[6] PHA6131 LAB - Activity 5: Onion Root Tip Assay

onion root tip assay

• commonly used in pharmaceutical toxicology to determine whether a chemical or drug can damage the cell or the DNA.

The onion root tip is a good model because

its cells divide rapidly, making it easier to observe cell division under the microscope.

Somatic growth in plants and animals takes place by

the increase in the number of cells

A cell divides mitotically to form two daughter cells wherein

the number of chromosomes remains the same (i.e., unchanged) as in the mother/parent cell. 

in plants, cell divisions

• rapidly take place in meristematic tissues of root and shoot apices, where the stages of mitosis can be easily observed. 

In animals, mitotically dividing cells can be easily viewed in the

bone marrow tissue of a vertebrate, epithelial cells from gills in fishes and the tail of growing tadpole larvae of frog

Cytotoxicity

refers to the ability of a substance to cause damage to cells, leading to cell dysfunction or death.

Cytotoxicity

Effects on Cells

It can result in apoptosis (programmed cell death), necrosis (uncontrolled cell death), or inhibition of cell growth.

Cytotoxicity

Causes

Toxic chemicals, radiation, certain drugs (like chemotherapy), or immune system responses

Cytotoxicity

assessment

Measured using assays like the MTT assay, Trypan Blue exclusion, or Lactate Dehydrogenase (LDH) release assay, Onion root tip assay (for preliminary screening)

CYTOTOXIC SUBSTANCES

Cause cell damage or death

CYTOTOXIC SUBSTANCES

Chemotherapeutic/Chemotherapy Drugs

• doxurubicin

• cisplatin

Doxorubicin

Kills rapidly dividing cancer cells but also harms healthy cells.

Cisplatin

Causes cell death by damaging proteins and membranes.

Heavy Metals (cytotoxic substances)

• lead (pb)

• mercury (Hg)

Lead (Pb)

Disrupts cellular functions and can cause oxidative stress.

Mercury (Hg)

Interferes with cell metabolism, leading to cell death.

[cytotoxic substance]

Toxins

• snake venoms

• bacterial toxins (like diptheria toxin)

Snake Venom

Some venom components destroy red blood cells and tissues.

Bacterial Toxins (e.g., Diphtheria Toxin)

Inhibit protein synthesis, leading to cell death.

[cytotoxic substances] industrial chemicals

• benzene

• pesticides (paraquat)

Benzene

Disrupts bone marrow cells, leading to blood disorders.

Pesticides (e.g., Paraquat)

Causes oxidative stress, leading to cell death.

Genotoxicity

refers to the ability of a substance to damage the genetic material (DNA) within a cell, leading to mutations or chromosomal alterations.

Genotoxicity

• effects on cells

It may cause mutations, chromosomal breaks, or abnormal recombination, potentially leading to cancer or hereditary diseases.

Genotoxicity

• causes

Exposure to radiation, chemicals, environmental pollutants, or certain pharmaceuticals.

Genotoxicity

• assessment

Tested using assays like the Ames test, Comet assay, or Micronucleus test (animals) or Onion Root tip assay (plants)

GENOTOXIC SUBSTANCES

• Damage DNA and cause mutations

GENOTOXIC SUBSTANCES

• radiation

• Ultraviolet (UV) Radiation

• Ionizing Radiation (X-rays, Gamma rays)

GENOTOXIC SUBSTANCES

• chemicals

• Benzene

• Aflatoxins (from mold)

GENOTOXIC SUBSTANCES

• drugs

Cisplatin

Cyclophosphamide

GENOTOXIC SUBSTANCES

• environmental pollutants

Polycyclic Aromatic Hydrocarbons (PAHs, e.g., from cigarette smoke)

Asbestos

Ultraviolet (UV) Radiation

Causes DNA damage, leading to skin cancer.

Ionizing Radiation (X-rays, Gamma rays)

Induces DNA breaks and mutations.

Benzene

Not only cytotoxic but also genotoxic, causing DNA damage in bone marrow cells.

Aflatoxins (from mold)

Causes mutations in liver cells, leading to liver cancer.

Cisplatin

While used in chemotherapy, it also causes DNA crosslinking, leading to mutations.

Cyclophosphamide

Induces DNA breaks, leading to potential secondary cancers.

Polycyclic Aromatic Hydrocarbons (PAHs, e.g., from cigarette smoke)

Cause DNA adduct formation leading to mutations.

Asbestos

Induces oxidative stress and chromosomal damage, increasing lung cancer risk.

key difference

• Cytotoxicity affects overall cell survival and function, it damages or kill the cells. Cytotoxic substances kill or harm cells by disrupting their normal functions

• Genotoxicity specifically targets the DNA, potentially leading to mutations and long-term genetic effects, it damages or targets the DNA. Genotoxic substance specifically damage DNA, leading to mutations and cancer

A genotoxic substance can also be cytotoxic, but

NOT ALL cytotoxic substances are genotoxic. Some substances (e.g., benzene, cisplatin) are both cytotoxic and genotoxic.

APPLICATION IN PHARMACEUTICAL TOXICOLOGY

• cytotoxicity assessment

• genotoxicity testing

• drug development & screening

• environmental & regulatory applications

Cytotoxicity Assessment

The assay helps determine the toxicity of pharmaceutical compounds by analyzing changes in the mitotic index. 

Mitotic index

- the number of cells undergoing cell division

Cytotoxicity Assessment

• If a drug has a reduced mitotic index

• indicates cytotoxic effects

Cytotoxicity Assessment

• this helps researchers

• evaluate the safety of a new pharmaceutical compound

Genotoxicity Testing

• Detect genotoxic effects

• The assay detects chromosomal aberrations such as breaks, bridges, and micronuclei.

• If these abnormalities increase, it indicates that the substance causes genetic damage or mutations.

Genotoxicity Testing

• useful in _

 dentifying potential mutagenic and carcinogenic effects of drugs.

Drug Development and Screening

• Provides preliminary screening for drug-induced toxicity before advanced mammalian studies. 

Drug Development and Screening

• helps in

assessing the safety profile of newly developed pharmaceutical compounds

Preliminary toxicity screening is

performed first before testing drugs on animals or humans

Environmental and Regulatory Applications

• Used to evaluate the impact of pharmaceutical pollutants on plant systems.

• Recognized in environmental toxicology and regulatory compliance studies.

• It can determine pharmaceutical waste or industrial chemicals that could cause genetic damage in the plants

Advantages

• Cost-effective and easy to perform.

• Does not require sophisticated equipment.

• Ethical alternative to animal testing in preliminary toxicology studies.

Limitations

• Limited to plant cells, which may respond differently than human cells.

• Requires confirmation with mammalian cell assays for comprehensive toxicological evaluation.

ONION ROOT TIP ASSAY

• widely used method in cytogenetics to study cell division, chromosome structure,  and assess cytotoxicity and genotoxicity.

ONION ROOT TIP ASSAY

• This assay is particularly valuable in pharmaceutical toxicology to

• evaluate the effects of drugs and chemicals on cellular processes.

Interphase (first stage of mitosis)

prophase

metaphase

anaphase

telophase

• Irregular metaphase

• Chromosomes are not properly aligned

sticky anaphase

• sticky chromosome at prophase

• chromosomes that clump together

• anaphase bridge

• chromosomes remain attached while separating

condensed nucleus prophase

• polyploidy

• extra sets of chromosomes

sticky metaphase

• telophase with vagrant chromosome

nuclear buds

• binucleated cell

• small extra nuclei caused by DNA damage

delayed anaphase

chromosome missegregation and fragmentation

Mitotic index (%) 

(number of dividing cells/ total cells counted) x100

A low mitotic index indicates

cytotoxicity

high mitotic index

may indicate cell proliferation/ division

Types of Abnormalities Observed

• Chromosomal breaks

• Bridges and laggards

• Micronuclei formation

• Disturbed metaphase and anaphase

An increase in chromosomal aberrations suggests

genotoxic effects of the tested pharmaceutical compound

Higher concentrations of a toxic compound generally lead to a

decrease in mitotic index and an increase in chromosomal aberrations.

Establishing a dose-response curve helps in

determining the safe and toxic threshold for pharmaceutical compounds.

Nuclear clumping and lesions

(Black arrow shows the feature)

Binucleate

Sticky chromosomes at metaphase

Disturbed metaphase

Anaphase bridge and laggard chromosome

Disturbed telophase