1. General Toxicology
Our research activities focus on in-life acute (single dose) and sub-acute (28 days repeated dose) toxicity studies using rodents as animal models. Generally, an acute toxicity study is conducted to determine the Lethal dose (LD50) of the herbal extract/product before deciding on a series of concentrations to be further examined in the repeated dose studies (low, medium, and high). In repeated dose studies, our unit further analyses the results of biochemical, haematological, and histopathological samples to determine the Non-Observed Adverse Effect Levels (NOAEL) of the herbal extract/product in animal models.
Figure 1: In vivo general toxicity studies using rats. Equipment to
2. Genotoxicity Testing
Herbal medicinal plants possess several characteristics that differentiate them from other conventional medicinal products. The purpose of genotoxicity testing is to improve the risk characterisation of medicinal plants for potential carcinogenic effects.
Figure 2: In vitro and vivo genotoxicity testing
Ames test or bacteria reversed mutation assay is used to assess the mutagenic potential of plant/herbal extracts. This test uses several Salmonella typhimurium (S. typhi) and Escherichia coli (E. coli) strains that carry mutations. Medicinal plant or herbal extracts with mutagenic properties will cause an increase in the number of revertant colonies. Our unit also conducts in vitro micronucleus test to detect small membrane-bound DNA fragments (known as micronuclei) in the cytoplasm of interphase cells. Cells are exposed to herbal extracts with and without the presence exogenous source of metabolic activation (S9) and then treated with Cytochalasin B to arrest cell division. Chromosomal aberration is detected by identifying the presence of micronuclei in the treated cell. Genotoxicity risk is also assessed through a mammalian in vivo micronucleus test. Chromosomal damage is detected from sampled erythrocytes obtained from the bone marrow of treated rodents. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals indicates induced chromosomal damage.
3. Reproductive and Developmental Toxicity Study
A reproductive and developmental toxicity study is used to evaluate and identify a potential toxicity effect of chemical agents which can interfere with fertility in both male and female reproductive systems. In 2010 our unit researched reproductive toxicology, focusing on the prenatal developmental toxicity risk on selected Malaysian herbal plants. Prenatal in vivo developmental toxicity effect is determined by clinical observations of maternal toxicity signs and embryonic and fetal development. After being stained with Alizarin red, the fetuses' teratogenicity effect is further evaluated through skeleton evaluation and head sectioning.
In 2014 we conducted a reproductive toxicity study using Orthosiphon stamineus (Misai Kucing) by evaluating the sperm concentration, daily sperm production (DSP), the efficiency of DSP, epididymis transit time and sperm morphology using Computer-assisted sperm analysis (CASA).
We continued to expand our capacity in conducting reproductive toxicity by adopting an embryotoxicity study methodology using the whole embryo culture (WEC) system. This technique was established by our unit in 2018 and we became the first unit in Malaysia to conduct embryotoxicity assessment using this procedure after our researchers went through comprehensive training at Tohoku University, Japan. The rat embryos are cultured in ex-vivo conditions using BTC Precision Incubator (Cambridge, UK), accessible during the early stages of organogenesis. The WEC system is part of the alternative models advocated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL-ECVAM) to reduce, refine and replace the animals’ use for safety and efficacy testing. This testing will precede gene expression responses corresponding to differentiation and morphological changes to elucidate the mode of the toxicological action of the herbal plants.
|Figure 3: In vivo prenatal developmental toxicity. The foetus was stained
with Alizarin Red S and the level of skeletal ossification and deformation were determined
Figure 4: Postimplantation rat embryos are cultured with treatment from embryonic day 10 to 12
The toxicity effects of herbal extracts/products can be investigated overall (also known as general toxicity), or specifically to target organs like the liver, lung, kidney, heart, and brain. The liver is considered to be one of the most important organs in drug toxicity testing. Our unit conducted the hepatotoxicity study of Dioscorea hispida in rodents by looking at the oxidative stress level and biochemical and histopathological changes.
In addition to using Sprague Dawley rats as our animal model, our latest research ventures include the use of Zebrafish (Danio rerio), a teleost freshwater fish from South Asia are also used in specific organ toxicology research. Its advantages are small size, very high fecundity, low cost and easy husbandry, transparent embryos and larvae, rapid development, and similarity of genetic homology to humans. Based on these features, zebrafish is a globally accepted alternative model for drug efficacy, as well as safety and toxicology assessments in the drug discovery and development of pharmaceutical and natural products. Zebrafish toxicology evaluation can be concluded in a few weeks and it can provide early readouts of potential off-target effects or safety liabilities to detect toxic effects that may be missed in the cell culture or other animal models. The zebrafish toxicity assay is considered as a useful pre-filter to support the selection of the safest potential drug candidates as early as possible in the drug discovery process, eventually reducing procedural delays and costs of the drug development pipeline.
|Figure 5: The use of zebrafish model in specific organ toxicology|
Our unit provides diagnostic services to all government hospitals in the field of toxicology. Our unit is the only laboratory in the Ministry of Health, Malaysia that provides tests for determining the levels of copper and lead in biological fluids using Graphite Furnace Atomic Absorption Spectroscopy. Copper testing aims to help in the differential diagnosis of various diseases, such as Wilson disease, chronic liver disease, acute hepatitis, Menkes disease, and copper toxicity due to long term exposure to high levels of copper through contaminated food and water. In addition, our unit also actively participates in the Trace Elements External Quality Assessment Scheme organised by the Royal College of Pathologists of Australasia (RCPA).
Figure 6: Heavy metal testing using PinAAcle™ 900T with Fumace Autosampler AS900