1. Induced Expression of CYP1A by Triclosan in Zebra Fish
Mujtaba M. Qureshi
Department of Biochemistry
Rutgers School of Environmental and Biological Sciences
mmq10@scarletmail.rutgers.edu
732-649-9070

2. Abstract
Triclosan, an antibacterial and antifungal agent found in household consumer products
(from toothpaste to soaps), is a chemical studied to form TCDD, a dioxin that has toxicological
effects on the environment, aquatic life, and mammals. We study the effects of triclosan on
zebrafish as our model organism, by observing induced expression of protein. Two genes in
particular are observed: CYP1a and TCF3a. CYP1a produces cytochrome p450 and TCF3a
acts as a transcriptional repressor; both genes are theorized to be correlated with toxicological
endpoints. We use the sensitive biomarker assay EROD (enzyme 7-ethoxy-resorufin-O-
deethylase) to test induction for CYP1a, and we use RT-qPCR to isolate RNA and observe
induced expression for both genes. Using qPCR, we observe induction of CYP1a in our treated
samples. The study shows the need to further test the effects of ecological concentrations of
triclosan on endpoints related to CYP1a on aquatic life, as well as on human life in countries
with significant levels of triclosan in their bodies (China, Vietnam, and more).
Introduction
The toxicology of chemicals found in everyday household items is not always studied in
full detail. One such chemical is triclosan, an antibacterial and antifungal agent found in
household consumer products, from toothpaste to soaps (i.e. hand sanitizers). Research shows
that at least 1500 tons of Triclosan were used in 2015 in China alone (Zhang et al., 2015).
Triclosan has been detected in human urine as well as plasma (Hovander et al., 2002), which
may be due to seafood consumption or due to exposure to household consumer products.
Furthermore, research has indicated triclosan by itself has adverse endocrinological effects in

3. human cancer patients (Gee et al., 2007). It has also been found that Triclosan has the capacity
to undergo chlorination in seawater, forming TCDD (2,3,7,8-tetrachlorodibenzopdioxin), a dioxin
(Kanetoshi et al., 1987). This dioxin has been researched and studied to cause a number of
toxicity endpoints, such as auditory neuropathy in mice for instance (Safe et al., 2016).
Research to understand the toxicity of triclosan on health impacts of organisms and
environmental effects remains limited in the molecular level for a number of toxicological
endpoints. We use Zebrafish as the model of our study for its features as a good predictive
model to that of human development since there is much overlap in biological processes. For
example, all proteins studied have similar functions in both mammals and in fish, with their
functions often conserved. The Zebrafish model is also valued for its optical transparency, which
aids in visually monitoring mutations, lesions, and any or all abnormalities, and thus the impact
of treatments or diseases are easily observed. Furthermore, fish are easier to handle,
reproduce, grow, and monitor than most other mammalian model organisms. We focus on
zebrafish in both their early developmental stages, larvae & adult, since the lc50 and endpoints
remain similar from early stages to adult stages, despite growth and development (Oliveria et
al., 2009).
In this study, we focus on the effects of Triclosan on two genes: CYP1a and TCF3a.
CYP1a codes for cytochrome p450 1A, a xenobiotic metabolizing enzyme found in the smooth
endoplasmic reticulum within the cell. One study has shown that induction of CYP1a has an
effect seen and correlated with liver tumors (Spitsbergen et al., 2000). The second gene,
TCF3a, functions as a transcriptional repressor. A recent study has shown TCF3 plays a crucial
role in regulating the timing of neurogenesis (Gribble et al., 2009). We aim to test if treatment
with triclosan will show induced expression of the CYP1a and TCF3a genes for the larvae, on
the molecular level, through an increase in RNA production. We also aim to test if treatment with
triclosan will show induced expression for ethoxyresorufin in adult zebrafish through an increase
in EROD activity.

4. We use two methods to observe these endpoints: real time qualitative PCR and EROD
assay (enzyme 7-ethoxy-resorufin-O-deethylase). RT-qPCR is using fluorescence to detect
PCR products, namely cDNA. We use qPCR to observe amount of RNA produced from the two
genes in question, using reverse transcriptase to form cDNA from RNA. The second method,
EROD, is a sensitive biomarker, where the reaction activity is catalyzed by cytochrome p450,
since EROD generally is catalyzed by halogenated and polycyclic aromatic hydrocarbons. As
CYP1a is induced, cytochrome p450 is produced, and the EROD reaction is catalyzed.
Following the rate of reaction of EROD indirectly allows us to follow the rate of production of
cytochrome p450.
Methods
In order to perform RNA isolation, zebrafish embryos were collected in microcentrifuge
tubes. 100uL of RNAZOL was added to the embryos. Once in the larvae stage, the zebrafish
were homogenized using a pestle and grinder. 400uL of RNAZOL was added to the embryos
once again, and mixed. 200uL of H2O was then added, and the solution was vortexed. The
solution remained at room temperature for 15 minutes, and then centrifuged for 15 minutes at
12,000xg. 600uL of the supernatant was transferred into a new tube, followed by addition of
600uL of isopropanol. The supernatant-solution sat at room temperature for 15 minutes and
then spun for 10 minutes at 12,000xg. The solution was then rinsed with 75% ethanol and then
spun again for 3 minutes 8,000xg. This step was repeated twice more. The ethanol was then
removed and the solution was left under a hood until the pellet was dry. The solution was then
resuspended in 25uL of nuclease-free-water and incubated at 55-60 degrees C for 10 minutes.
1uL of sample was taken and mixed with water to measure OD at 260nm using a
spectrophotometer, to determine RNA concentration. cDNA templates were then generated
using reverse transcriptase through the following procedure. To each reverse transcriptase
reaction, 1ug of RNA was added, followed by 4uL of 5X iScript Reaction mix, 1uL of iScript

5. Reverse Transcriptase, and then enough nuclease-free H2O to end up at a total volume of
20uL. The reactions were then placed in the PCR machine and run for 45 minutes. Next, real
time qPCR was carried out. Each sample (control and treatment) was performed in triplicates,
for three primer sets (B-actin, CYP1a, and TCF3a). Each sample was made of 1uL of cDNA,
1uL for primer sets, 6.25uL of iCyclerQ SYBR mix, and 4.25uL of H2O. The plate was spun at
4,000rpm for 5minutes and then ran under the BioRad iCYCLERQ program.
A standard lowry assay was then carried out using aliquots from the zebrafish samples
prior to generating cDNA templates. BSA was used as the standard, and absorbance was
measured at 750nm.
An EROD activity assay was carried out using two different methods: a continuous
assay and a stop assay, in order to determine the activity of CYP1a using 7-Ethoxyresorufin.
For each run of the triplicate for each assay, 10ug of zebrafish sample was added with 5uM of
ethoxyresorufin, 5mM of NADPH, and enough EROD buffer to have a total volume of 200uL.
For the continuous assay, fluorescence was measured at an excitation/emission wavelength of
544nm/590nm. Fluorescence was read every 10 minutes for 90 minutes. For the stop assay, the
reaction was run only for 30 minutes at 23 degrees C and then terminated using 500uL of
methanol. Fluorescence was measured using a spectrophotometer at 572 nm. A standard curve
was also made using 1:10 serial dilutions of Resorufin stock, starting at 100uL, in order to
measure the concentration of resorufin from the relative fluorescence units obtained from the
stop-assay.
Statistical analysis was done using an Excel add-on called ‘xtoolbox by sourceforge.
ANOVA tests and t-tests were taken for control and treatment data from the qPCR and the two
EROD assays.
Results
The experiment was carried out for the EROD activity assay, and the RNA
concentrations were both observed and measured using qPCR as well.

6. Using qPCR to isolate RNA and measure concentration, the number of DNA copies of
CYP1A and TCF3A were obtained. Comparing CYP1A control samples and treated samples,
the control samples had 20,891 copies, while the treated samples had 51,802 copies. The
treated samples thus showed a 250% increase in RNA production. Using the ANOVA test, a p-
value of 0.043879 was obtained, showing statistical significance.
Figure #1: Number of DNA copies for CYP1A gene in control and triclosan-treated samples
obtained using qPCR.
# of DNA Copies -qpcr CYP1A: Posthoc test, Bonferroni-Holm
Group 1 Group 2 Critical P Significant?
Control Treatment 0.05 0.043879 Yes

7. Figure #2: ANOVA, Posthoc test Bonferroni-Holm, for CYP1A, comparing # of DNA copies
obtained from qPCR for control group and triclosan-treated group. Statistical significance is
shown.
Comparing TCF3A control samples and treated samples, the control had 684,657
copies, while the treated samples had 750,287 copies. The treated samples thus showed a 9%
increase. Using the ANOVA test, a p-value of 0780454 was obtained, showing no statistical
significance.
Figure #3: Number of DNA copies for TCF3A gene in control and triclosan-treated samples
obtained using qPCR.
# of DNA Copies -qpcr TCF3A: Posthoc test, Bonferroni-Holm
Group 1 Group 2 Critical P Significant?
Control Treatment 0.05 0.780454 No

8. Figure #4: ANOVA, Posthoc test Bonferroni-Holm, for TCF3a gene, comparing # of DNA copies
obtained from qPCR for control group and triclosan-treated group. Statistical significance is not
found.
Next, the lowry assay was successful, except there is one outlier as shown in figure 5.
The total protein content for control and treatment were both calculated using absorbancies
measured at 750nm for 4 groups of control and treatment samples. Group 1 showed a 4x fold
increase in protein concentration for treated samples, group 2 showed over an 8x fold increase,
and group 4 showed a near 2.5x fold increase. Group 3 showed a decrease in protein content
for the treated samples, which was the outlier in the data amongst the 4 groups. Errors may be
due to dissecting additional GI content in the control samples, instead of dissecting just the liver
alone. This experimental error would increase the amount of protein obtained in the control
samples.
Protein Content - Lowry Assay
Group Control [mg] Treatment [mg] Ratio T/C
1 0.0134 0.05285 3.94403
2 0.0081 0.068 8.395062
3 0.012325 0.009725 0.789047
4 0.00425 0.0105 2.470588
Figure #5: Cytochrome p450 concentration measurement using lowry assay carried out in
replicates of 4 groups.

9. The EROD assay was carried out for both the stop-assay and the continuous-assay.
Figure 6 and 8 show the rate of EROD reaction measured. In the stop assay, the control rate
was 7.4x10^-9 and the treatment rate was 4.78x10^-9, as displayed in figure 6. Statistical
analysis shows no significance between the control and treatment data, as seen in figure 7.
Figure #6: EROD stop assay was used to measure relative fluorescence units (RFU), which is
used to calculate rate of reaction for control and treated samples.
Rate of Reaction - Stop Assay: Posthoc test, Bonferroni-Holm
Group 1 Group 2 Critical P Significant?
control treatment 0.05 0.187088 No
Figure #7: ANOVA, Posthoc test Bonferroni-Holm, comparing rate of ethoxyresorufin production
obtained from EROD stop assay for control group and triclosan-treated group. Statistical
significance is not found.
In the continuous assay, the control rate was 3.7x10^-11 and the treatment rate was
1.8x10^-11. Statistical analysis shows no significance between the control and treatment data

10. for this assay as well. There were errors in the assay, where sample groups had shown
opposite results. One group had a significant decrease in rate for the treated samples, while two
groups had near equal rates for control and treated samples. These opposing data recorded
thus effected the statistical calculations.
Figure #8: EROD continuous assay was used to measure relative fluorescence units (RFU),
which is used to calculate rate of reaction for control and treated samples.
Rate of Reaction - Continuous Assay: Posthoc test, Bonferroni-Holm
Group 1 Group 2 Critical P Significant?
Control Treatment 0.05 0.150599 No
Figure #9: ANOVA, Posthoc test Bonferroni-Holm, comparing rate of ethoxyresorufin production
obtained from EROD continuous assay for control group and triclosan-treated group. Statistical
significance is not found.
Discussion

11. Three end-points were tested for the effects of triclosan on zebrafish: 1) increase in
CYP1a and TCF3a RNA, 2) increase in total protein content, and 3) increase in EROD activity.
The results from the qPCR support the hypothesis that triclosan-treated samples have an
increase in CYP1a RNA. The results show no significant increase for TCF3a RNA, which leads
to a conclusion that TCF3a is not significantly affected by triclosan. If TCF3a is not being
induced or reduced, then it’s not a factor in the enlarged liver, let alone a target of triclosan.
CYP1a specifically does show a clear increase, which concludes that it is a target of triclosan as
a toxicological endpoint. Similarly, total protein content does show an increase in treated
samples, as determined by the lowry assay, which would explain enlarged livers observed in
treated adult zebrafish after dissection as well.
Whether or not this increase in protein concentration correlates with cytochrome p450 is
not answered by the lowry assay; instead, the EROD assay is meant to test that
question/endpoint. An increase in EROD activity was not observed. There was a decrease in
rate observed from control to treated, although statistically it was not shown to be significant.
This error in the assay conflicts with observations from the qPCR. It is more probable that the
result obtained from qPCR is accurate and that the EROD data is faulty. The standard
deviations seen on the rates for both the continuous assay and the stop assay are equal in
value to almost half of the rates, which shows extreme outliers and inconsistencies in data. This
would mean that the rates are inaccurate, and the lack of statistical significance is inconclusive.
The results from the qPCR and the lowry suggest that triclosan causes induced
expression for cytochrome p450. In zebrafish, other studies have shown that induction of
CYP1a is an effect that is seen and correlated with liver tumors (Spitsbergen et al., 2000).
Similarly, CYP1a has been shown to be a player in circulation failure when treated with dioxins
(Teraoka et al., 2003). This study has shown that CYP1a, a key player in toxicological endpoints
in zebrafish and other aquatic life, can be induced by triclosan as well, further supporting

12. triclosan as a hazardous toxin. With CYP1a as a target induced, the relevance of triclosan as a
harmful chemical to ecological systems and to human health risks only grows greater.
Alternatively, in order to confirm whether or not samples or aquatic life have been
affected by triclosan or dioxins, EROD assays may not always be the most sensitive. This study
has shown reliability in qPCR as a method of analyzing exposure to triclosan treatment, over
that of the EROD assay. Other studies have also shown that zebrafish is not the top fish that’s
induced by triclosan in the strongest manner. According to one study, the rainbowfish trout
species shows a much greater induction for CYP1a induction than that of zebrafish (Jonnson et
al., 2009). Although zebrafish is a good model organism for the effects of toxins and has shown
effects on several genes, attempting an EROD assay on rainbow fish trout may provide more
significant results than what was obtained from the zebrafish.
Furthermore, triclosan has been suggested in some studies to affect estrogen-
dependent cancer growth (Dinwiddie et al., 2014). Longer studies for effects of triclosan on
human health are even more so needed to give a better understanding on the chemical’s
relation to cancer. With increasing amount of studies on triclosan as a possible growth inhibitor
for cancer, alternative treatment methods may be found potentially. Additionally, with conflicting
results in studies that show cancer inhibition and others hinting towards tumor inception, the
need for studies in human cancer patients is needed to better clarify what exactly the
relationship is between triclosan, triclosan-derivatives, and cancer-related pathways.
References
1. Spitsbergen et al., 2000
J.M. Spitsbergen, H.W. Tsai, A. Reddy, T. Miller, D. Arbogast, J.D. Hendricks, G.S. Bailey
Neoplasia in zebrafish (Danio rerio) treated with 7,12-dimethylbenz[a]anthracene by two
exposure routes at different developmental stages
Toxicol. Pathol., 28 (2000), pp. 705–715

13. 2. Teraoka et al., 2003
Hiroki Teraoka, Wu Donga, Yoshikazu Tsujimoto, Hiroyuki Iwasa, Daiji Endohb, Naoto Uenoc,
John J Stegemand, Richard E Petersone, Takeo Hiragaa
Induction of cytochrome P450 1A is required for circulation failure and edema by 2,3,7,8-
tetrachlorodibenzo-p-dioxin in zebrafish
Biochem. and Biophys. Research Comm., 304 (2003), pp. 223-228
3. Safe et al., 2016
Theresa M. Safe, Anne E. Luebke
Prenatal low dosage dioxin (TCDD) exposure impairs cochlear function resulting in
auditory neuropathy
Hearing Research, 331 (2016), pp. 7-12
4. Oliveira et al., 2009
Oliveira R, Domingues I, Koppe Grisolia C, Soares AM
Effects of triclosan on zebrafish early-life stages and adults.
Environmental Science and Pollution Research, 16 (2009), pp 679-688
5. Gribble et al., 2009
Suzanna L. Gribble, Hyung-Seok Kim, Jennifer Bonner, Xu Wang, and Richard I. Dorsky
Tcf3 inhibits spinal cord neurogenesis by regulating sox4a expression
Development. 2009 March 1; 136(5): 781–789. Published online 2009 January 28. doi:
10.1242/dev.027995
6. Zhang et al., 2015
Qian-Qian Zhang, Guang-Guo Ying , Zhi-Feng Chen, Jian-Liang Zhao, You-Sheng Liu
Basin-scale emission and multimedia fate of triclosan in whole China
Environmental Science and Pollution Research, 22 (2015), pp. 10130-10143
7. Hovander et al., 2002
Identification of hydroxylated PCB metabolites and other phenolic halogenated pollutants
in human blood plasma
Hovander, L., Malmberg, T., Athanasiadou, M., Athanassiadis, I., Rahm, S., Bergman, A.,
Wehler, E.K.
Archives of Environmental Contamination and Toxicology, 42 (2002), pp. 105-117
8. Gee et al., 2007
R. H. Gee, A. Charles, N. Taylor and P. D. Darbre
Oestrogenic and androgenic activity of triclosan in breast cancer cells
Journal of Applied Toxicology28 (2008), pages 78–91
9. Kanetoshi et al., 1987
Akio Kanetoshi, Hiroshi Ogawa, Eiji Katsura, Hiroyasu Kaneshima
Chlorination of irgasan DP300 and formation of dioxins from its chlorinated derivatives
Journal of Chromatography A, 389 (1987), Pages 139-153

14. 10. Jonsson et al., 2009
Maria E. Jönsson, Björn Brunström, Ingvar Brandt
The zebrafish gill model: Induction of CYP1A, EROD and PAH adduct formation
Aquatic Toxicology, 91 (2009), Pages 62–70
11. Dinwiddie et al., 2014
Michael T. Dinwiddie, Paul D. Terry, and Jiangang Chen
Recent Evidence Regarding Triclosan and Cancer Risk
Int J Environ Res Public Health. 2014 Feb; 11(2): 2209–2217.