Marta Kot

The relative contribution of human cytochrome P450 isoforms to the four caffeine oxidation pathways : An in vitro comparative study with cDNA-expressed P450s including CYP2C isoforms

The aim of the present study was to estimate the relative contribution of cytochrome P450 isoforms (P450s), including P450s of the CYP2C subfamily, to the metabolism of caffeine in human liver. The experiments were carried out in vitro using cDNA-expressed P450s, liver microsomes and specific P450 inhibitors. The obtained results show that (1) apart from the 3-N-demethylation of caffeine - a CYP1A2 marker reaction and the main oxidation pathway of caffeine in man - 1-N-demethylation is also specifically catalyzed by CYP1A2 (not reported previously); (2) 7-N-demethylation is catalyzed non-specifically, mainly by CYP1A2 and, to a smaller extent, by CYP2C8/9 and CYP3A4 (and not by CYP2E1, as suggested previously); (3) C-8-hydroxylation preferentially involves CYP1A2 and CYP3A4 and, to a smaller degree, CYP2C8/9 and CYP2E1 (and not only CYP3A, as suggested previously) at a concentration of 100 microM corresponding to the maximum therapeutic concentration in humans. At a higher caffeine concentration, the contribution of CYP1A2 to this reaction decreases in favour of CYP2C8/9. The obtained data show for the first time the contribution of CYP2C isoforms to the metabolism of caffeine in human liver and suggest that apart from 3-N-demethylation, 1-N-demethylation may also be used for testing CYP1A2 activity. Moreover, they indicate that the C-8-hydroxylation is not exclusively catalyzed by CYP3A4.

Cytochrome P450 is regulated by noradrenergic and serotonergic systems

The aim of the present study was to ascertain whether the noradrenergic or serotonergic systems may affect the expression of liver cytochrome P450 (CYP). Rats were injected intraperitoneally with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4, a noradrenergic neurotoxin) or p-chloroamphetamine (PCA, a serotonergic neurotoxin) or p-chlorophenylalanine (PCPA, an inhibitor of serotonin synthesis). One week after neurotoxin injection the levels of neurotransmitters (noradrenaline, dopamine, serotonin) and their metabolites were measured in brain structures, and the activity and protein levels of CYP isoforms were measured in the liver. In the brain, DSP-4 or PCA and PCPA selectively decreased noradrenaline or serotonin levels, respectively. In the liver, the applied neurotoxins evoked decrease in the activity of CYP2B, CYP2C11 and CYP3A (DSP-4, PCA, PCPA) and increase in the activity of CYP1A (PCA, PCPA), while the activity of CYP2A, CYP2C6 and CYP2D was not affected by the applied neurotoxins. Since the affected isoforms (CYP1A/2B/2C11/3A) are regulated by endogenous hormones (growth hormone, corticosterone, thyroid hormones), the latter being under control of the central nervous system, it is postulated that the brain noradrenergic and serotonergic systems are involved in the physiological regulation of liver CYP expression.

Disposition of 1,2,3,4,-tetrahydroisoquinoline in the brain of male Wistar and Dark Agouti rats

Direct evidence for accumulation of 1,2,3,4-tetrahydroisoquinoline (TIQ), an endo- and exogenous substance suspected of producing Parkinsonism in humans, has not yet been shown. This study aimed to examine TIQ disposition in the whole rat brain and in the striatum and substantia nigra (SN). TIQ was administered to male Wistar and Dark Agouti rats (20, 40 and 100 mg/kg i.p.) alone or jointly with specific CYP2D inhibitor quinine (20, 40, 80 mg/kg i.p.), acutely or chronically. TIQ concentration in brain of both strains was several-fold higher than in plasma. The level of its metabolite, 4-OH-TIQ, was very low in the brain and plasma of TIQ-treated Wistar while in those receiving additionally quinine or in Dark Agouti rats, 4-OH-TIQ was absent or negligible. Inhibition of CYP2D catalyzing TIQ 4-hydroxylation in the liver had no influence on TIQ accumulation in the brain. Exogenous TIQ was actively transported from periphery into the brain by the organic cation transporter system, mainly OCT3, and quickly eliminated from it by P-glycoprotein. TIQ accumulation after chronic injection to Wistar rats was short-lasting and limited to SN. High concentration of TIQ in SN induces while in the liver inhibits the nigral and hepatic activity CYP2D, respectively.

The cytochrome P450 2D-mediated formation of serotonin from 5-methoxytryptamine in the brain in vivo: a microdialysis study

The cytochrome P450 2D (CYP2D) mediates synthesis of serotonin from 5‐methoxytryptamine (5‐MT), shown in vitro for cDNA‐expressed CYP2D‐isoforms and liver and brain microsomes. We aimed to demonstrate this synthesis in the brain in vivo. We measured serotonin tissue content in brain regions after 5‐MT injection into the raphe nuclei (Model‐A), and its extracellular concentration in rat frontal cortex and striatum using an in vivo microdialysis (Model‐B) in male Wistar rats. Naïve rats served as control animals. 5‐MT injection into the raphe nuclei of PCPA‐(tryptophan hydroxylase inhibitor)‐pretreated rats increased the tissue concentration of serotonin (from 40 to 90% of the control value, respectively, in the striatum), while the CYP2D inhibitor quinine diminished serotonin level in some brain structures of those animals (Model‐A). 5‐MT given locally through a microdialysis probe markedly increased extracellular serotonin concentration in the frontal cortex and striatum (to 800 and 1000% of the basal level, respectively) and changed dopamine concentration (Model‐B). Quinine alone had no effect on serotonin concentration; however, given jointly with 5‐MT, it prevented the 5‐MT‐induced increase in cortical serotonin in naïve rats and in striatal serotonin in PCPA‐treated animals. These results indicate that the CYP2D‐catalyzed alternative pathway of serotonin synthesis from 5‐MT is relevant in the brain in vivo, and set a new target for the action of psychotropics.

The role of the dorsal noradrenergic pathway of the brain (locus coeruleus) in the regulation of liver cytochrome P450 activity.

Our previous study conducted after intracerebroventricular DSP-4 injection showed an important stimulating role of a brain noradrenergic system in the neuroendocrine regulation of liver cytochrome P450 (CYP) expression. The aim of the present research was to study involvement of the dorsal noradrenergic pathway of the brain (originating from the locus coeruleus) in the expression of liver cytochrome P450. The experiment was carried out on male Wistar rats. Local injection of 6-hydroxydopamine to the locus coeruleus selectively decreased noradrenaline level in the brain (e.g. in the hypothalamus). The serum concentration of the growth hormone rose, while that of the thyroid hormones or corticosterone remained unchanged. A comparative study into cytochrome P450 isoform activity revealed significant increases in the activity of liver CYP2C11 and CYP3A after administration of 6-hydroxydopamine. The observed increase in the activity of CYP2C11 positively correlated with that in CYP protein level, while the enhanced activity of CYP3A was not accompanied with a simultaneous change in the enzyme protein. A 5-day-injection of noradrenaline into the lateral ventricles produced opposite effects on the CYP isoforms. It is concluded that damage to or activation of the dorsal noradrenergic innervation of the periventricular nucleus of the hypothalamus containing somatostatin (a growth hormone release-inhibiting factor) may be responsible for the changes observed in the activity of isoforms CYP2C11 and CYP3A that are regulated by the growth hormone. The obtained results indicate that the dorsal noradrenergic pathway plays an inhibitory (but not a crucial) role in the neuroendocrine regulation of cytochrome P450.

Simultaneous alterations of brain and plasma serotonin concentrations and liver cytochrome P450 in rats fed on a tryptophan-free diet.

Our previous study suggested involvement of the brain serotonergic system in the regulation of liver cytochrome P450 (CYP). The aim of the present study was to demonstrate simultaneous responsiveness of liver CYP and the peripheral and brain serotonergic systems to a tryptophan deficient diet during three days and one or three weeks of ingestion. The concentrations of serotonin, noradrenaline, dopamine and their metabolites were measured in blood plasma, the hypothalamus and brain stem of male rats. The enzyme activity and protein levels in the liver were determined for isoforms CYP1A, CYP2A, CYP2B, CYP2C6, CYP2C11, CYP2D and CYP3A. A three-day tryptophan-free diet increased serotonin content in the hypothalamus (but not in the brain stem or plasma). After one week, the level of serotonin was not changed in the brain, but was markedly increased in the plasma. A three week tryptophan restriction significantly reduced the concentration of serotonin in the brain and plasma. Changes in CYP2C6 and CYP2C11 (an increase and a decrease, respectively) were maintained throughout the experiment, while those found in other CYP isoforms varied, which usually resulted in a gradual increase in the enzyme activity within three weeks. The observed alterations in liver CYPs suggest involvement of both central and peripheral serotonin in the regulation of liver CYP expression whose mechanism is discussed. In conclusion, a deficit in tryptophan in the diet may be responsible for very serious food-cytochrome P450 and food-drug metabolism interactions. Interactions of this type may also refer to drugs acting via serotonergic system.

Disruption of glucocorticoid receptors in the noradrenergic system leads to BDNF up-regulation and altered serotonergic transmission associated with a depressive-like phenotype in female GRDBHCre mice

Recently, we have demonstrated that conditional inactivation of glucocorticoid receptors (GRs) in the noradrenergic system, may evoke depressive-like behavior in female but not male mutant mice (GR(DBHCre) mice). The aim of the current study was to dissect how selective ablation of glucocorticoid signaling in the noradrenergic system influences the previously reported depressive-like phenotype and whether it might be linked to neurotrophic alterations or secondary changes in the serotonergic system. We demonstrated that selective depletion of GRs enhances brain derived neurotrophic factor (BDNF) expression in female but not male GR(DBHCre) mice on both the mRNA and protein levels. The possible impact of the mutation on brain noradrenergic and serotonergic systems was addressed by investigating the tissue neurotransmitter levels under basal conditions and after acute restraint stress. The findings indicated a stress-provoked differential response in tissue noradrenaline content in the GR(DBHCre) female but not male mutant mice. An analogous gender-specific effect was identified in the diminished content of 5-hydroxyindoleacetic acid, the main metabolite of serotonin, in the prefrontal cortex, which suggests down-regulation of this monoamine system in female GR(DBHCre) mice. The lack of GR also resulted in an up-regulation of alpha2-adrenergic receptor (α2-AR) density in the female but not male mutants in the locus coeruleus. We have also confirmed the utility of the investigated model in pharmacological studies, which demonstrates that the depressive-like phenotype of GR(DBHCre) female mice can be reversed by antidepressant treatment with desipramine or fluoxetine, with the latter drug evoking more pronounced effects. Overall, our study validates the use of female GR(DBHCre) mice as an interesting and novel genetic tool for the investigation of the cross-connected mechanisms of depression that is not only based on behavioral phenotypes.

Effect of diethyldithiocarbamate (DDC) and ticlopidine on CYP1A2 activity and caffeine metabolism: an in vitro comparative study with human cDNA-expressed CYP1A2 and liver microsomes

The aim of the present study was to test the effect of diethyldithiocarbamate (DDC), which is regarded as a cytochrome P450 (CYP) CYP2A6 and CYP2E1 inhibitor, and ticlopidine, an efficient CYP2B6, CYP2C19 and CYP2D6 inhibitor, on the activity of human CYP1A2 and the metabolism of caffeine (1-N-, 3-N- and 7-N-demethylation, and C-8-hydroxylation). The experiment was carried out in vitro using human cDNA-expressed CYP1A2 (Supersomes) and human pooled liver microsomes. The effects of DDC and ticlopidine were compared to those of furafylline (a strong CYP1A2 inhibitor). A comparative in vitro study provides clear evidence that ticlopidine and DDC, applied at concentrations that inhibit the above-mentioned CYP isoforms, potently (as compared to furafylline) inhibit human CYP1A2 and caffeine metabolism, in particular 1-N- and 3-N-demethylation.

The Effect of Chronic Treatment with Lurasidone on Rat Liver Cytochrome P450 Expression and Activity in the Chronic Mild Stress Model of Depression

Recent studies indicated an important role of the monoaminergic nervous systems (dopaminergic, noradrenergic, and serotonergic systems) and stress in the regulation of cytochrome P450 (CYP) expression and activity in the liver. The aim of our present research was to determine the effect of the novel atypical neuroleptic drug with antidepressant properties lurasidone, on the expression (mRNA and protein level) and activity of liver CYP isoforms involved in the metabolism of drugs and endogenous steroids, in the chronic mild stress (CMS) model of depression. Male Wistar rats were subjected to CMS for 7 weeks. Lurasidone (3 mg/kg per os per day) was administered to nonstressed or stressed animals for 5 weeks (weeks 3–7 of CMS). It has been found that 1) CMS moderately affects CYP (CYP2B, CYP2C11, and CYP3A), and its effects are different from those observed after other kinds of psychologic stress, such as repeated restraint stress or early-life maternal deprivation; 2) chronic lurasidone influences the expression and/or activity of CYP2B, CYP2C11, and CYP3A isoforms; and 3) CMS modifies the action of lurasidone on CYP expression and function, leading to different effects of the neuroleptic in nonstressed and stressed rats. Based on the obtained results, it can be suggested that the metabolism of endogenous substrates (e.g., steroids) and drugs, catalyzed by the isoforms CYP2B, CYP2C11, or CYP3A, may proceed at a different rate in the two groups of animals (nonstressed and stressed) in the rat CMS model.

Gender-dependent activity of CYP3A is indirectly modified by GR in the noradrenergic system

BACKGROUND The noradrenergic system is involved in the regulation of cytochrome P450 activity in the liver. We investigated the effect of selective ablation of the glucocorticoid receptor in the noradrenergic systemon the activity of the CYP3A isoform in mouse liver. METHODS The activity of CYP3A was studied by measuring the rate of testosterone 6β-hydroxylation in liver microsomes. RESULTS In mutant mice, the activity of CYP3A was reduced to 68% of the control in females, but remained unchanged in males. Chronic restraint stress increased CYP3A activity in mutant mice only. CONCLUSIONS The total basal activity of mouse CYP3A may be indirectly modulated by the glucocorticoid receptor in the noradrenergic system during a pubertal period.