SNAPSHOT

Evidence for Perturbation by Stressor

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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

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.

Domain of Applicability

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).

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.

Domain of Applicability

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.

Vallender, E.J., Xie, Z., Westmoreland, S.V., and Miller, G.M. (2010). Functional evolution of the trace amine associated receptors in mammals and the loss of TAAR1 in dogs. BMC Evol Biol 10, 51.

 

 

Domain of Applicability

Key Event Description

Sexual behavior in male bird is characterized by components such as crowing, strutting, and mounting, whereas the newt exhibits a tail-vibrating behavior (Hutchison, 1978). In both species, sexual behavior varies on a daily (photoperiod) and seasonal (breeding) basis. A decrease in sexual behavior is defined by a reduction in the frequency of these typical behaviors.

 

How it is Measured or Detected

Since sexual behavior varies along the day and the season, timing is an important component of the measurement. Light exposure, endocrine disruptors and season should all be considered in the protocol design in order to limit the bias in the measurement.

Sexual behavior in male is measured in presence of a sexually receptive female. To limit the risk of bias induced by differences in female receptivity, it is important to repeat the experiment later/the day after with a different female for each male (Halldin et al., 1999).In bird, the frequency of chasing, pecking, head grabbing, and mounting for a X minutes observation can be measured (Halldin et al., 1999; Ogura et al., 2016). 

For newt, sexual behavior is characterized by a tail-vibrating behavior and can be measured by counting the frequency and incidence of this behavior during X minutes.  Incidence and frequency are expressed as the percentage of animals exhibiting the behavior and the mean number of times the behavior was recorded per test animal over the test period, respectively (Toyoda et al., 1983). 

References

Adkins, E. K. and N. T. Adler. 1972. Hormonal control of behavior in the Japanese quail. J. Comp. Physiol. Psychol.81:27-36.

Halldin, K., Berg, C., Brandt, I., and Brunstrom, B. (1999). Sexual behavior in Japanese quail as a test end point for endocrine disruption: effects of in ovo exposure to ethinylestradiol and diethylstilbestrol. Environ Health Perspect 107, 861-866.

Hutchison, R.E. (1978). Hormonal differentiation of sexual behavior in Japanese quail. Horm Behav 11, 363-387.

Ogura, Y., Haraguchi, S., Nagino, K., Ishikawa, K., Fukahori, Y., and Tsutsui, K. (2016). 7alpha-Hydroxypregnenolone regulates diurnal changes in sexual behavior of male quail. Gen Comp Endocrinol 227, 130-135.

Sachs, B.D. (1967). Photoperiodic control of the cloacal gland of the Japanese quail. Science 157, 201-203.

Toyoda, F., Ito, M., Tanaka, S., and Kikuyama, S. (1993). Hormonal induction of male courtship behavior in the Japanese newt, Cynops pyrrhogaster. Horm Behav 27, 511-522.

Key Event Component

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List of Adjacent Key Event Relationships

List of Non Adjacent Key Event Relationships