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SSRI Effect on Neurotransmitters Associated with Sexual Dysfunction

A selective serotonin reuptake inhibitor (SSRI) is the most commonly prescribed drug to

treat depression. Unfortunately, a common side effect is sexual dysfunction, such as erectile

impotence, absence of orgasm, delay of ejaculation, pain, loss of sensation, and decreased

pleasure.1 It may be the most clinically significant SSRI side effect among patients; however, the

mechanism of the drug in regards to sexual function is poorly understood.2 It also tends to be

overlooked by clinicians during inquiry and that many patients do not discuss these side effects

with their physicians (80% of women).3 Sexual dysfunction occurs in as many as 67% of

patients. Even when drug administration decreases depression, the sexual symptoms do not

recover to pre-drug behavior. Instead the SSRI increases the severity of the symptoms.4 The

increase in severity of side effect is more prevalent in men but also in women. Because of these

sexual side effects, people have opted to cancel treatment, thus not helping with the depression.5

Previous research has shown that SSRIs can induce sexual dysfunction in male rats.6

The purpose of this experiment is to determine the quantity of the neurotransmitters

affected in U937, SH-SY5Y and COLO 320DM cell lines that are either known or thought to

influence sexual function upon treatment with Celexa® or Serotonin. The drug used in this

experiment is citalopram hydrobromide (Celexa®) and the cell lines tested are U937 (white

blood cells), SH-SY5Y (neuroblastoma) and COLO 320DM (colonicadenocarcinoma).

Serotonin was also administered to the cells because since serotonin increases as an effect of

SSRI administration, administering serotonin should have similar effects to the

neurotransmitters/hormones. It is hypothesis that Celexa® will negatively affect the

neurotransmitters/hormones; dopamine and oxytocin will be decreased while serotonin and

morphine will be increased.

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SSRIs have been known to cause sexual dysfunction, which includes a whole number of

sexually related symptoms. Of them includes loss of libido, anorgasmia, erectile dysfunction,

dyspareunia, loss of desire and a general decrease in sexual activity.2,3,7,8 When tested with lab

rats, sexual behavior has been shown to decrease in both males and females. Females display a

decrease in lordosis behavior (mounting position) while males have an increased latency in

copulation (ejaculation) and an increase in intromission (thrust movement).6,7,8,9 In both male

and female humans, cases of sexual side effects have been reported.2,5,7,8,10

SSRIs are known to affect the levels of serotonin, oxytocin, and dopamine.1, 2, 5,8,11 These

neurotransmitters or hormones are known to effect sexual behavior positively or negatively.2, 3, 5,

6, 7, 8, 12 Extensive experimentation on rat modules has shown that affecting these

neurotransmitters or hormones can negatively or positively affect sexual function.9

Neurotransmitters that are thought to play a role in maintain normal sexual function include

dopamine, serotonin, oxytocin, and nitric oxide. They have the most important roles in the

pathophysiology and treatment of sexual dysfunction arising from antidepressant and

antipsychotic drugs.

Serotonin is the main neurotransmitter affected by the treatment of SSRIs.2, 5, 7, 12 The

drug works by blocking the serotonin transporters, thus having an overall increase in the amount

of serotonin in the nerve synapses.5, 6 As early as the 1960’s, it was hypothesized that serotonin

inhibited sexual receptivity.6 Serotonin has been linked to play a main role in sexual dysfunction.

Studies show that SSRIs can induce sexual dysfunction because of an increase of serotonin

among other effects.3, 5, 6, 7, 8 Serotonin agonists can exacerbate these effects while serotonin

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antagonists can help alleviate these effects.7, 8 Increased serotonin levels have been shown to

inhibit ejaculation in lab animals.

Dopamine is known to be beneficial for sexual function.5 Dopamine antagonists can

interfere with copulation while dopamine agonists facilitate copulation. Increased levels of

dopamine can increase sexual arousal and enhance penile erection.7, 8 Dopamine has been shown

to help facilitate sexual behavior in animals.8 Centrally acting dopaminergic compounds have

been shown to facilitate sexual behavior in animal models.

Oxytocin, like dopamine, is believed to help sexual function. High amounts have been

shown to facilitate sexual behavior.11, 12 Oxytocin is released during sexual behavior and is

involved in uterine smooth muscle contraction. Plasma oxytocin levels are known to be increased

during sexual arousal, erection, and at orgasm.12 It is linked with feelings of companionship as

well.3 Oxytocin is known to induce penile erection when administered to male rats.

Administration of oxytocin also increases nitric oxide production by activating its own

receptors.11 Depletion of oxytocin does not seem to decrease copulation but it causes mild effects

on the sexual behavior of male rats. Oxytocin antagonists increase the intromission latency and

intromissions, decrease the number of mounts, and abolish ejaculation in rats, which is part of

both erectile and ejaculatory dysfunction.12 Oxytocin seems to play at least a modulating role in

erection and ejaculation.

Since nitric oxide plays a role and it is a precursor for morphine, morphine is believed to

play a small role in sexual function. Morphine may help induce penile erection through nitric

oxide production in a manner similar to Sildenafil (Viagra).3 Morphine would then also be

involved in the dopaminergic and oxytocin pathways since nitric oxide plays a role in the

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forming of those neurotransmitters.12 So it may influence those neurotransmitters/hormones

depending on how it is affected by a drug, or in this case, SSRIs.

METHODS

Cell Culture

The cell lines tested were the SH-SY5Y (neuroblastoma), U937 (white blood cells), and

COLO 320DM (colonicadenocarcinoma) cell lines. Cells were split using Tryple from Invitrogen

and seed cells into plates at a density of 500,000 cells/mL. Three groups were made to be tested

for one neurotransmitter at a time; serotonin, morphine, oxytocin, or dopamine. Each cell line

was treated with either Celexa® or serotonin. Four micro molars of each diluted treatment were

added to each cell line. A third group was created for a control group where nothing was added

to the cells. All neuroblastoma trials were treated with the treatments for 24 hours, while most

trials in only the U937 cell line were treated with treatments for 4 hours and 24 hours. Each cell

line would be cultured with treatments in human like conditions (98.6 degrees Fahrenheit) in a

sterile environment.

After treatment, RNA would be isolated from the cells to be later converted to cDNA.

Once RNA was isolated, a reverse transcription would be performed to convert the RNA to

cDNA for use in a polymerase chain reaction (PCR). Using specific primers for serotonin,

oxytocin, or morphine, a conventional PCR would be performed to amply the desired genes

which would then later be used to determine the levels of oxytocin, morphine, and serotonin

through the use of an electrophoresis gel. A specific primer for dopamine was used in a real time

PCR to determine levels of dopamine and dopamine pathway activations. For all cell lines,

GAPDH was used as a reference gene.

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Taqman low density arrays

A Taqman low density array (Applied Biosystems) consisting of 11 genes of interest and

1 reference gene (GAPDH) was used to evaluate the effects of morphine and epinephrine on

gene expression in the COLO 320DM cell line. The array contained selected genes of the

proposed morphine biosynthetic pathway. Table 1 lists the genes in the array with their

corresponding gene symbol. GAPDH was selected as the most stable reference gene. For the

arrays, 2ug of total RNA was reverse transcribed and 1uL of cDNA was diluted with 49ul of

H2O. This diluted cDNA was mixed with 50 ul of 2X universal master mix (Applied

Biosystems). The resulting mixtures were added to the appropriate sample ports on the array (8

samples per array). Arrays were run on the 7900HT fast real time PCR system (Applied

Biosystems) using the default thermal cycler settings. Gene expression fold changes were

determined by the software using the ΔΔCt method. Each PCR was performed in quadruplicate.

Electrophoresis Gel

After the conventional PCR’s were completed, a 1% agarose gel was created for use in

electrophoresis. This was used to visually represent the amplified genes to determine an up or

down regulation based on band intensity.

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RESULTS

Key

S=Serotonin

C=Celexa

Co=Control

MMW = Molecular Weight Marker

Figure 3 U937 (Celexa® and Serotonin treatment) - morphine presence

Figure 1 U937 (Celexa®treatment only) - morphine presence

Figure 2 COLO 320DM (Celexa® and serotonin treatment) - oxytocin presence

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Table 1 Genes in the custom Taqman low density array.

*All graphs of Real Time PCR are read in the same order of Table 1.

Gene Symbol Gene Name

GAPDH glyceraldehyde-3-phosphate dehydrogenase

ALDH2 aldehyde dehydrogenase 2 family (mitochondrial)

AOF2 amine oxidase (flavin containing) domain 2

COMT catechol-O-methyltransferase

CYP2D6 cytochrome P450, family 2, subfamily D, polypeptide 6

DBH dopamine beta-hydroxylase (dopamine beta-monooxygenase)

DDC dopa decarboxylase (aromatic L-amino acid decarboxylase)

GCH1 GTP cyclohydrolase 1 (dopa-responsive dystonia)

OCA2 oculocutaneous albinism II (tyrosine transporter)

PNMT phenylethanolamine N-methyltransferase

SLC6A3 solute carrier family 6 (neurotransmitter transporter, dopamine), member 3

TH tyrosine hydroxylase

Figure 4: 24hr Treatment Celexa®(SH-SY5Y) - Custom Taqman Array

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Figure 5: 24hr Celexa®(COLO320DM) - Custom Taqman Array

Figure 7: Celexa®and Serotonin treatment (SH-SY5Y) - Serotonin Presence

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Figure 6: 24hr Serotonin (COLO320DM) - Custom Taqman Array

Figure 8: Celexa®and Serotonin (U937) - Morphine Presence

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Figure 9: Celexa® and Serotonin - Oxytocin Presence

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Figures 1-3 all show electrophoresis gels that were done to show expressed genes. Figure

1 shows expressed morphine in the U937 cell line after treatment with Celexa®. Figure 2 shows

the expression of oxytocin in COLO 320DM cells after Celexa® and serotonin treatments.

Figure 3 shows the morphine presence after treatment with Celexa® and serotonin treatments in

the U937 cell line.

Figure 4-6 are graphs derived from Real Time PCRs used to determine changes in the

genes involved in the morphine pathway. All of the data are compared to the reference gene

GAPDH. Table 1 shows all the genes involved in the morphine pathway. Figure 4 shows how

much of each gene in the morphine pathway was expressed in comparison to the reference gene

after treatment of Celexa® in the SH-SY5Y cell line. Figure 5 shows the expression of each gene

in comparison to the reference gene after treatment of Celexa® in the COLO 320DM cell line.

Figure 6 shows the expression of each gene in comparison to the reference gene after treatment

of serotonin in the COLO 320DM cell line.

Figures 7-9 are graphs that show the mean intensity of the bands displayed in the

electrophoresis gels in the U937, COLO 320DM, and SH-SY5Y cell lines after treatment of

Celexa® or serotonin. Figure 7 shows the expression of serotonin after treatment of Celexa® or

serotonin in the SH-SY5Y cell line. Figure 8 shows the expression of morphine after treatment of

Celexa® or serotonin in the U937 cell line. Figure 9 shows the expression of oxytocin after

treatment of Celexa® or serotonin in the U937, SH-SY5Y, and COLO 320DM cell lines.

Morphine and Dopamine

When treated with Celexa® and serotonin, there was a slight down regulation of

morphine in the U937 in general, according to Figure 1 and 8. With Celexa® there would be a

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slightly greater down regulation compared to that of serotonin’s effect on morphine. For

Celexa®, compared to the 24 hour treatments, the 4 hour treatments were greater in the down

regulation of morphine. Overall, morphine had a general decrease in the U937 cell line when

comparing the treatment of Celexa® and serotonin to the control.

Since dopamine is involved in the morphine pathway, the effects of SSRIs on both

dopamine and morphine would be suggested to be similar, but since dopamine is involved in a

normal sexual function, it can also be suggested that an SSRI would decrease the levels of

dopamine in the body. Using a custom Taqman array in a Real Time PCR, the genes involved in

the morphine pathway were able to be determined. In Figure 4, there was an overall significant

decrease in the gene expressions of the morphine pathway. As for Figure 6, there was a general

increase in the gene expressions of the morphine pathway. And for Figure 5, there was a slight

increase in the gene expressions, but nothing significant.

Serotonin

When treated with Celexa® or serotonin, there was a general up regulation of serotonin

in the cells in the SH-SY5Y cell line. Compared to that of serotonin, Celexa® generally had a

more significant increase in the up regulation of serotonin in the SH-SY5Y cell line and

compared to that of the control, Celexa® had a significant increase in the up regulation of

serotonin, as shown in Figure 7. This data suggests that the administration of SSRIs is associated

with an increase in the levels of serotonin.

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Oxytocin

As shown in Figure 9, in the COLO 320DM cell line after treatment with Celexa® or

serotonin, Celexa® had shown the greatest down regulation compared to serotonin’s down

regulation of oxytocin. Both treatments have shown a down regulation of oxytocin compared to

that of the control groups.

According to Figure 2, the band that is brighter is the group that expressed more of

oxytocin. A brighter band means it has a higher mean intensity, which means the same thing; the

gene was expressed more. In this case, the control groups had a higher mean intensity. Referring

to Figure 7, oxytocin was not expressed as much as the control group, thus suggesting that

oxytocin levels are decreased after administration of an SSRI.

DISCUSSION/LIMITATIONS

SSRIs are associated with sexual dysfunction.1, 2, 5, 6, 8 The neurotransmitters/hormones

linked with sexual function are serotonin, dopamine, morphine, and oxytocin. After treatment

with SSRI, levels of serotonin increased and levels of morphine exhibited no significant change.

The mean intensities of the bands of serotonin expressed in the electrophoresis gels for the

treatments were 17.24 for the treatment of Celexa® and 11.64 for the control group in the SH-

SY5Y cell line. As a result, high levels of serotonin may induce sexual dysfunction. It is believed

that SSRIs affect these said neurotransmitters. The data suggests that SSRIs cause sexual

dysfunction by decreasing oxytocin and dopamine in cells. Low amounts of dopamine and

oxytocin can negatively affect sexual behavior.13

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Because of these effects, sexual dysfunction can occur in people. Oxytocin is known to

help facilitate ejaculation.9 With low levels, it would inhibit rather than facilitate. Serotonin is

known to inhibit sexual behaviors. With higher levels, it is more likely to do so. Along with

dopamine and morphine, when you have the levels of the neurotransmitter/hormone where it is

possible to cause a sexual side effect, it is more prevalent in doing so.

Time was crucial part of this project and more time would have been able to provide for

more data to support conclusions more effectively. Also, using human subjects rather than just

controlled cell lines would have given a more concrete idea of how precisely neurotransmitters

inside the body would be effected by SSRIs.

CONCLUSION

Overall, Celexa® and serotonin decrease concentrations of oxytocin and dopamine and

increases serotonin; however, the treatments have no significant effect on morphine. Since these

neurotransmitters are linked with sexual function, this data supports the idea that treatment

emergent sexual dysfunction is associated with the administration of SSRIs.1, 2, 3, 7 Through the

these effects of the neurotransmitters/hormones, SSRIs tend to induce sexual dysfunction to up to

67% of patients who are prescribed antidepressants to treat depression.14

Future investigations would focus on clinical trials on patients that are treated with an

SSRI. Administer either Celexa® or a placebo to depressed patients to see how they are affected

in terms of their sexual behavior. If sexual dysfunction becomes present during treatment,

dopamine or oxytocin agonists or serotonin and morphine antagonists may alleviate the side

effect. Investigation on other types of drugs that work with these neurotransmitters/hormones

would be necessary to see if they have similar effects on patients.

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Since nitric oxide plays a role in the morphine pathway15, using a nitric oxide probe in

cells that were treated with Celexa® or serotonin can be used to see their effects on nitric oxide

levels. With more time, a future application of this project would be to combine certain types of

drugs to see if there would be an effective combination of drugs that may be able to counteract

the sexual side effects induced by SSRIs.

ACKNOWLEDGEMENTS

I acknowledge Dr. Kirk Mantione, lecturing research faculty of biology and

neuroscience, for aiding me in my research and paper. I would also like to acknowledge Mrs.

Renee Barcia for assisting me with my paper.

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