Aims
This study aims to evaluate the gene expression of CYP 1A1, 1B1, and 3A of the Nile tilapia
from the Philippines. This study will determine the liver mRNA expression of selected CYPs
as toxicity or stress biomarkers.
Procedure
Sample collection
Nile tilapias were collected from February 22 – March 4, 2013 in Laguna Lake (Sta. Rosa andLos Banos sites), and in freshwater fisheries in Bilar, Bohol, Philippines. The fish samples
were immediately dissected on board after measurement of biometry (body length, bodyweight, etc.). Organ (liver, spleen, muscle, and kidney) samples were removed and total organ
weight was measured. The subsamples were frozen in liquid nitrogen, transported to Ehime
University, Japan, and stored at -80°C.
Quantitative PCR
To quantify mRNA expression levels of different CYPs, two step real-time RT-PCR was performed.
Total RNA were isolated from the liver samples in each sampling sites and then treated with DNase.
After reverse transcription by a High Capacity cDNA Reverse Transcription Kit (AppliedBiosystems), the cDNA were applied for a real-time PCR using SYBR® Premix Ex Taq TM (TliRNaseH Plus) Master Mix and analyzed using StepOnePlusTM Software (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 15 s 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 triplicate and normalization
was performed.

RESULTS AND DISCUSSION
Laguna Lake is among the largest and one of the economically important water system in thePhilippines. It is the largest lake in the Philippines and ranked as the 3rd largest in the whole of South
East Asia and that twenty- two rivers and streams gutter into the lake (Laguna Lake Development
Authority, 2016). While the bay provided a lot of resources in the country, it is continuouslyendangered by pollutants.
Our previous study (LaMer Project 28-08, 2016) showed that cytochrome P450 content of the Niletilapia from two sampling sites in Laguna lake and one sampling site in Bilar, Bohol ranged from223.988 to 297.641 pmol CYP450/ mg microsome. In various studies, high CYP450 content had beenhighly correlated with the occurrence of pollutants in the environment (Bainy et al. 1999; Chen et al.
2001). CYP450 is the major biochemical marker in pollution studies as its expression is induced by
the presence of endogenous and exogenous chemicals and drugs.
To confirm the results of our previous study, gene expression of CYP 1A1, 1B1, and 3A wasperformed.

RNA Isolation and Concentration
Total RNA were isolated from the liver samples in each sampling sites and the concentration of each®

was determined using NanoDrop ND-1000 Spectrophotometer. The RNA concentrations ranged
from 447.09 to 2412.86 ng/ul (Table 1).

All samples were checked for purity using the ratio ofabsorbance at 260 and 280, and were used for cDNA synthesis.
Table 1. Liver RNA concentrations of Oreochromis niloticus collected in three sampling sites.

Primer Design
Primers for O. niloticus CYP genes and β -actin cDNA as an internal standard (Table 2) were designed
using Primer Blast with melting temperatures ranging from 56 to 60 °C, and amplicon lengths of 122429bp.

Table 2. Real-Time PCR primers of Oreochromis niloticus CYP and β-actin genes.
Primer
Gene name

Descirption

Sequence

Amplicon Length

CYP1A

F

CCGTGACATCACAGACTCCC

429bp

R

CACAGCTCCGGGTCATGATTFTTACGTCATGGCCTTCATCTACR

ATGACTGTGTTCTTTGGTACGGF

CACCTCTGGGAGATTGAAAGAG

R

TGCTGGTTACCACATCCATACFGGGTCAGAAAGACAGCTACGTT

R

CTCAGCTCGTTGTAGAAGGTGTCYP1B1CYP3Aβ-actin

122bp
149bp
143bp

Primer Specificity
The melting curves for each primer pair of CYP1B and CYP3A were very specific as there was anobserved single temperature peak (Figure 3B and C).

Meanwhile, melting curve for CYP1A (Figure3A) shows melting curves with different peaks at different temperatures. This might be a manifestationof a higher-Tm peak and may be the result of amplicon concantenation (end-to-end annealing) in early
PCR cycles. It is also possible that high concentration cDNA template was used and saturated thereaction resulting in primers binding non-specifically to other targets. It is suggested that running anagarose gel should be done to verify whether there are other, non-expected by-products.

AFigure 1.

BC

Melting curves of Nile tilapia CYP1A (A), CYP1B (B) and CYP3A (C) by qRT-PCR.

CYP1B mRNA expression
Results revealed that the transcript levels of cyp1b in the liver of Nile tilapia were higher in Los Banos
and Bohol sites than in the Sta Rosa site (Figure 2). The expression of CYP1B1 gene is regulated byAhR, which forms an active transcription factor heterodimer with the AhR nuclear translocator(ARNT) after ligand-binding such as pesticides, and consequently induces the expression of theCYP1B1. Observed induction of CYP1B1 in liver of Nile tilapia in the two sampling sites provided
a defensive mechanism against the pollutants entering from the external environment.

Figure 2. Nile tilapia CYP1B1 mRNA normalized to β-actin mRNA.

CYP3A mRNA expression
Similar to the cyp1b, the relative gene expression of cyp3a of Nile tilapia Los Banos and Bohol sites
were higher (Figure 2). Fish from Sta Rosa expressed significantly lower mRNA. The higher
concentrations of OCPs and altered levels of CYP3A in fish from Los Banos and Bohol may be
associated with impacts from the agricultural activity performed in lands adjacent to the sampling sites.

Figure 3. Nile tilapia CYP1B1 mRNA normalized to β-actin mRNA.

Agro-chemicals are one of the major sources of pollution in Laguna Lake since some of surroundingparts of the lake are agricultural areas. A study of Varca (2012) measured the concentration of pesticide
residues on the surface waters of Pagsanjan and Lumban, Laguna catchment. These two rivers aremajor tributaries of Laguna Lake and this may load considerable amounts of pesticides since
agricultural activities from both sites are active. Above the recommended level of WHO, malathion(up to 3.3 ug/L) and profenofos (upto 15.3 ug/L) were detected in Pagsanjan and Lumban,respectively.

Xenobiotic compound are being metabolized by Nile tilapia and continuous exposure to these may
disrupt metabolic pathways of the fish and other organisms in the environment. Several studies
suggested that Nile tilapia is the best model organism in water pollution studies because of its greatsensitivity to organic pollutants.

On a serious note, Nile tilapia is freely cultured in Laguna lakeadjacent to Los Banos and is a common fish food source of the people living nearby. In this study,molecular responses as predictors of fish health may serve as strong tools to observe effects notdetected by classical biomarkers. Although changes in gene expression are not always directly related
to a final effect, protein and gene expression and other parameters should be considered to evaluate
fish health.
Perspective in Future
By far, this is among the first studies in the Philippines that revealed induction of cytochrome P450
monooxygenase genes in Nile tilapia that may be due to environmental pollutants in the Laguna deBay. Hence, continuous monitoring of the lakes ecosystem by using the Nile tilapia as a model ishighly recommended. The findings from this study can be a basis for future studies on pollutant
assessment of the Philippine water system as well as measures in preserving and conserving thesewater systems.
Results from this study will be presented in international conferences such as SETAC World Congressin September 2020, and in national conferences such as Philippine Society for Freshwater Science inNovember 2020.
References:
BAINY ACD, WOODIN BR, STEGEMAN JJ. 1999. Elevated levels of multiple cytochrome P5450
forms in tilapia from Billings Reservoir-São Paulo, Brazil. Aquatic Toxicology. 44:289-305
CHEN CM, LIU MC, SHIH ML, YU SC, YEH CC, LEE ST, YANG TY and HUNG SJ. 2001.
Microsomal monooxygenase activity in Tilapia (Oreochromis mossambicus) exposed to a
bleached kraft mill effluent using different exposure systems. Chemosphere. 45:581-588
LAGUNA LAKE DEVELOPMENT AUTHORITY (LLDA). 2016. Annual Report.
<http://llda.gov.ph/2016-annual-report/>
VARCA, L. M. 2012. Pesticide residues in surface waters of Pagsanjan-Lumban catchment of Lagunade Bay, Philippines. Agricultural Water Management, 106, 35-41.