Glioblastoma cells express crucial enzymes involved in androgen synthesis: 3ß-hydroxysteroid dehydrogenase, 17-20α-hydroxylase, 17ß-hydroxysteroid dehydrogenase and 5α-reductase.

Glioblastoma (GB) is the most common and aggressive primary brain tumour in adult humans. Therapeutic resistance and tumour recurrence after surgical removal contribute to poor prognosis for glioblastoma patients. Men are known to be more likely than women to develop an aggressive form of GB, and differences in sex steroids have emerged as a leading explanation for this finding. Studies indicate that the metabolism and proliferation of GB-derived cells are increased by sex steroids, the expression of androgen receptors (ARs) and the synthesis of androgens and oestrogens, suggesting that these hormones have a role in the tumour pathogenesis. The expression of aromatase, the enzyme that converts androgens to oestrogens, has been reported in glial cells and GB cell lines. Thus, it was necessary to test whether the steroidogenic enzymes involved in androgen synthesis are expressed in GB cells. Therefore, here, we investigated the expression of four key enzymes involved in androgen synthesis in human-derived GB cells. U87 cells were cultured in Dulbecco's modified Eagle medium plus foetal bovine serum and antibiotics on slides for immunocytochemistry or immunofluorescence. U87, LN229 and C6 cells were also cultured in multi-well chambers to obtain proteins for Western blotting. We used primary antibodies against 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17α-hydroxilase/17,20-lyase (P450c17), 17ß-hydroxysteroid dehydrogenase (17ß-HSD) and 5α-reductase. Immunocytochemistry, and immunofluorescence results revealed that glioblastoma cells express 3ß-HSD, P450c17, 17ß-HSD and 5α-reductase proteins in their cytoplasm. Moreover, Western blot analyses revealed bands corresponding to the molecular weight of these four enzymes in the three GB cell lines. Thus, glioblastoma cells have the key enzymatic machinery necessary to synthesize androgens, and these enzymes might be useful targets for new therapeutic approaches.

Another abnormal trait in the serotonin metabolism path in intrauterine growth-restricted infants.

BACKGROUND: The present study was aimed to obtain information on the interaction kinetics of L-tryptophan (L-Trp) with plasma albumin from normal, intrauterine growth-restricted (IUGR) and nutritionally recovered (NR) newborn infants. METHODS: A case study cohort was planned in 37 newborns during the first 3 months of life. At birth two groups were formed. The first group included 20 newborns with IUGR. The control group (C) included 17 appropriate for gestational age newborns. At 30 days of age, 9 infants of the IUGR group showed a return to normal of the anthropometric parameters, these infants formed the NR group. Free, bound and total L-Trp were measured. To assess binding kinetics albumin was freed of fatty acids and tested in mole to mole samples from IUGR, NR and control babies. RESULTS: Plasma free L-Trp was increased, K(d) (dissociation constant) elevated and B(max )(maximal binding)decreased in IUGR patients up to postnatal day 90. These changes remained even after nutritional recovery. CONCLUSIONS: Abnormal kinetics of L-Trp binding to albumin explains the increased availability of this precursor amino acid in the plasma of IUGR infants. This finding corroborates previous results in IUGR rats and newborn babies, indicating enhanced potential for brain serotonergic synthesis.

A functional disturbance in the auditory cortex related to a low serotonergic neurotransmission in women with type 2 diabetes.

BACKGROUND/AIMS: To determine if the slope of the amplitude/stimulus intensity function (ASF) of the N1/P2 component of the auditory evoked potential was increased in women with type 2 diabetes reflecting a low brain serotonergic activity in the auditory cortex. METHODS: In a comparative study in women with type 2 diabetes and controls, we measured free, bound and total plasma L-tryptophan (L-Trp), neutral amino acids (NAA) and free fatty acids (FFA) and recorded the N1/P2 component of the auditory evoked potential. RESULTS: The diabetic patients were overweight and FFA and NAA in plasma were significantly elevated. The free, bound to albumin and total L-Trp were decreased. The values of free/total L-Trp and free/NAA ratios were significantly lower. The latencies of N1 and P2 at all intensities and the slope ASF of the N1/P2 component significantly increased. CONCLUSION: The decrease of the free fraction of L-Trp in plasma and the increase of the ASF slope of the N1/P2 component reflect a functional relationship between the brain serotonergic activity and the N1/P2 changes in the auditory cortex, suggesting a cortical impaired activity associated with anomalies of brain serotonergic neurotransmission in women with type 2 diabetes. We proposed the ASF slope together with measurement of the plasma FFT as noninvasive clinical indicators of serotonergic neurotransmission in the brain in these as well as in other types of patients.

A low brain serotonergic neurotransmission in children with type 1 diabetes detected through the intensity dependence of auditory-evoked potentials.

OBJECTIVE: To determine in children with type 1 diabetes the plasma free fraction of L-tryptophan (FFT) and the intensity-dependent auditory-evoked potentials (IDAEPs) as indicators of possible changes in brain serotonergic neurotransmission. RESEARCH DESIGN AND METHODS: A prospective and comparative study was performed in children with type 1 diabetes and normal control subjects. We measured FFT, bound and total plasma L-tryptophan, neutral amino acids (NAAs), albumin, free fatty acids (FFAs), glucose, and HbA1c(A1C) and recorded IDAEPs with four intensities (40, 60, 90, and 103 dB). RESULTS: The glycemia, A1C, FFAs, and NAAs in plasma were significantly elevated. The FFT and the FFT-to-total L-tryptophan and FFT-to-NAA ratios were reduced. The latencies of N1 and P2 increased at all intensities and the slope of the amplitude/stimulus intensity function (ASF slope) of the N1/P2 component significantly increased. CONCLUSIONS: The decrease of the FFT in plasma and increase in the N1/P2 component amplitude may reflect a functional relationship between the brain serotonergic activity with the N1/P2 changes. The increase of the ASF slope in children with type 1 diabetes suggests that the response of the auditory cortex to sound intensity stimulus may be regulated by the serotonergic tone and that decreased serotonergic neurotransmission may provoke a different behavior of sensory cortices. Therefore, the IDAEP (N1/P2 component) may be an electrophysiological indicator of brain changes of serotonergic neurotransmission in children with type 1 diabetes. These changes may be related to psychoemotional manifestations observed in diabetic children such as anxiety and depression.

Prenatal impairment of brain serotonergic transmission in infants.

OBJECTIVE: To evaluate whether the free fraction of L-tryptophan (L-Trp) and the N1/P2 component of the auditory evoked potentials (AEPs) are associated with impaired brain serotonin neurotransmission in infants with intrauterine growth restriction (IUGR). STUDY DESIGN: We measured free, bound, and total plasma L-Trp and recorded the N1/P2 component of AEP in a prospective, longitudinal, and comparative study comparing IUGR and control infants. RESULTS: Plasma free L-Trp was increased and the amplitude of N1/P2 component was significantly decreased in IUGR relative to control infants. The free fraction of L-Trp and N1/P2 component had a negative association. CONCLUSIONS: In newborns with IUGR, the changes in measured plasma free fraction of L-Trp and in the amplitude the N1/P2 component of the AEP suggest an inverse association between free L-Trp and components of the AEP. The changes observed in the free fraction of L-Trp and AEP may be causally associated with brain serotonergic activity in utero. In IUGR, epigenetic factors such as stress-induced disturbances in brain serotonin metabolism or serotonergic activity, identifiable by alterations in AEP, influence cerebral sensory cortex development and may be causally associated with serotonin-related disorders in adulthood.

Inhibition and kinetic changes of brain tryptophan-5-hydroxylase during insulin-dependent diabetes mellitus in the rat.

In the present study we report results on the possible mechanism of inhibition of tryptophan-5-hydroxylase activity induced by insulin-dependent diabetes mellitus (IDDM). Kinetic experiments were done with different L-tryptophan (L-Trp) concentrations in the rat brain at different days of evolution of the disease. Additionally, different activation conditions of the enzyme were evaluated, to gain information on the mechanism of the activity changes. Diabetes state was induced in normal male rats, by the administration of 75 mg/kg body weight of streptozotocin (STZ). The results showed an increase of the Km value and a decrease in the Vmax in the diabetic's brain as compared to controls. Interestingly, in the diabetic group, the response capacity to phosphorylation is significantly reduced. These shifts in the activity of tryptophan-5-hydroxylase developed during IDDM may not be explained only by a decrease of L-Trp, but also by a possible change in the enzyme itself, reflected in a diminished affinity for the substrate and a decreased response to phosphorylating conditions.

N1/P2 component of auditory evoked potential reflect changes of the brain serotonin biosynthesis in rats.

It is known that L-tryptophan stimulates serotonin synthesis in the brain and serotonergic neuronal activity. Also, the N1/P2 component of auditory evoked potential (AEP) is a good indicator of this activity in the auditory cortex. In the present work, we examined the effect of the L-tryptophan administration on electric activity of the auditory cortex recorded as the N1/P2 component of the AEP in adult male rats. The effect of serotonergic agonists or antagonists was also tested. The results showed that indeed L-tryptophan was able to induce a drastic change in auditory cortex electric activity, reducing very significantly the amplitude of the N1/P2 component of the AEP. Quipazine maleate had a similar effect as L-tryptophan and the serotonergic antagonist spiperone induced an increase in the N1/P2 amplitude. These results show how an isolated nutrient is able to induce significant changes in brain auditory cortical function, through stimulation of serotonin synthesis. Besides, they add evidence about the important role of serotonergic neurotransmission modulating sensory cortical activity and that the N1/P2 component of AEPs represent a useful noninvasive indicator of brain serotonin tone.

Serotonergic receptors in the brain of in utero undernourished rats.

In this study, we report that 5-HT(1A) receptors are already present in fractions of axonal growth cones, from the normal rat fetal brain (E-17). Also, in utero undernourished (UN) rat pups at birth show a noteworthy enhancement in the B(max) of [3H]5-hydroxytryptamine (5-HT) and [3H]8-hydroxy-(2-N,N-dipropilamin)-tetralin (([3H])8-OH-DPAT), in the brainstem and cerebral cortex up to the second week after birth. Afterwards, there is a significant decrease in the binding of these ligands. [125I]Cyanopindolo binding in the cerebral cortex only showed a decrease in the same period. An elevation of brain serotonin in both regions was also present. These findings together, suggest that the mechanisms of regulation of serotonergic receptors' expression during the period studied, may not depend on the amount of neurotransmitter in the synaptic cleft, because in the early UN brain it would be expected only a lower receptor's density due to the chronic serotonin increase. On this basis, we propose that developmental activation of brain serotonin biosynthesis observed in early UN animals may disrupt the mechanism regulating the expression of 5-HT receptors during development.

Serotonin-related tryptophan in children with insulin-dependent diabetes.

In the course of the present research in school children with insulin-dependent diabetes mellitus, we observed that the free fraction of L-tryptophan, the free fraction of L-tryptophan/total L-tryptophan, and the free fraction of L-tryptophan/neutral amino acids ratios, are significantly reduced. The decrease of free fraction of L-tryptophan in plasma with a concomitant decrease of the free fraction of L-tryptophan/neutral amino acids ratio suggest a decrease in the transport of the precursor amino acid to the brain and in the serotonin synthesis rate, similar to that observed in diabetic animals. This finding may be of relevance in the pathophysiology and in the clinical picture, which could be related to an alteration of serotonin metabolism and neurotransmission in the brain and may be possibly related to neuropsychiatric disorders in diabetic school children. Thus we propose that the free fraction of L-tryptophan and the free fraction of L-tryptophan/neutral amino acids ratios may be clinically useful as indicators of brain serotonergic activity in these patients. In our laboratory, we are currently obtaining additional data on the functional role of the brain serotonergic system in humans to further support the relevance of our results.