and immunofluorescence binding localization assays were
employed in the study to further analyze the hemolytic activity of HALT-1 and its potential as
a toxin moiety candidate in future immunotoxins.
3. GENOTOXICITY OF HYDRA
The ability of a substance to modify a cell's genetic code and potentially lead to cancerous
mutations is known as genotoxicity. The genetic material within the cells is harmed by the
genotoxic substances' interactions with the structure and sequence of DNA. Because all
mutagenic chemicals are genotoxic, genotoxicity and mutagenicity are commonly confused,
even though not all genotoxic compounds are mutagenic.
Genotoxicity testing is a rapid and cost-effective way to discover chemicals that may interact
with DNA or cause chromosomal loss. In order to certify small molescules as contaminants
and to start clinical trials, regulatory organizations require these tests. Pharmaceutical
companies routinely use these tests, modified versions, or other indicator tests during the drug
discovery and development process to rule out chemical series that may be genotoxic, verify
that development candidates have negligible genotoxic liability, and clarify the risk to human
health in the event that one or more assays yield a positive result.
The initial round of in vitro experiments should address the three levels of genotoxic effects:
gene mutations, chromosomal abnormalities, and DNA damages. The mouse spot test, the in
vivo cytogenetic tests - chromosomal analysis, the rat dominant lethal tests, the mammalian
germ cell cytogenetic assay, and the micronucleus test are the tests used to determine in vivo
genotoxicity. The most used in vivo genotoxicity test is the mouse micronucleus test, albeit not
all of them are required. In accordance with the biomaterial's known composition, genotoxicity
experiments are conducted with appropriate extracts or dissolved components in appropriate
media.
.Numerous genotoxicity assays have been devised that employ DNA integrity as an ambiguous
marker of genotoxicity. Thus, many methods are employed to quantify DNA fragmentation
resulting from breaks in DNA strands. Alkaline DNA denaturing conditions, alkali-labile
locations, double strand breaks, single strand breaks, and incisions mediated by repair enzymes
are among the test methods.
3.1 TECHNIQUES TO ASSES GENOTOXICITY OF HYDRA
3.1.1 COMET ASSAY
The Comet assay's rising appeal can be referred to its ease of use and adaptability to a variety
of species and tissues. Singh et al. created an alkaline version of the comet assay in 1988. Cells
are typically combined with slowly melting agarose, placed on microscope slides, then lysed
with an alkaline buffer including ionic detergents. In an electrophoresis chamber, the DNA is
resolved, dyed, and examined under fluorescence microscopy. Cells that have experienced
greater DNA damage migrate from the nuclear area to the anode, according Singh et al. (1988).
To quantify the comet-like structure, one must measure the tail length and/or tail moment,
which are calculated by multiplying the intensity of migrating DNA by the tail length (integral)
with regard to the nucleus DNA.
In their 1998b review, Mitchelmore and Chipman examined the application of the comet assay
to environmental monitoring. The test has been applied to a variety of aquatic organisms, such
as fish (Devaux et al. 1997, Risso-de Faverney et al. 2001) algae (Erbes et al. 1997), mussels
(Mitchelmore et al. 1998, Pavlica et al. 2001), amphibians (Ralph and Petras 1998), and
mussels (Mitchelmore et al. 1998).The test provides a number of benefits, such as the capacity
to use non-proliferating cells, choose from a wide variety of organisms and tissues, and obtain
results in a single day.
3.1.2. ALKALINE DNA ELUTION ASSAY
The alkaline elution experiment is capable of detecting both single and double strand breaks in
DNA. The test calculates the rate at which tissue is digested and denatured in a protease and
detergent-containing solution, at which point DNA elutes via a membrane filter.
Fluorimetry is used to quantify the amount of DNA present in the filtrate. When DNA
fragments' molecular weight drops, elution rates rise. The test has been applied to waste water
from a paraquat manufacturing plant using Chinese hamster cell line V79 (Kuo and Lin ,1993)
and wild live clams (Corbicula fluminea) to identify genotoxic potentials in surface waters.
3.1.3. MICRONUCLEUS ASSAY
A "micronucleus" is the small nucleus that results from the breakage of a chromosome or
fragment. During cell division, the chromosome is not incorporated into one of the daughter
nuclei. Human red blood cells that are only beginning to develop have Howell-Jolly bodies.
When humans and other mammals without nuclei have red blood cells, the spleen swiftly
removes micronuclei from them. Excessive levels of micronuclei in peripheral blood from
humans suggest a ruptured or absent spleen. The premise of the in vivo Micronucleus test is
that mice do not get rid of them A 24-hour prior injection of 100 mg/kg of cyclophosphamide
was administered. The experimental animals were sacrificed by cervical dislocation. Surgical
techniques were used to remove the animals' tibia and femur. Slice the top of the femur till a
tiny opening is revealed in order to harvest the marrow. To make sure the upper end was open,
a needle was utilized. Insert the needle at the lower end of the epiphyseal region after filling a
syringe with 0.5 ml of suspending medium. A sanitized cavity block was injected full of bone
marrow. Solid marrow was aspirated and flushed with a syringe to distribute it. Tibial marrow
was procured in a similar manner. The amount of suspending media required to extract bone
marrow from the tibia and femur was 2.0 ml. The suspension of bone marrow was centrifuged
at 1000 rpm for eight minutes. After the supernatant was extracted, a drop was smeared on a
sterile glass to make marrow films. After that, slides were left to air dry.
Staining:
For ten minutes, blots were fixed in pure methanol. Pure methanol is necessary to prevent
artifacts on the slide. Slides were stored for 15 minutes in coupling jars containing freshly
diluted May Grunwald's stain (PH = 6.8) in an equal volume of phosphate buffer. After being
moved to Giemsa, the slides were diluted once more with phosphate buffer (1:6) and stored for
ten minutes. Washing the slides in three or four changes of distilled water was done swiftly.
The slides were then air-dried after being left undisturbed in deionized water for five minutes
to allow for distinction.
4.
Hydra attenuata cells are used in the cytogenetic (genotoxicity) study
of chemical and inorganic harmful substances.
A comprehensive approach for testing drinking water quality by a series of Flora and fauna is
now regarded as a high-priority task. H. attenuata. were selected for the study. To evaluate
genotoxicity following measurements were done, quantitative properties of nucleoli, mitotic
index MI, and frequency of cells with micronuclei (MN) and double nuclei (2N). A marker of
subcellular processes like induced chromosomal breakage (clastogenesis) or malfunctioning
cell spindles (aneugenesis) is the micronucleus assay (Reddy et al., 1995). The micronucleus
test is generally used to detect substances suspected of having genotoxic activity. The
micronucleus test is known to provide the same and sometimes more information than does the
test for chromosomal aberrations. The advantages of this test include less artefacts and more
accuracy in counting under the microscope, which is also less laborious (Ilinskikh et al., 1992;
Hurna et al.,1997). Moreover, the size of micronuclei allows one to discriminate between toxic
substances as being either clastogens or aneugens (Reddy et al., 1995).For these indices, 3000
to 5000 cells were analyzed for every sample. Nucleolar characteristics were studied on cells
of adult hydras without buds (air-dried slides). employing the standard procedure and staining
with a 50% AgNO3 solution in accordance with method 3 (Howell and Black 1980 and Dev
and Tantravahi 1982). According to earlier descriptions (Arkhipchuk, 1999), the nucleolar
quantitative analysis was performed. (Arkhipchuk and others, 2000)
The percentage of PNhet and the number of nucleoli in each Hydra were not significantly
affected by either of the two most harmful chemicals: organic pentachlorophenol or inorganic
mercury.(Figure 3)
Fig.3 - Nucleoli in hydra cells (Arkhipchuk et al,2000)
5. Utilizing a Comprehensive Method to Evaluate the Genotoxicity of
Acetylsalicylic Acid and Metamizole Sodium
Multicenter Evaluation of In Vitro Cytotoxicity (MEIC), International Program on Chemical
Safety (IPCS), Gene-Tox, are some of the international programmes that aid in the evaluation
of drug toxicity using techniques such as bioassays on plants, invertebrates, and nonmammalian vertebrates as well as their cells.Under the MEIC program, 68 bioassays were used
to study 50 reference chemicals, mostly medicines, in order to assess the usefulness of toxicity
tests in predicting human toxicity (Clemedson et al., 1996a). Following a number of studies on
both organic and inorganic materials (Arkhipchuk and Garanko, 2002; Arkhipchuk and
Malinovskaya, 2002).The following series of bioassays was found to be the most successful in
examining various aspects of the detrimental influence on organisms and their cells: fish (such
as crucian carp, Carassius auratus gibelio), invertebrates (such as Hydra attenuata), and plant
representatives (such as onion, Allium cepa).However, only the genotoxicity of hydra is
discussed here.
Two chemicals: analgin (50 percent solution in ampoules: 500 mg of metamizole sodium) and
aspirin (500 mg of acetylsalicylic acid).The medication solutions in water were used to
incubate the test organisms. The materials were dissolved in control water to create stock
solutions. Acute toxicity was assessed using a variety of species from various taxonomic
groupings. Here are hydra cultures that were obtained from natural water bodies and kept alive
for a few weeks in lab aquariums.The drug solutions' toxicity was evaluated in a lab setting (20
± 2 °C, 16-hour daylight cycle) using the same procedures as the hydra 48-hour sublethal and
lethal effects test.
By raising the number of cells with double nuclei, which denote harm to cell division, and
micronuclei resulting from chromosomal anomalies, the genotoxicity of the drug was assessed.
A positive control for genotoxicity tests was a 10.0 mg/L concentration of copper ions(Cu2+
from CuSO4•5H2O) in water ,In two bioassays using invertebrate (hydra) and vertebrate (fish)
animals the aspirin concentration under examination (1.6 mg of acetylsalicylic acid per 1 ml of
water) did not exhibit any toxicity. Onions and ceriodaphnia experiments, however,
demonstrated the acute toxicity of this concentration: two-thirds of the animals died over the
course of 48 hours, and the root length shrank.
Sodium metamizole acute toxicity Research on the toxicity of drugs revealed that all test
organisms showed acute toxicity at a concentration of 6.25 percent. Within the first 24 hours,
there was 100% animal fatality rate. Acute toxicity was seen for all test species in the
metamizole sodium solution at the dose hazardous for mammalian cells (ic50). Furthermore,
the solution's toxicity was greater than the acetylsalicylic acid concentration under
investigation's toxicity. The fast death of the organisms used in animal tests made it impossible
to assess the genotoxicity of analgesics.
The practicality of employing biotesting techniques to evaluate drug toxicity, cytotoxicity, and
genotoxicity was validated by this study. The results showed how effective the complicated
strategy that was suggested was. It could be used to gather more information on the toxicity of
currently available pharmaceutical compounds as well as to conduct preliminary screenings of
newly created medications and their constituent parts.
6. ROS dependent copper toxicity in Hydra-biochemical and molecular study
The heavy metal copper is a major and enduring pollutant in aquatic environments. Because
metals don't dissolve like other pollutants do, they stay in water bodies for a very long time
(Clark et al., 2001). Because of its redox activity, copper exposure can potentially lead to the
production of reactive oxygen species (ROS) (Valko et al., 2005).Lipid peroxidation (Barata
et al., 2005), disturbance of embryonic development (Kong et al., 2013), DNA damage (Bopp
et al., 2008), and apoptosis (Krumschnabel et al., 2005) have all been connected to ROS caused
by copper. This study looks at the molecular changes that ROS cause in Hydra, such as DNA
damage, changes to genes that serve as antioxidant biomarkers, and apoptosis activation. The
results show a correlation with population, regeneration, and morphological changes.
Freshly hatched Artemia salina nauplii were continuously fed to Hydra magnipapillata strain
105, which was cultivated at 18 °C under a 12-hour light-dark photoperiod.
6.1 Acute toxicity testing was carried out using dissolved cupric sulphate (CuSO4·5H2O) in
Hydra medium. At 24, 48, 72, and 96 hours after exposure to copper (0– 0.175 mg/L at 0.01
mg/L intervals), structural changes was recorded as a score of 10 indicates that the animal is
healthy; scores of 9–6 indicate morphological changes at a non-lethal grade; scores of 5 and
lower indicate mortality.The median lethal concentration (LC50) was found via probit analysis
using the median scores that were computed at 24, 48, 72, and 96 hours. There were two
sublethal doses: 0.06mg/L and 0.1 mg/L,moderate and no morphological alterations.
Significant morphological alterations in hydra exposed to a 48-hour T2 dosage of copper were
not seen in those treated to a T1 dose. The development of clubbed tentacles (scoring 8) was
the first prominent morphological alteration. This was followed by the body and tentacles
becoming shorter (score 6), and lastly, the entire body disintegrating (score 0).The body and
tentacles then started to get shorter (scoring 6), and finally, the entire body started to
disintegrate (score 0).(figure 4)
Fig.4 - Morphology describing the toxic effect of copper in Hydra. A) Images of Hydra
showing progressive morphological changes on exposure to copper. (M. Zeeshan et
al.,2016)
6.2 Regeneration assay
Effect of Copper on Hydra regeneration was investigated by allowing contact the
gastrointestinal region to copper. Polyps were treated with 0.06 and 0.1 mg/L copper, known
as T1 and T2, respectively. Untreated animals completely regenerated the lost portions after 72
hours. T1 exposure slowed the embryonic process, and after 96 hours, To stop the polyp from
clinging to the vessel's bottom, the creatures evolved a mouth that was encircled by four short
tentacles and did not have a peduncle. When the tentacles and hypostome had fully developed
after 120 hours, the structures were reconstructed.T2 exposure impacted the regeneration
process (p < 0.05), resulting in 60% mortality after 96 hours of exposure. There was some
regeneration during the first 48 hours, but eventually the copper toxicity exceeded the capacity
for regeneration. There was some regeneration during the first 48 hours, but eventually the
copper toxicity exceeded the capacity for regeneration (Figure 5).
Fig.5 –A) Sample pictures of the stomach area of Hydra during wound healing(M.
Zeeshan et al.,2016)
6.3 Study of the tentacle structure
Nematocyte-specific staining dye toluidine blue O was used to detect changes in tentacle
structure. After being incubated for 48 hours, both treated and untreated Hydra were cleaned
in xylene, mounted in DPX, relaxed in 2% urethane, fixed in 70% ethanol, and their tentacles
were examined under a bright-field microscope. The toluidine blue O stain was applied in 10
Mm Tris-HCl (Ph 7.5). The neurons and nematocytes are adorned with ring-like structures all
the way around the tentacles. Because there were a lot of nematocytes in the tentacles of the
untreated animals, they looked thick and had the distinctive ring-like BCCs. This ring structure
was changed by the T1 treatment, and the tentacles showed less density. (Figure 6.)
Fig.6 - Effect of tentacle structure in Hydra.(M. Zeeshan et al.,2016)
6.4 Intracellular ROS generation
Using 2,7-dichlorodihydrofluoresce in diacetate, the formation of intracellular ROS in treated
and untreated polyps was investigated (Zeeshan et.,al2017).There was little DCHFDA
fluorescence in the untreated animals.Intense fluorescence was observed in the mice who
received treatment. Most of the time, the treated animals' epidermis displayed green punctae
representing the ROS. The creation of ROS was dependent on both dosage and duration. After
48 hours of exposure, the T2 animals showed the highest amount of generation (1.5 times
greater than the group under control, p < 0.001).(figure 7).
Fig.7 -The DCHFDA fluorescence in untreated animals was minimal (Fig. A and B).
The treated mice showed a strong fluorescence (Fig C and D)(M. Zeeshan et al.,2016)
6.5 Genotoxicity assessment: Comet assay
The comet test was used on dissociated cells of cobalt-treated Hydra to determine the amount
of DNA damage. The DNA damage was characterised by calculating the percentage of DNA
in the comet's tail. Untreated mice showed some DNA damage at 24 hours, with the majority
of the cells falling into the "zero" category. T2 caused the greatest amount of DNA damage at
48 hours, as 60% of the cells fell into the "high" category (p < 0.001). As a result, the damage
to DNA depended on both dosage and time.
This study outlined the organismal-level changes brought about by cobalt, which included
histological alterations, delayed development, and population decline in Hydra as a result of
poor nutrition. Using a combination of molecular research and whole-animal bioassays, Hydra
is an excellent tool for evaluating the consequences of heavy metal contamination in freshwater
habitats.
7.
DISCUSSION
The current study synthesizes extensive literature and highlights experimental observations
regarding the cytotoxic and genotoxic responses of Hydra species to various environmental
and pharmaceutical agents. As a simple freshwater cnidarian, Hydra demonstrates exceptional
regenerative abilities, mitotic activity, and morphological plasticity, making it a sensitive and
efficient model for toxicity testing (Bode, 2003; Loomis and Lenhoff, 1956; Gierer et al.,
1972).While zebrafish and Drosophila offer system-wide toxicological insights, Hydra
uniquely combines simplicity, regeneration, and cost-efficiency, making it ideal for initial
high-throughput screening.
7.1.1. Cytotoxicity: Nanoparticles and Heavy Metals
Hydra’s cytotoxic responses to zinc oxide (ZnO) and copper oxide (CuO) nanoparticles were
particularly evident in morphological degeneration, tentacle clubbing, and inhibition of
regenerative capabilities. ZnO NP20 showed higher toxicity compared to ZnO NP100,
suggesting that smaller particles may penetrate tissues more effectively and disrupt cellular
function (Valdiglesias et al., 2013). This finding aligns with previous studies demonstrating
apoptosis, DNA fragmentation, and cell cycle arrest induced by ZnO nanoparticles in
mammalian and aquatic models (Blaise et al., 2008; Zeeshan et al., 2016).
Copper-induced toxicity, as reported in Hydra magnipapillata, supports the hypothesis that
reactive oxygen species (ROS) generation plays a central role in cytotoxicity. Progressive
morphological changes and impaired regeneration observed at sub-lethal concentrations
indicate ROS-mediated lipid peroxidation and mitochondrial dysfunction (Valko et al., 2005;
Barata et al., 2005). Such responses correlate with earlier findings in fish and other aquatic
invertebrates (Bopp et al., 2008).
7.1.2. Hydra-Derived Toxins as Cytotoxic Agents
An emerging area of interest is the application of Hydra-derived actinoporins, particularly
HALT-1, in targeted cancer therapy. The cytolytic activity of wtHALT-1 against various
cancer cell lines via membrane disruption and apoptosis induction is significant, while mutated
forms reduce cytotoxicity, confirming the role of membrane-binding domains (Liew et al.,
2015; Tejuca et al., 2009). This opens avenues for developing immunotoxins with improved
specificity and lower systemic toxicity (Becker & Benhar, 2012; Aruna, 2006).
7.1.3. Genotoxicity: Assay-Based Insights
Multiple genotoxic assays, including the comet assay, micronucleus test, and alkaline DNA
elution, confirmed DNA damage under various exposures. The alkaline comet assay, widely
used due to its sensitivity and simplicity, showed clear evidence of strand breaks in hydra
exposed to heavy metals and drugs (Singh et al., 1988; Mitchelmore & Chipman, 1998).
Similarly, the micronucleus assay in Hydra attenuata allowed the detection of chromosomal
aberrations, aligning with previous environmental genotoxicity assessments (Reddy et al.,
1995; Arkhipchuk et al., 2000).
Gene expression analysis further revealed differential regulation of genes involved in organic
cyclic compound binding, hydrolase activity, and protein-ligand interactions following
nanoparticle exposure. This suggests early cellular stress responses and functional disruption
at the molecular level (Zeeshan et al., 2016; Zeeshan et al., 2017).
7.1.4. Hydra as a Model Organism: Relevance and Limitations
Hydra stands out as an ethical and cost-effective alternative to vertebrate models for
ecotoxicological studies and pharmaceutical screening. Its transparency, ease of culture, and
regenerative capacity allow real-time observation of toxicity-induced changes (Pollino &
Holdway, 1999; Pascoe et al., 2003). However, despite its simplicity, Hydra lacks organ-level
complexity, which limits its applicability in modeling systemic pharmacokinetics and
metabolism seen in higher organisms.
7.1.5. Future Directions
To further establish Hydra as a mainstream model for toxicology:

Quantitative dose-response models should be established.

Multi-omics approaches (transcriptomics, proteomics) can provide mechanistic
insights.

Comparative studies with other invertebrate and vertebrate models can validate findings
and enhance translational relevance.
8. CONCLUSION
This review highlights Hydra as a robust and sensitive model organism for assessing
cytotoxicity and genotoxicity of environmental pollutants and pharmacological agents. Its
simple body plan, high regenerative ability, and ease of maintenance make it a valuable
alternative to vertebrate models, particularly in the context of ethical and cost-effective toxicity
testing.
The reviewed literature shows that Hydra exhibits clear morphological, developmental, and
molecular responses to toxicants such as zinc oxide and copper oxide nanoparticles, as well as
heavy metals like cobalt. These agents induce cellular stress responses including oxidative
damage, impaired regeneration, and alterations in gene expression. Furthermore, the study of
Hydra-derived toxins like HALT-1 opens new avenues for targeted cancer therapies via
immunotoxins, underscoring Hydra’s relevance in biomedical research.
Various bioassays—including morphological observation, regeneration inhibition, comet
assay, and micronucleus testing—demonstrate Hydra’s utility in evaluating acute and sublethal effects. The organism’s transparency and suitability for molecular techniques allow realtime tracking of cellular and subcellular changes.
In conclusion, Hydra represents a versatile and ethically favorable model system with dual
applications in ecotoxicology and drug development. Future studies should focus on expanding
the use of Hydra in omics-based approaches and high-throughput screening platforms to
deepen our mechanistic understanding of toxicant action and to validate its translational
potential in human health risk assessment.
Acknowledgement: Thanks to JSS Medical College and Hospital, JSSAHER, Mysuru, and
Kristu Jayanti Deemed to be University, Bangalore, India
Funding Declaration: This study did not receive any funding
Clinical Trial Registration: Clinical Trial: Not applicable
Ethics declarations
Ethics approval and consent to participate: Not applicable.
Consent for publication: Not applicable.
Statement of informed consent, human/animal rights: No conflicts, informed consent,
human or animal rights applied to this study.
Competing interests: The authors declare no competing interests.
Authorship contribution statement
Jagadisha Tavarekere Venkataravanappa: Writing – original draft, Data curation, review
& editing.
Yuvaraj Shapur Gopalkrishnashetty, Parvathi Soman, Nayana Mitta Lakshminarayana
Gupta, Shreya Srivastava, Saraswathi Saraswathi, Shambhavi Kumari,Konakanchi
Suresh, Venkateswarlu raavi :Conceptualisation, Writing, Reviewing & editing.
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