THE INFLUENCE OF BRAIN SEROTONERGIC SYSTEM ON THE EXPRESSION AND ACTIVITY OF

CYTOCHROME P450 IN THE LIVER

Marta Rysz, Ewa Bromek, Anna Haduch, Władysława Anna Daniel Institute of Pharmacology, Polish Academy of Sciences, Smętna 12, PL 31-343 Kraków, Poland

1. INTRODUCTION

Studies conducted so far have indicated that hormones acting as ligands of cytoplasmic and nuclear receptors (growth hormone, glucocorticosteroids, thyroid hormones) influence transcriptional level of genes encoding cytochrome P450 (CYP) isoenzymes [1-3]. All the above-mentioned hormones are controlled by the hipothalamo-pituitary axis. It is also known that the hypothalamus is den-sely innervated by serotonergic axons projecting from raphe nuclei (dorsal nuclei B6, B7 and median nuclei B5, B8). Serotonergic neurons reach hypothalamic nuclei forming parvocellular neurosecretory system (the paraventricular and arcuate nuclei), Thus, it can be assumed that brain serotonergic projections to the hypothalamus influence hepatic cytochrome P450 expression via the above-mentioned hormones. The aim of our study was to investigate the effect of damage to the serotonergic system on the expression of liver cytochrome P450 and serum level of the key hormones (growth hormone, thyroid hormones and corticosterone) that contribute to this process.

2. MATERIALS AND METHODS

The experiments were carried out on male Wistar rats. The 5,7-DHT (5,7-

dihydroxytryptamine), a selective serotonergic neurotoxin was injected into dor-

sal and median raphe nuclei (in a dose of 10 μg/raphe nucleus) or intracere-

broventricularly (in a dose of 70μg/ventricle). Ten days after the neurotoxin in-

jection, brain structures, liver tissue and blood were collected and prepared for

further analysis. The levels of noradrenaline (NA), dopamine (DA) and serotonin

(5-HT) in the brain structures were determined by a high pressure liquid chro-

matography (HPLC) with an electrochemical detection.

The activity of individual cytochrome P450 isoenzymes was determined in mi-

crosomal fraction of the liver, based on the velocity of reactions specific for in-

dividual isoenzymes: caffeine 3-N-demethylation (catalyzed by CYP2C11 and

CYP1A) and caffeine 8-hydroxylation (catalyzed by CYP1A), testosterone hy-

droxylation at positions: 2, 6 (catalyzed by CYP3A); 2, 16 (catalyzed by

CYP2C11), 7 (catalyzed by CYP2A) and 16 (catalyzed by CYP2B); warfarin 7-

hydroxylation (catalyzed by CYP2C6) and bufuralol 1’-hydroxylation (catalyzed

by CYP2D). Specific metabolites formed in vitro were assayed using HPLC with

UV of fluorescence detection [4]. Serum concentrations of hormones and inter-

leukines were estimated using ELISA kits.

Fig. 1. The effect of injection of 5,7-DHT into the dorsal and median raphe nuclei or

intracerebroventricularly on the level of serotonin in rat brain structures

3. RESULTS %

of

co

ntr

ol

CYP1A

1/2

CYP2C

11

CYP3A

0

50

100

150

200

*

* *

Fig. 3. The effect of injection of 5,7-DHT into dorsal and median

raphe nuclei on the protein level of liver CYP isoenzymes

Fig. 4. The effect of injection of 5,7-DHT into dorsal and me-

dian raphe nuclei on the hormones and cytokines level

CYP1A CYP2A

CYP2B

CYP2C6

CYP2C11

CYP2D

CYP3A

5,7-DHT

GROWTH HOR-MONE

TST

CORT T4

T3 IL-2 IL-6

5,7-DHT

Enzyme activity Protein level

SUMMARY

ACKNOWLEDGMENTS

This work was financially supported by the project Interdisciplinary PhD Studies „Molecular Sciences for Medicine (co-financed by the Eu-

ropean Social Fund within the Human Capital Operational Programme) and by statutory funds from the Institute of Pharmacology, PAS

CONCLUSIONS

1. Either mode of lesion (intracerebroventricular or intrastructural) reduced the serotonin level in all the brain structures ex-amined. In the hypothalamus, the 5-HT level fell to 35% and 18% of the control value (after injection to the raphe nuclei and lateral ventricles, respectively).

2. In the liver, similar effects were observed after either mode of lesion: 5,7-DHT increased the activity of CYP1A, CYP3A and CYP2C11, while the activity of CYP2A, 2B, 2C6 and 2D remained unchanged.

3. The increased activity of CYP1A, 3A and 2C11 correlated positively with the enhanced enzyme protein levels.

4. Simultaneously, a significant rise in the serum concentration of the growth hormone, testosterone and corticosterone and drop of triiodothyronine, but no change in thyroxin and cytokine levels (IL-2 and IL-6) were observed.

5. The results obtained indicate that brain serotonergic system contributes to the regulation of liver cytochrome P450. Ho-wever, its effect on the main male isoforms (CYP2C11, CYP3A) is opposite to that observed for dopaminergic or noradre-nergic systems.

REFERENCES 1. Monostory K., Pascussi J.M., Kobori L., Dvorak Z., Hormonal regulation of CYP1A expression. Drug Metabolism Reviews, 2009, 41, 547-72. 2. Waxman D.J., Holloway M.G., Sex differences in the expression of hepatic drug metabolizing enzymes. Molecular Pharmacology, 2009, 76, 215-228 3. Wójcikowski J., Gołembiowska K., Daniel WA., The regulation of liver cytochrome P450 by the brain dopaminergic system. Curr Drug Metab., 2007,

8, 631-8. 4. Sadakierska-Chudy A., Haduch A., Rysz M., Gołembiowska K., Daniel WA; The role of brain noradrenergic system in the regulation of liver cyto-

chrome P450 expression, Biochemical Pharmacology, 2013, 86, 800-807

Fig. 2. The effect of injection of 5,7-DHT into dorsal and median raphe nuclei or

intracerebroventricularly on the activity of liver CYP isoenzymes

Ht—Hypothalamus;

Hp—Hippocampus;

BS—Brain Stem;

FCx—Frontal cortex;

St—Striatum;

Na—Nucleus accum-

bens;

Th—Thalamus;

Mo—Medulla oblongata

Cb—Cerebellum;

Rcx– Rest of cortex

co

ncen

trati

on

(p

g/m

g o

f ti

ssu

e)

0

200

400

600

800

***

***

*

***

*

*

*

*

***

*

*

SHAM control

5,7-DHT

Naive

SEROTONIN - after i.raphe injection of 5,7-DHT

Ht Hp BS FCx St Na Th Mo Cb Rcx Ht Hp BS FCx St Na Th Mo Cb Rcx

co

ncen

trati

on

(p

g/m

g o

f ti

ssu

e)

0

200

400

600

800

1000

Naive

Sham control

5,7-DHT

SEROTONIN - after icv. injection of 5,7-DHT

***

***

***

*** *** ******

***

***

% o

f co

ntr

ol

1A 1A

1A 1A

2A

2B

2C11

2C11 3A

3A 2C

62D

0

50

100

150

2001-N 3-N 7-N 8-OH

caffeine warfarin bufuralol

*

*

7 16 2 16 2 6

* *

testosterone

*

*

7-OH -OH

% o

f co

ntr

ol

1A 1A 1A 1A 2A 2B2C

11

2C11 3A 3A

0

50

100

150

200

250caffeine testosterone

**

*

* * *

1-N 3-N 7-N 8-OH 7 16 2 16 2 6

1) After i.raphe injection of 5,7-DHT 2) After icv. injection of 5,7-DHT

% o

f co

ntr

ol

GH TST CORT. T4 T3 IL-2 IL-60

50

100

150

1000

1500

2000

2500

3000

*

**

Hormones Cytokines

*