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Created at: 2018-04-30 15:45

AOP ID and Title:

AOP 218: Inhibition of CYP7B activity leads to decreased reproductive success via decreased locomotor activity
Short Title: Inhibition of CYP7B leads to decreased locomotor activity

Graphical Representation

Authors

Florence Pagé-Larivière

Laval University, Québec, Qc, Canada

florence.page-lariviere.1@ulaval.ca

 


Status

Author status OECD status OECD project SAAOP status
Under development: Not open for comment. Do not cite Under Development

Abstract

This AOP details the downstream events of CYP7B inhibition leading to a decreased locomotor activity that adversely impacts reproductive success. CYP7B is expressed in the brain and catalyzes the conversion of pregnenolone to 7α-hydroxypregnenolone, a neurosteroid that stimulates the release of dopamine in the telencephalon. When released through this pathway, dopamine binds D2 receptor which is involved in locomotor activity induction. Ketoconazole and other azole fungicides are potent inhibitor of cytochrome P450s, including CYP7B. They bind to the heme site of the enzyme preventing its catalytic activity. Exposure to one of these molecules induces a decrease in 7α-hydroxypregnenolone synthesis which, in turn, reduces dopamine release in the telencephalon and limits locomotor activity. Since locomotor activity is closely associated to reproductive success through courtship enhancement (newt), expansion of territory (bird) and homing migration (salmon), its inhibition negatively affects the fitness of animals. 

7α-hydroxypregnenolone was recently discovered and its function and regulation remain unclear. The few studies that focused on this neurosteroid and that were used for this AOP are based on in vitro and in vivo experiments in salmon, quail and newt. At present, it is believed that the function of this neurosteroid differs in mammals, which suggest that this AOP is only applicable to non-mammalian vertebrates. Also, the sex applicability of the AOP varies according to species.  


Background

The stressor identified for this AOP is used as fungicide both in the field for crop protection and in animal against fungus infection. Because it can inhibit various cytochrome P450 enzymes activity, a family of enzymes involved in a plethora of pathways including steroidogenesis, it has the potential to induce many different side effects for animal exposed indirectly through the environment or directly through medical treatment. This AOP targets one of these side effects. 


Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Sequence Type Event ID Title Short name
1 MIE 1386 CYP7B activity, inhibition CYP7B activity, inhibition
2 KE 1387 7α-hydroxypregnenolone synthesis in the brain, decreased 7α-hydroxypregnenolone synthesis in the brain, decreased
3 KE 1388 Dopamine release in the brain, decreased Dopamine release in the brain, decreased
4 KE 1389 Locomotor activity, decreased Locomotor activity, decreased
5 KE 1141 Decreased, Reproductive Success Decreased, Reproductive Success
6 AO 442 Decreased, Population trajectory Decreased, Population trajectory

Key Event Relationships

Upstream Event Relationship Type Downstream Event Evidence Quantitative Understanding
CYP7B activity, inhibition adjacent 7α-hydroxypregnenolone synthesis in the brain, decreased High Moderate
7α-hydroxypregnenolone synthesis in the brain, decreased adjacent Dopamine release in the brain, decreased Low Low
Dopamine release in the brain, decreased adjacent Locomotor activity, decreased High Moderate
Locomotor activity, decreased adjacent Decreased, Reproductive Success
Decreased, Reproductive Success adjacent Decreased, Population trajectory
CYP7B activity, inhibition non-adjacent Locomotor activity, decreased Low Low
7α-hydroxypregnenolone synthesis in the brain, decreased non-adjacent Locomotor activity, decreased High Moderate

Stressors


Name Evidence
Ketoconazole High

Ketoconazole

Conazole is a class of fungicide that inhibits CYP51 14α-lanosterol demethylase activity in yeats and moults, thereby preventing ergosterol synthesis (Hof et al., 2006). In animals, conazoles are known to be less specific than in fungi since they can interfere with various cytochromes P450 activity.  For instance, it is clearly demonstrated that ketoconazole directly inhibits CYP7B activity which induces a decrease in 7α-hydroxypregnenolone (Matsunaga et al., 2004, Tsutsui et al., 2008; Toyoda et al., 2012; Ogura et al., 2016).

Overall Assessment of the AOP

 

 

Domain of Applicability

Life Stage Applicability
Life Stage Evidence
Adult, reproductively mature High
Taxonomic Applicability
Term Scientific Term Evidence Links
Japanese quail Coturnix japonica NCBI
Cynops pyrrhogaster Cynops pyrrhogaster NCBI
Sex Applicability
Sex Evidence
Male High

Taxons: This AOP is supported with evidence from studies conducted with newt, quail, and salmon. Based on anticipated conservation of the biology associated with the KEs and KERs described, it is presumed to be applicable to all amphibian, bird and migratory teleost fish. 

Previous evidence suggest that this AOP is not applicable to mammal. All the key events of this AOP are described or are biologically plausible in mammal, but the relationship between them might differ, as suggested by Yau et al. (2006). 

Sex: The sex applicability of this AOP is species-specific. Female quail and newt are insensitive to this MIE in regard to locomotor activity whereas male are highly sensitive. In salmon, both male and female exhibit a decreased locomotor activity with induction of the MIE.   

Life Stage: This AOP applies to sexually mature animals since the endpoint is related to reproduction. However, all the key events except “reproductive success, decreased” (event 1141) and and the adverse outcome (event 442) are known to occur in juveniles which suggest that an AOP connecting CYP7B inhibition and decreased locomotor activity in juvenile to an endpoint not sexually-oriented could be built.

Essentiality of the Key Events

Few studies measured multiple key events of this AOP.  For this reason, the evidence for essentiality of the key events is mainly indirect and provided by a series of antagonist/exogenous supplementation experiments. The animal models used for these investigations were newt, quail, and salmon. 

 

Key event

Essentiality

Rational

MIE

Inhibition of CYP7B

Moderate

At present, no CYP7B knock-out experiments were conducted in species of interest. However, several indirect evidences linking CYP7B inhibition to a decreased locomotor activity suggest an important correlation between the two events.

  • Inhibition of CYP7B with intracranial injection of ketoconazole decreased 7α-hydroxypregnenolone synthesis and prevented locomotor activity in newt and quail (Tsutsui et al., 2008, Toyoda et al., 2012). Ketoconazole is a non-specific inhibitor of cytochromes P450 activity known to bind to and inhibit CYP7B both in vitro and in vivo.
  • In salmon, decreased locomotor activity was observed following a depletion of CYP7B substrate (pregnenolone) with intracranial injection of aminoglutethimide, an inhibitor of cytochrome P450 ssc (Haraguchi et al., 2015).
  • Penguins exposed orally to voriconazole, an azole molecule with the same effects as ketoconazole on CYPs activity were lethargic and weak. The side effects dissipated or resolved with discontinuation or dose reduction of voriconazole (Hyatt et al., 2015).

 

KE1

7α-hydroxypregnenolone, decreased

Strong

Numerous direct evidences connecting this neurosteroid to locomotor activity were described.

  • Intracerebroventricular injection of 7α-hydroxypregnenolone in male chick, salmon, quail, and newt induced spontaneous locomotor activity in a dose-dependent manner. The same treatment had no effect on female. The experiments were conducted during the season (salmon, newt) or the time of the day (chick, quail) with the lowest endogenous locomotor activity (Matsunaga et al., 2004; Tsutsui et al., 2008; Hatori et al., 2011; Toyoda et al., 2012; Haraguchi et al., 2015).
  • Salmon treated with aminoglutethimide to deplete pregnenolone concentration in the brain exhibited increased locomotor activity following intracerebroventricular injection of 7α-hydroxypregnenolone (Haraguchi et al., 2015).

 

KE2

Dopamine release, decreased

Moderate

There is strong evidence demonstrating the involvement of dopamine in locomotor activity among all vertebrates. However, only indirect evidence relates CYP7B inhibition to a decreased dopamine release. The rational is stronger for 7α-hydroxypregnenolone in relation to dopamine release, although this neurosteroid receptor remains to be identified. 

  • Locomotor activity was stimulated in male newt with intracerebroventricular injection of 7α-hydroxypregnenolone. Newt treated with a dopamine D2-like receptor antagonist (haloperidol or sulpiride) prior to receiving 7α-hydroxypregnenolone exhibited no increase in locomotor activity.
  • Inhibition of 7α-hydroxypregnenolone synthesis with aminoglutethimide (pregnenolone depletion) decreases dopamine concentration in the salmon brain. Supplementation with physiological concentration of 7α-hydroxypregnenolone restored dopamine concentration to normal (Haraguchi et al., 2015).

KE3

Locomotor activity, decreased

Strong

All the previous key events can decrease locomotor activity in salmon and male quail, chicken, and newt.  

Weight of Evidence Summary

Biological plausibility

This AOP connects the cyp7b catalyzed synthesis on an important neurosteroid to a well characterized sequence of events. For instance, the involvement of dopamine in locomotor activity that in turn impacts on reproductive success is well described and undisputed (Bardo M.T. et al., 1999; Levens et al., 2000). What is less characterized is the relation between 7α-hydroxypregnenolone and dopamine release. Since the neurosteroid receptor has yet to be identified, no direct interaction between 7α-hydroxypregnenolone and dopaminergic neuron has been demonstrated. It is thus possible that an intermediate event takes place in between to indirectly connect the neurosteroid to dopamine release.

In terms of structural plausibility, the brain expresses the steroidogenic enzymes required for pregnenolone synthesis, the main substrate of CYP7B. It also expresses CYP7B which synthesizes high concentration of 7α-hydroxypregnenolone in the diencephalon. This region of the brain is populated by neurons projecting into the striatum which is known to express a high quantity of D1- and D2-like dopamine receptor and control motor activity (Orgen S. et al., 1986; Mezey S. et al., 2002; Callier S. et al., 2003).

 

Uncertainties or inconsistencies

At present, there are no inconsistencies reported in the literature, but some gaps remain to be filled.

The most important ones are 7α-hydroxypregnenolone receptor localization and the connection between 7α-hydroxypregnenolone and dopamine release discussed in the previous section.

In addition, mammalian CYP7B not only catalyzes the 7α-hydroxylation of pregnenolone but also that of dehydroepiandrosterone (DHEA). Although no clear information reported this enzymatic reaction in the bird, it is plausible that CYP7B catalyzes the hydroxylation of DHEA. Thus, the phenotypic effect of CYP7B inhibition in the brain cannot be uniquely attributed to a depletion in 7α-hydroxypregnenolone. Additionally, ketoconazole is known to inhibit a variety of CYPs, which suggest that animal exposed to it are likely to have several other enzymes inhibited. It is plausible that the impacts of ketoconazole are the result of multiple CYPs inhibition that all converge towards the same phenotype. These off target effects greatly limit the investigations on 7α-hydroxypregnenolone since its concentration cannot be specifically decreased.

If a CYP7B knock-out in the brain was to be performed in an animal species, 7α-hydroxyDHEA supplementation would be required to properly study 7α-hydroxypregnenolone function.

Quantitative Consideration

This information is not available for the moment. 

References


Appendix 1

List of MIEs in this AOP

Event: 1386: CYP7B activity, inhibition

Short Name: CYP7B activity, inhibition

Stressors

Name
Ketoconazole
Tebuconazole
Propiconazole
Tioconazole
Miconazole
Fluconazole
Voriconazole
Clotrimazole

Biological Context

Level of Biological Organization
Molecular

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

The binding of inhibitors to CYP7B is demonstrated in vitro with purified recombinant protein in presence of the inhibitor. Ligand-induced spectral changes is analyzed using spectrophotometric titration as a shift of the heme (Yantsevich et al., 2014). 

Ketoconazole and other conazole are known to bind to CYPs preventing its enzymatic activity.

  • CYP7B inhibitor (ketoconazole, 10-4 M) decreased the synthesis of 7α-hydroxypregnenolone
  • CYP7B inhibitor (intracerebroventricular injection of ketoconazole) decreased the synthesis of 7α-hydroxypregnenolone in the male quail and newt brain, in vivo (Matsunaga et al., 2004; Rose et al., 1997; Tsutsui et al., 2008). 
  • The heme prosthetic group (catalytic site) of human recombinant CYP7B thightly bound to various imidazole- and triazole-based drugs in an in vitro spectrometric titration assay. The drugs with the highest affinities were the industrial pesticides tebuconazole (0.11 μm), propiconazole (0.13 μm) and the antifungal drugs tioconazole (0.15 μm) and miconazole (0.23 μm). Voriconazole and metyrapone (non-azole compound) also interacted with CYP7B (Yantsevich et al., 2014). 




Ketoconazole

It is clearly demonstrated that ketoconazole directly inhibits CYP7B (Matsunaga et al., 2004). It is expected for the other members of the conazole family to have the same effect.

Some other azoles such as clotrimazole can also inhibit CYP7B activity (Liu et al., 2011; Rose et al., 1997). 


Tebuconazole

In vitro, tebuconazole was shown to bind to the catalytic site of the human recombinant CYP7B and to inhibit its catalytic activity (Yantsevich et al., 2014). 


Propiconazole

In vitro, propiconazole was shown to bind to the catalytic site of the human recombinant CYP7B and to inhibit its activity (Yantsevich et al., 2014).


Tioconazole

In vitro, tioconazole was shown to bind to the catalytic site of the human recombinant CYP7B and to inhibit its activity (Yantsevich et al., 2014).


Miconazole

In vitro, miconazole was shown to bind to the catalytic site of the human recombinant CYP7B and to inhibit its activity (Yantsevich et al., 2014).


Fluconazole

In vitro, fluconazole was shown to bind to the catalytic site of the human recombinant CYP7B and to inhibit its activity (Yantsevich et al., 2014).


Voriconazole

In vitro, voriconazole was shown to bind to the catalytic site of the human recombinant CYP7B and to inhibit its activity (Yantsevich et al., 2014).


Clotrimazole

Clotrimazoles can inhibit CYP7B activity (Liu et al., 2011; Rose et al., 1997). 


Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
Japanese quail Coturnix japonica NCBI
Cynops pyrrhogaster Cynops pyrrhogaster NCBI
Oncorhynchus keta Oncorhynchus keta NCBI
Life Stage Applicability
Life Stage Evidence
All life stages
Sex Applicability
Sex Evidence
Mixed

CYP7B is known to be conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, zebrafish, and frog. https://www.ncbi.nlm.nih.gov/homologene/3544


Key Event Description

Site of action:

CYP7B is expressed in different organs including liver, prostate and brain.

How does it work :

CYP7B is a member of the cytochrome P450 family of enzymes. It is involved in steroidogenic pathways as well as in the synthesis of bile acids. In the brain, it is involved in neurosteroids synthesis.

In the brain, the reactions catalyzed by CYP7B are : 

  • Probably in all vertebrates: Pregnenolone into 7α-hydroxypregnenolone and its stereoisomer 7β-hydroxypregnenolone (bird only) (R08943) (Matsunaga et al., 2004; Rose et al., 1997; Tsutsui et al., 2008)
  • Proven in mouse and human: Dehydroepiandrosterone (DHEA) to 7α-hydroxy-DHEA and its stereoisomer 7β-hydroxy-DHEA (Martin et al., 2004; Weihua et al., 2002). 

In the human and mouse liver, CYP7B is responsible for (Toll et al., 1994): 

  • 5-cholesten-3-beta, 25(S)-diol into Cholest-5-ene-3 beta-7 alpha, 25-thiol (R07209 R08723),
  • Cholest-5-ene-3 beta, 26-diol into 7 alpha, 27-dihydroxycholesterol (R07372 R08724),
  • 3 beta-hydroxy-5-cholestenoate into 3 beta, 7 alpha-dihydroxy-5-cholestenoate (R08727 R08728).
  • It is expressed in the chicken liver and is probably involved in the same reactions (Handschin et al., 2005). 

In the prostate:

  • Proven for human and rat: Dehydroepiandrosterone (DHEA) to 7α-hydroxy-DHEA and 7β-hydroxy-DHEA (Martin et al., 2001; Martin et al., 2004). 

Inhibitors prevent the metabolism of pregnenolone into 7-alpha-hydroxypregnenolone, thereby decreasing the concentration of the neurosteroid. 


How it is Measured or Detected

In vitro

To measure CYP7B activity in vitro, different experiments based on HPLC and GS-MS analysis can be performed.

  • An assay in liver microsome followed by HPLC analysis of the metabolites (Souidi et al., 2000). 
  • Labeled steroid conversion in vitro with cell or tissue extract in presence of NADPH followed by GS-MS analysis (Rose et al., 1997; Tsutsui et al., 2008). 
  • CYP7B can be cloned in bacteria to produce an active protein in vitro. In presence of adequate precursor and cofactors, the enzymatic activity of the protein can be measured and analyzed using HPLC.  
  • CYP7B can be transfected in a cell line unable to synthesize 7α-hydroxypregnenolone in order to measure with HPLC the ability of the protein to catalyze the enzymatic reaction in presence of the appropriate substrate and cofactor (Tsutsui et al., 2008)

In vivo

Experiments may include knock-out of mice (followed by RNA, protein blotting and enzymatic activity to confirm knock-out) (Li-Hawkins et al., 2000) followed by the measurement of substrate and metabolites of CYP7B in plasma and tissues (Rose., 2001). 


References

Dulos, J., van der Vleuten, M.A., Kavelaars, A., Heijnen, C.J., and Boots, A.M. (2005). CYP7B expression and activity in fibroblast-like synoviocytes from patients with rheumatoid arthritis: regulation by proinflammatory cytokines. Arthritis Rheum 52, 770-778.

Handschin C., Gnerre C., Fraser DJ., Martinez-Jimenez C., Jover R., Mever UA., (2005) Species-specific mechanisms for cholesterol 7α-hydroxylase (CYP7A1) regulation by drugs and bile acids, Archives of Biochemistry and Biophysics, Vol 434-1, pp75-85

Haraguchi, S., Koyama, T., Hasunuma, I., Okuyama, S., Ubuka, T., Kikuyama, S., Do Rego, J.L., Vaudry, H., and Tsutsui, K. (2012). Acute stress increases the synthesis of 7alpha-hydroxypregnenolone, a new key neurosteroid stimulating locomotor activity, through corticosterone action in newts. Endocrinology 153, 794-805.

Haraguchi, S., Yamamoto, Y., Suzuki, Y., Hyung Chang, J., Koyama, T., Sato, M., Mita, M., Ueda, H., and Tsutsui, K. (2015). 7alpha-Hydroxypregnenolone, a key neuronal modulator of locomotion, stimulates upstream migration by means of the dopaminergic system in salmon. Sci Rep 5, 12546.

Li-Hawkins, J., Lund, E.G., Turley, S.D., and Russell, D.W. (2000). Disruption of the oxysterol 7alpha-hydroxylase gene in mice. J Biol Chem 275, 16536-16542.

Liu, C., Yang, X.V., Wu, J., Kuei, C., Mani, N.S., Zhang, L., Yu, J., Sutton, S.W., Qin, N., Banie, H., et al. (2011). Oxysterols direct B-cell migration through EBI2. Nature 475, 519-523.

Martin, C., Bean, R., Rose, K., Habib, F., and Seckl, J. (2001). cyp7b1 catalyses the 7alpha-hydroxylation of dehydroepiandrosterone and 25-hydroxycholesterol in rat prostate. Biochem J 355, 509-515.

Martin, C., Ross, M., Chapman, K.E., Andrew, R., Bollina, P., Seckl, J.R., and Habib, F.K. (2004). CYP7B generates a selective estrogen receptor beta agonist in human prostate. J Clin Endocrinol Metab 89, 2928-2935.

Matsunaga, M., Ukena, K., Baulieu, E.E., and Tsutsui, K. (2004). 7alpha-Hydroxypregnenolone acts as a neuronal activator to stimulate locomotor activity of breeding newts by means of the dopaminergic system. Proc Natl Acad Sci U S A 101, 17282-17287.

Rose, K., Allan, A., Gauldie, S., Stapleton, G., Dobbie, L., Dott, K., Martin, C., Wang, L., Hedlund, E., Seckl, J.R., et al. (2001). Neurosteroid hydroxylase CYP7B: vivid reporter activity in dentate gyrus of gene-targeted mice and abolition of a widespread pathway of steroid and oxysterol hydroxylation. J Biol Chem 276, 23937-23944.

Rose, K.A., Stapleton, G., Dott, K., Kieny, M.P., Best, R., Schwarz, M., Russell, D.W., Bjorkhem, I., Seckl, J., and Lathe, R. (1997). Cyp7b, a novel brain cytochrome P450, catalyzes the synthesis of neurosteroids 7alpha-hydroxy dehydroepiandrosterone and 7alpha-hydroxy pregnenolone. Proc Natl Acad Sci U S A 94, 4925-4930.

Souidi, M., Parquet, M., Dubrac, S., Audas, O., Becue, T., and Lutton, C. (2000). Assay of microsomal oxysterol 7alpha-hydroxylase activity in the hamster liver by a sensitive method: in vitro modulation by oxysterols. Biochim Biophys Acta 1487, 74-81.

Toll, A., Wikvall, K., Sudjana-Sugiaman, E., Kondo, K.H., and Bjorkhem, I. (1994). 7 alpha hydroxylation of 25-hydroxycholesterol in liver microsomes. Evidence that the enzyme involved is different from cholesterol 7 alpha-hydroxylase. Eur J Biochem 224, 309-316.

Tsutsui, K., Inoue, K., Miyabara, H., Suzuki, S., Ogura, Y., and Haraguchi, S. (2008). 7Alpha-hydroxypregnenolone mediates melatonin action underlying diurnal locomotor rhythms. J Neurosci 28, 2158-2167.

Weihua, Z., Lathe, R., Warner, M., and Gustafsson, J.A. (2002). An endocrine pathway in the prostate, ERbeta, AR, 5alpha-androstane-3beta,17beta-diol, and CYP7B1, regulates prostate growth. Proc Natl Acad Sci U S A 99, 13589-13594.

Yantsevich, A.V., Dichenko, Y.V., Mackenzie, F., Mukha, D.V., Baranovsky, A.V., Gilep, A.A., Usanov, S.A., and Strushkevich, N.V. (2014). Human steroid and oxysterol 7alpha-hydroxylase CYP7B1: substrate specificity, azole binding and misfolding of clinically relevant mutants. FEBS J 281, 1700-1713.


List of Key Events in the AOP

Event: 1387: 7α-hydroxypregnenolone synthesis in the brain, decreased

Short Name: 7α-hydroxypregnenolone synthesis in the brain, decreased

Stressors

Name
Ketoconazole

Biological Context

Level of Biological Organization
Cellular

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
Japanese quail Coturnix japonica High NCBI
Cynops pyrrhogaster Cynops pyrrhogaster High NCBI
mouse Mus musculus High NCBI
human Homo sapiens High NCBI
salmonid fish salmonid fish High NCBI
Life Stage Applicability
Life Stage Evidence
During development and at adulthood
Sex Applicability
Sex Evidence
Unspecific

The enzyme synthesizing 7α-hydroxypregnenolone is known to be conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, zebrafish, and frog. https://www.ncbi.nlm.nih.gov/homologene/3544


Key Event Description

7α-hydroxypregnenolone is an active neurosteroid synthesized in the brain from pregnenolone via a reaction catalyzed by CYP7B (R08943). Pregnenolone can also be synthesized in most vertebrate brain by CYP11A from cholesterol (Tsutsui and Yamazaki, 1995; do Rego et al., 2016).

Compared to other brain regions of the male quail and newt, 7α-hydroxypregnenolone concentration is higher in the diencephalon. In the brain of both salmon and newt, the peak concentrations are measured in the hypothalamus and optic tectum (Matsunaga et al., 2004; Tsutsui et al., 2008; Haraguchi et al., 2015). 

7α-hydroxypregnenolone synthesis in the brain is cyclic and driven by a different mechanism according to the specie.

  • In male quail, a diurnal animal, it is inhibited by a melatonin-receptor mechanism after melatonin secretion from the pineal gland (Tsutsui et al., 2008). 
  • In male newt, a nocturnal animal, melatonin secretion stimulates its synthesis in the brain.
  • Another regulating mechanism is observed in male newt where 7α-hydroxypregnenolone concentration peaks during the breeding period in response to prolactin signal (Matsunaga et al., 2004). 
  • In salmon, 7α-hydroxypregnenolone stays high during homing migration (Haraguchi et al., 2015). The endogenous factor regulating its synthesis has yet to be determined.

Thus, 7α-hydroxypregnenolone synthesis is regulated by the circadian cycle and/or by seasonal factors such as breeding and migration.


How it is Measured or Detected

Detection and quantification of 7α-hydroxypregnenolone can be performed using GC-MS and/or HPLC analysis.

In vitro

  • Cell not expressing CYP7B can be transfected with CYP7B cDNA and incubated in presence of pregnenolone and NADPH. Concentration of 7α-hydroxypregnenolone can be measured by HPLC analysis (Haraguchi et al., 2015). 
  • To distinguish 7α- and 7β-hydroxypregnenolone, HPLC analysis was performed (Tsutsui et al., 2008). Brain homogenates can be incubated in presence of pregnenolone and NADPH. Concentration of 7α-hydroxypregnenolone can be measured by HPLC analysis Haraguchi et al., 2015).  

In vivo

The extracted steroids derived from brain homogenates and plasma can be measured using GC-MS analysis (Tsutsui et a;., 2008). 

 


References

Haraguchi, S., Koyama, T., Hasunuma, I., Vaudry, H., and Tsutsui, K. (2010). Prolactin increases the synthesis of 7alpha-hydroxypregnenolone, a key factor for induction of locomotor activity, in breeding male Newts. Endocrinology 151, 2211-2222.

Haraguchi, S., Yamamoto, Y., Suzuki, Y., Hyung Chang, J., Koyama, T., Sato, M., Mita, M., Ueda, H., and Tsutsui, K. (2015). 7alpha-Hydroxypregnenolone, a key neuronal modulator of locomotion, stimulates upstream migration by means of the dopaminergic system in salmon. Sci Rep 5, 12546.

Matsunaga, M., Ukena, K., Baulieu, E.E., and Tsutsui, K. (2004). 7alpha-Hydroxypregnenolone acts as a neuronal activator to stimulate locomotor activity of breeding newts by means of the dopaminergic system. Proc Natl Acad Sci U S A 101, 17282-17287.

Petkam, R., Renaud, R.L., Freitas, A.M., Canario, A.V., Raeside, J.I., Kime, D.E., and Leatherland, J.F. (2003). In vitro metabolism of pregnenolone to 7alpha-hydroxypregnenolone by rainbow trout embryos. Gen Comp Endocrinol 131, 241-249.

Tsutsui, K., Inoue, K., Miyabara, H., Suzuki, S., Ogura, Y., and Haraguchi, S. (2008). 7Alpha-hydroxypregnenolone mediates melatonin action underlying diurnal locomotor rhythms. J Neurosci 28, 2158-2167.

Tsutsui, K., and Yamazaki, T. (1995). Avian neurosteroids. I. Pregnenolone biosynthesis in the quail brain. Brain Res 678, 1-9.

Yau, J.L., Noble, J., Graham, M., and Seckl, J.R. (2006). Central administration of a cytochrome P450-7B product 7 alpha-hydroxypregnenolone improves spatial memory retention in cognitively impaired aged rats. J Neurosci 26, 11034-11040.


Event: 1388: Dopamine release in the brain, decreased

Short Name: Dopamine release in the brain, decreased

Biological Context

Level of Biological Organization
Tissue

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
Vertebrates Vertebrates NCBI
Life Stage Applicability
Life Stage Evidence
All life stages
Sex Applicability
Sex Evidence
Mixed High

Dopamine is used as a neurotransmitter in multicellular animals (Barron et al., 2010). Across a wide range of vertebrates, dopamine has an "activating" effect on behavior-switching and response selection, comparable to its effect in mammals. 


Key Event Description

Dopamine is a monoamine, catecholaminergic neurotransmitter synthesized in the brain and the kidney from precursor L-DOPA (Carlsson et al., 1957). It is synthesized in neuron cells, stored in vesicules nearby the synaps, and is released into the synaptic cleft after excitation of the neuron. Once released, it can bind D1-like or D2-like G protein receptor which have different effects (Stoof and Kebabia, 1984; Vallender et al., 2010).

It is conserved among vertabrates and regulates neural activity, behavior and gene expression. The main impacts are related to voluntary movement, feeding, and reward.   

In birds, fish, and other vertebrates, dopaminergic neurons located in mesencephalic region (VTA, SN) project to the telencephalon, a region of the brain rich in D1 and D2 receptors (Hara et al., 2007; Ball et al., 1995; Levens et al., 2000).  


How it is Measured or Detected

In vitro

To measure the ability of a molecule to stimulate dopamine release, brain can be incubated in physiological saline in presence of a presumptive activator (e.g. 7α-hydroxypregnenolone, a neurosteroid) and dopamine concentration in saline is measured by HPLC-ECD (Matsunaga et al., 2004). 

In vivo

To measure the concentration of dopamine in the brain in vivo, freshly collected brain can be homogenized and dopamine concentration can be analyzed using HPLC-ECD (ECD-300, Eicom).


References

Barron, A.B., Sovik, E., and Cornish, J.L. (2010). The roles of dopamine and related compounds in reward-seeking behavior across animal phyla. Front Behav Neurosci 4, 163.

Carlsson, A., Lindqvist, M., and Magnusson, T. (1957). 3,4-Dihydroxyphenylalanine and 5-hydroxytryptophan as reserpine antagonists. Nature 180, 1200.

Matsunaga, M., Ukena, K., Baulieu, E.E., and Tsutsui, K. (2004). 7alpha-Hydroxypregnenolone acts as a neuronal activator to stimulate locomotor activity of breeding newts by means of the dopaminergic system. Proc Natl Acad Sci U S A 101, 17282-17287.

Stoof, J.C., and Kebabian, J.W. (1984). Two dopamine receptors: biochemistry, physiology and pharmacology. Life Sci 35, 2281-2296.

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Event: 1389: Locomotor activity, decreased

Short Name: Locomotor activity, decreased

Biological Context

Level of Biological Organization
Individual

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
Vertebrates Vertebrates NCBI
Sex Applicability
Sex Evidence
Mixed High

Measurement of locomotor activity can be performed on any motile animal. 


Key Event Description

Vertebrate move for a variety of reasons including reproduction, search for food or suitable microhabitat, and escape predator. In birds, newt, and other vertebrates, locomotor activity is cyclic and follows the circadian and/or seasonal rhythm (Saper et al., 2005; Binkley et al., 1971; Chabot  and  Menaker,  1992).

  • Locomotor activity is elevated in quail under daylight and decreases at night, following a circadian cycle. It was shown in bird that locomotor activity was mainly related to maintenance of territory (Wada, 1981; Watson, 1970). 
  • In newt, locomotor activity is high during breeding season and night time (Nagai et al., 1998).
  • In salmon, the maximum locomotor activity is observed during homing migration where fishes swim against the water flow (Gowans et al., 2003).

How it is Measured or Detected

Locomotor activity is a measurement of distance per unit of time. Experiment design should take into account the normal seasonal and daily variation of locomotor activity.

To measure locomotor activity, animals can be placed individually in a water-filled aquarium (newts) marked with parallel lines to define sectors. Quantification of total number of lines crossed during a certain amount of time is then measured (Lowry et al., 2001; Moore et al., 1984).  

Birds can be put in a soundproof box with a telemetry system implanted to calculate their total distance during the experiment ( or in a box with wire-mesh floor and ceilings and photobeams activated when the animal break the beam (Levens et al., 2001; Tsutsui et al., 2008). 


References

Gowans A. R. D., Armstrong J. D., Priede I. G. & Mckelvey S. (2003). Movements of Atlantic salmon migrating upstream through a fish-pass complex in Scotland. Ecol. Freshw. Fish 12, 177–189.

Lowry, C.A., Burke, K.A., Renner, K.J., Moore, F.L., and Orchinik, M. (2001). Rapid changes in monoamine levels following administration of corticotropin-releasing factor or corticosterone are localized in the dorsomedial hypothalamus. Horm Behav 39, 195-205.

Moore, F.L., Roberts, J., Bevers, J. (1984). Corticotropin-releasing factor (CRF) stimulates locomotor activity in intact and hypophysectomized newts (Amphibia). J Exp Zool 231, 331-333.

Nagai, K., T. Oishi. T. (1998). Behavioral rhythms of the Japanese newts, Cynops pyrrhogaster, under a semi-natural condition. Int. J. Biometeorol. 41: 105–112.

Levens N., Akins C.K. (2001). Cocaine induces conditioned place preference and increases locomotor activity in Japanese quail. Pharmacol Biochem Behav. 68-1, 71-80

Tsutsui, K., Haraguchi, S., Fukada, Y., and Vaudry, H. (2013). Brain and pineal 7alpha-hydroxypregnenolone stimulating locomotor activity: identification, mode of action and regulation of biosynthesis. Front Neuroendocrinol 34, 179-189.

Tsutsui, K., Inoue, K., Miyabara, H., Suzuki, S., Ogura, Y., and Haraguchi, S. (2008). 7Alpha-hydroxypregnenolone mediates melatonin action underlying diurnal locomotor rhythms. J Neurosci 28, 2158-2167.

Wada, M. (1981). Effects of photostimulation, castration, and testosterone replacement on daily patterns of calling and locomotor activity in Japanese quail. Horm Behav 15, 270-281.

Watson, A. (1970). Territorial and reproductive behaviour of red grouse. J Reprod Fertil Suppl 11, Suppl 11:13-14.


List of Adverse Outcomes in this AOP

Event: 442: Decreased, Population trajectory

Short Name: Decreased, Population trajectory

Key Event Component

Process Object Action
population growth rate decreased

Biological Context

Level of Biological Organization
Population

Appendix 2

List of Key Event Relationships in the AOP