Title of research project: Analysis of toxic/stress-responsive gene expression
in selected Philippine wildlife species
Names of Project Members
Maria Claret Tsuchiya (University of the Philippines Los Baños)
Tracy Maffett Prado (University of the Philippines Los Baños)
Hisato Iwata (Ehime University)
PurposesIn recent years, the escalating environmental concerns associated with persistentorganic pollutants (POPs) have prompted an intensified focus on understanding
their impact on diverse ecosystems. Among the affected organisms, cane toads(Rhinella marina) in the Philippines represent crucial subjects for investigation
due to their ecological significance and potential vulnerability to environmental
stressors.
Our previous study observed the accumulation of 2,2’,4,4’,5,5’Hexachlorobiphenyl (PCB153), 2,2’,4,4’-Tetrabromodiphenyl ether (PBDE47)
and 4,4’ – Dichlorodiphenyl dichloroethylene (4’4DDE) in the tissue of organisms
in Laguna Bay influencing the expression of some stress-related proteins andcytochrome P450 (CYP) enzymes in cane toads as revealed by proteomics
analysis. Also, our previous study using molecular docking showed that PCB153,PBDE47, and 4,4DDE exhibited hydrophobic interactions with the binding sitesof proteins. These observed direct interaction provide valuable insights into thepotential disruption of expression of genes involved in xenobiotic metabolism andstress response.
Hence, this study aims to analyze the expression of cyp1a1 and cyp1b1 genes
involved in metabolism of toxic substances, and stress-related genes in canetoads from the Philippines.
Methods
RNA Isolation, cDNA Synthesis, and Quantitative Real Time-PCR
Total RNA were isolated from the liver tissues of cane toads stored at − 80°C inCMES, Ehime University. To quantify mRNA expression levels of different genes,
two step real-time RT-PCR was performed. Single-stranded cDNA from 200ug oftotal RNA were synthesized using High-capacity cDNA reverse transcription (RT)kit (Applied Biosystems) and ran with GeneAmp PCR System 9700 (AppliedBiosystems). The cDNA were used for mRNA expression using TB Green ®
Premix Ex Taq II (Tli RNaseH Plus) Master Mix and analyzed using QuantStudio3 Real-time PCR System (Applied Biosystems). The optimization of the real-time
PCR reaction was performed according to the manufacturer’s instructions(Applied Biosystems). The PCR conditions were 10 min at 95 ◦C, 40 cycles of 15s at 95 ◦C and 60 s at 95 ◦C. The calibration curves were generated by plotting
Ct values against logarithmic value of template concentration. Each reaction was
run in triplicates and normalization was against the gadph gene.
Results
Xenobiotic Metabolism and Cytochrome P450s
This study reveals varying expression levels of cyp1a1 and cyp1b1 in cane toads

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from two sites in Laguna Bay, Philippines (Figure 1). cyp1a1 expression is higherin Los Baños, while cyp1b1 shows greater mRNA expression in Sta. Rosa. These
cytochrome P450 enzymes serve as biomarkers for environmental pollution,responding to pollutants like PAHs, dioxins, dibenzofurans, and PCBs. The
induction of these enzymes is linked to the formation of the AhR-Arnt complex in
the cell nucleus (Goksøyr 1995), resulting in increased production of carcinogenic
intermediates (Buters et al. 2002), and altered steroid metabolism and decreased
reproductive success in fish (Arinç et al. 2000). Limited research exists onamphibian CYP1 genes beyond cyp1a1, emphasizing the significance of ourinvestigation.

Figure 1.
Hepatic mRNA
expression of cyp1a1 and cyp1b1
from Laguna de Bay.

The use of cyp1a1 as a biomarker is further justified by its correlation withcontaminant levels in the environment. Concentrations of various contaminantsin water samples and fish species in Laguna de Bay may contribute to theexpression of cyp1a1 in cane toads. This correlation extends to the induction of
cyp1b1 in both study sites. Differences in the regulation of these enzymes areattributed to the presence or absence of specific agonists, with factors likedevelopmental stage, sex, diet, behavior, and environmental heterogeneity
playing crucial roles. Additionally, the nomadic behavior of cane toads, influenced
by environmental factors and body conditions (Seebacher and Alford 1999) adds
complexity to the interpretation of biomarker responses in these adaptableamphibians. Nonetheless, these results help enhance our understanding ofenvironmental pollutants' effects on crucial cellular processes, emphasizing thepivotal role of cytochrome P450 enzymes in the metabolism of toxic compounds.
Stress Responses
p53
The p53 gene, often referred to as the "guardian of the genome," plays a crucialrole in preventing the development and progression of cancer. In response tostress, beyond DNA damage, p53 responds to hypoxia, nutrient deprivation, andoncogene activation. In these situations, p53 activation can lead to cell cycle
arrest, allowing cells to cope with stress and preventing the survival andproliferation of potentially harmful cells.
The increased expression of p53 (Figure 2) in cane toads in Sta Rosa site revealthat these organisms are exposed to variety of stressors and may lead tomodifications of the p53 protein, such as phosphorylation and acetylation, which
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in turn increase its levels and activation (Lakin and Jackson 1999).

Figure 2. mRNA expression of p53 genein Rhinella marina.

Angiopoietin-like proteins (ANGPTLs) and matrix metalloproteinase-1(MMP-1)ANGPTLs and MMP-1 are involved in various cellular processes andunderstanding their roles in response to cellular stress is crucial for elucidating
the complex interplay of molecular pathways involved in tissue homeostasis,repair, and adaptation to various stressors. These proteins contribute to the finetuning of cellular responses under stress conditions, ultimately influencing tissue
function and overall organismal health.
According to Rosal (2018), the protein showing the highest upregulation in canetoads from Los Baños and Santa Rosa is angptl7, exhibiting fold change ratios of40.57 and 48.52, respectively. Our findings validate this by confirming that thegene expression levels of angptl7 are elevated in SR compared to LB (∆Ct SR =9.88, ∆Ct LB = 8.76) (Figure 3). Despite this distinction, statistical analysis reveals
no significant difference between the two sites (p = 0.29).

Figure 3. mRNA expression of angptl7
and mmp1 genes in Rhinella marina.

Annexins and integrins
Annexins and integrins play important roles in stress response and provide
insights into the intricate mechanisms by which cells cope with environmental
challenges and maintain homeostasis. Their involvement in anti-inflammatory
processes, membrane repair, and signaling makes them key players in thecellular responses to stress and injury.

6 / 8Although there is no significant difference in anxa2 and itgb1 expression between
the two sampling sites (Figure 4), these results imply comparable expression
levels of the two genes among the cane toad populations in LB and SR. Under
the studied circumstance, anxa2 and itgb1may be genes that are stably regulated
in response to the stressors or environmental factors examined.

Figure 4. mRNA expression ofanxa2 and itg1 genes in Rhinella
marina.

SWI/SNF-related matrix-associated actin-dependent regulator and Protein
Tyrosine Phosphatases
smarca2’s involvement in chromatin remodeling allows it to influence thetranscription of genes involved in stress response. It may regulate the expressionof genes related to cell survival, DNA repair, and other stress-related pathways.
Meanwhile, ptp4a3 is a protein tyrosine phosphatase implicated in cell migrationand invasion. In stress situations, such as tissue damage or inflammation, cells
may need to reposition themselves, and ptp4a3 could play a role in theseprocesses.
Except smarca2 expression in Los Banos, the gene expressions decreased(Figure 5). This may suggest a reduction in the activity of the associated proteins.
Genes involved in stress response often have roles in cellular adaptation andsurvival. A decrease in the expression of genes like smarca2 or ptp4a3 may
indicate a compromised ability of cells to respond to stress, potentially affecting
processes such as DNA repair, cell migration, or signaling.

Figure 5. mRNA expression ofsmarca2 and ptp4a3 genes inRhinella marina.

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In summary, POPs are known to disrupt various physiological processes withinorganisms, including pathways associated with xenobiotic metabolism,antioxidant defense, and stress response. By unraveling the intricacies of gene
expression in response to POPs, this study aims to validate and enhance ourunderstanding of how these pollutants influence critical biological pathways incane toads.
Future challenges
The Philippines, endowed with rich biodiversity, faces the imperative to safeguard
its ecosystems from the deleterious effects of environmental contaminants.
Through a comprehensive examination of gene expression patterns, further
research is needed to contribute essential knowledge that can inform
conservation efforts, guide environmental policies, and ultimately foster thesustainable coexistence of diverse species in the face of evolving environmentalchallenges.
References:Goksøyr A. 1995. Use of cytochrome P450 1A (CYP1A) in fish as a biomarker ofaquatic pollution. Arch Toxicol Suppl. 1995;17:80-95. doi: 10.1007/978-3-64279451-3_7.
Buters JT, Mahadevan B, Quintanilla-Martinez L, Gonzalez FJ, Greim H, BairdWM, Luch A. 2002. Cytochrome P450 1B1 determines susceptibility todibenzo[a,l]pyrene-induced tumor formation. Chem Res Toxicol. 2002
Sep;15(9):1127-35. doi: 10.1021/tx020017q.
Arinç, E., Alaattin Sen, and Azra Bozcaarmutlu. 2000. Cytochrome P4501A andassociated mixedfunction oxidase induction in fish as a biomarker for toxic
carcinogenic pollutants in the aquatic environment. Pure Appl. Chem., Vol. 72,
No. 6, pp. 985–994.
Seebacher, F., & Alford, R. A. 1999. Movement and Microhabitat Use of a Terrestrial
Amphibian (Bufo marinus) on a Tropical Island: Seasonal Variation andEnvironmentalCorrelates. JournalofHerpetology, 33(2),208–214.
doi.org/10.2307/1565716
Lakin ND, Jackson SP. Regulation of p53 in response to DNA damage. Oncogene.
1999 Dec 13;18(53):7644-55. doi: 10.1038/sj.onc.1203015.

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