Circadian ABCG2 Expression Influences the Brain Uptake of Donepezil across the Blood-Cerebrospinal Fluid Barrier.

Donepezil (DNPZ) is a cholinesterase inhibitor used for the management of Alzheimer's disease (AD) and is dependent on membrane transporters such as ABCG2 to actively cross brain barriers and reach its target site of action in the brain. Located in the brain ventricles, the choroid plexus (CP) forms an interface between the cerebrospinal fluid (CSF) and the bloodstream, known as the blood-CSF barrier (BCSFB). Historically, the BCSFB has received little attention as a potential pathway for drug delivery to the central nervous system (CNS). Nonetheless, this barrier is presently viewed as a dynamic transport interface that limits the traffic of molecules into and out of the CNS through the presence of membrane transporters, with parallel activity with the BBB. The localization and expression of drug transporters in brain barriers represent a huge obstacle for drug delivery to the brain and a major challenge for the development of therapeutic approaches to CNS disorders. The widespread interest in understanding how circadian clocks modulate many processes that define drug delivery in order to predict the variability in drug safety and efficacy is the next bridge to improve effective treatment. In this context, this study aims at characterizing the circadian expression of ABCG2 and DNPZ circadian transport profile using an in vitro model of the BCSFB. We found that ABCG2 displays a circadian pattern and DNPZ is transported in a circadian way across this barrier. This study will strongly impact on the capacity to modulate the BCSFB in order to control the penetration of DNPZ into the brain and improve therapeutic strategies for the treatment of AD according to the time of the day.

Circadian rhythmicity of amyloid-beta-related molecules is disrupted in the choroid plexus of a female Alzheimer's disease mouse model.

The choroid plexus (CP) is part of the blood-cerebrospinal fluid barrier (BCSFB) and was recently described as an important component of the circadian clock system. It is the principal source of cerebrospinal fluid (CSF) and responsible for the synthesis and secretion of various neuroprotective peptides including those involved in amyloid-ß (Aß) transport/degradation, contributing to Aß homeostasis. Inadequate Aß metabolic clearance and transport across the BCSFB have been associated with circadian dysfunctions in Alzheimer's disease (AD) patients. To investigate whether AD pathology influences Aß scavengers circadian expression, we collected CP at different time points from an AD mouse model (APP/PS1) (female and male animals, aged 6- and 12-months-old) and analyzed their mRNA expression by Real-time RT-PCR. Only angiotensin-converting enzyme (Ace) expression in 6-month-old female wild-type mice and transthyretin (Ttr) expression in 12-month-old female wild-type mice presented significant rhythmicity. The circadian rhythmicity of Ace and Ttr, prompt us to analyze the involvement of circadian rhythm in Aß uptake. A human CP papilloma (HIBCPP) cell line was incubated with Aß-488 and uptake was evaluated at different time points using flow cytometry. Aß uptake displayed circadian rhythmicity. Our results suggest that AD might affect Aß scavengers rhythmicity and that Aß clearance is a rhythmic process possibly regulated by the rhythmic expression of Aß scavengers.

The Role of Biological Rhythms in New Drug Formulations to Cross the Brain Barriers.

For brain protection, the blood-brain barrier and blood-cerebrospinal fluid barrier limit the traffic of molecules between blood and brain tissue and between blood and cerebrospinal fluid, respectively. Besides their protective function, brain barriers also limit the passage of therapeutic drugs to the brain, which constitutes a great challenge for the development of therapeutic strategies for brain disorders. This problem has led to the emergence of novel strategies to treat neurological disorders, like the development of nanoformulations to deliver therapeutic agents to the brain. Recently, functional molecular clocks have been identified in the blood-brain barrier and in the blood-cerebrospinal fluid barrier. In fact, circadian rhythms in physiological functions related to drug disposition were also described in brain barriers. This opens the possibility for chronobiological approaches that aim to use time to improve drug efficacy and safety. The conjugation of nanoformulations with chronobiology for neurological disorders is still unexplored. Facing this, here, we reviewed the circadian rhythms in brain barriers, the nanoformulations studied to deliver drugs to the brain, and the nanoformulations with the potential to be conjugated with a chronobiological approach to therapeutic strategies for the brain.

The Daily Expression of ABCC4 at the BCSFB Affects the Transport of Its Substrate Methotrexate.

The choroid plexuses (CPs), located in the brain ventricles, form an interface between the blood and the cerebrospinal fluid named the blood-cerebrospinal barrier, which, by the presence of tight junctions, detoxification enzymes, and membrane transporters, limits the traffic of molecules into the central nervous system. It has already been shown that sex hormones regulate several CP functions, including the oscillations of its clock genes. However, it is less explored how the circadian rhythm regulates CP functions. This study aimed to evaluate the impact of sex hormones and circadian rhythms on the function of CP membrane transporters. The 24 h transcription profiles of the membrane transporters rAbca1, rAbcb1, rAbcc1, rAbcc4, rAbcg2, rAbcg4, and rOat3 were characterized in the CPs of intact male, intact female, sham-operated female, and gonadectomized rats. We found that rAbcc1 is expressed in a circadian way in the CPs of intact male rats, rAbcg2 in the CPs of intact female rats, and both rAbcc4 and rOat3 mRNA levels were expressed in a circadian way in the CPs of intact male and female rats. Next, using an in vitro model of the human blood-cerebrospinal fluid barrier, we also found that methotrexate (MTX) is transported in a circadian way across this barrier. The circadian pattern of Abcc4 found in the human CP epithelial papilloma cells might be partially responsible for MTX circadian transport across the basal membrane of CP epithelial cells.

Adenosine inhibits human astrocyte proliferation independently of adenosine receptor activation.

Brain adenosine concentrations can reach micromolar concentrations in stressful situations such as stroke, neurodegenerative diseases or hypoxic regions of brain tumours. Adenosine can act by receptor-independent mechanism by reversing the reaction catalysed by S-adenosylhomocysteine (SAH) hydrolase, leading to SAH accumulation and inhibition of S-adenosylmethionine (SAM)-dependent methyltransferases. Astrocytes are essential in maintaining brain homeostasis but their pathological activation and uncontrolled proliferation plays a role in neurodegeneration and glioma. Adenosine can affect cell proliferation, but the effect of increased adenosine concentration on proliferation of astrocytes is not clarified and was addressed in present work. Human astrocytes (HA) were treated for 3 days with test drugs. Cell proliferation/viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay and by cell counting. Cell death was evaluated by assessing lactate dehydrogenase release and by western blot analysis of αII-Spectrin cleavage. 30 µM-Adenosine caused a 40% ± 3% (p < .05, n = 5) reduction in cell proliferation/viability, an effect reversed by 2U/ml-adenosine deaminase, but unchanged in the presence of antagonists of any of the adenosine receptors. Adenosine alone did not induce cell death. 100 µM-Homocysteine alone caused 16% ± 3% (p < .05) decrease in HA proliferation. Combined action of adenosine and homocysteine decreased HA proliferation by 76% ± 4%, an effect higher (p < .05) than the sum of the effects of adenosine and homocysteine alone (56% ± 5%). The inhibitory effect of adenosine on HA proliferation/viability was mimicked by two adenosine kinase inhibitors and attenuated in the presence of folate (100 µM) or SAM (50-100 µM). The results suggest that adenosine reduces HA proliferation by a receptor-independent mechanism probably involving reversal of SAH hydrolase-catalysed reaction.

Age, Sex Hormones, and Circadian Rhythm Regulate the Expression of Amyloid-Beta Scavengers at the Choroid Plexus.

Accumulation of amyloid-beta (Aß) in the brain is thought to derive from the impairment of Aß clearance mechanisms rather than from its overproduction, which consequently contributes to the development of Alzheimer's disease. The choroid plexus epithelial cells constitute an important clearance route for Aß, either by facilitating its transport from the cerebrospinal fluid to the blood, or by synthesizing and secreting various proteins involved in Aß degradation. Impaired choroid plexus synthesis, secretion, and transport of these Aß-metabolizing enzymes have been therefore associated with the disruption of Aß homeostasis and amyloid load. Factors such as aging, female gender, and circadian rhythm disturbances are related to the decline of choroid plexus functions that may be involved in the modulation of Aß-clearance mechanisms. In this study, we investigated the impact of age, sex hormones, and circadian rhythm on the expression of Aß scavengers such as apolipoprotein J, gelsolin, and transthyretin at the rat choroid plexus. Our results demonstrated that mRNA expression and both intracellular and secreted protein levels of the studied Aß scavengers are age-, sex-, and circadian-dependent. These data suggest that the Aß-degradation and clearance pathways at the choroid plexus, mediated by the presence of Aß scavengers, might be compromised as a consequence of aging and circadian disturbances. These are important findings that enhance the understanding of Aß-clearance-regulating mechanisms at the blood-cerebrospinal fluid barrier.

The choroid plexus as a sex hormone target: Functional implications.

The choroid plexuses (CPs) are highly vascularized branched structures that protrude into the ventricles of the brain, and form a unique interface between the blood and the cerebrospinal fluid (CSF). In recent years, novel functions have been attributed to this tissue such as in immune and chemical surveillance of the central nervous system, brain development, adult neurogenesis and circadian rhythm regulation. Sex hormones (SH) are widely recognized as modulators in several neurodegenerative diseases, and there is evidence that estrogens and androgens regulate several fundamental biological functions in the CPs. Therefore, SH are likely to affect the composition of the CSF impacting on brain homeostasis. This review will look at implications of the CPs' sex-related specificities.

Choroid plexus is an additional source of melatonin in the brain.

The cerebrospinal fluid melatonin is released from the pineal gland, directly into the third ventricle, or produced de novo in the brain from extrapineal melatonin sources leading to a melatonin concentration gradient in the cerebrospinal fluid. Despite the interest on this topic, the brain areas capable of producing melatonin are not yet clear. Bearing this in mind, we hypothesized that the choroid plexus (CP) could be one of these melatonin sources. We analyzed and confirmed the presence of the four enzymes required for melatonin synthesis in rat CP and demonstrated that arylalkylamine N-acetyltransferase shows a circadian expression in female and male rat CP. Specifically, this enzyme colocalizes with mitochondria in rat CP epithelial cells, an organelle known to be involved in melatonin function and synthesis. Then, we demonstrated that melatonin is synthesized by porcine CP explants, although without a circadian pattern. In conclusion, our data show that the CP is a local source of melatonin to the central nervous system, probably contributing to its high levels in the cerebrospinal fluid. We believe that in the CP, melatonin might be regulated by its endogenous clock machinery and by the hormonal background.

Histopathological and in vivo evidence of regucalcin as a protective molecule in mammary gland carcinogenesis.

Regucalcin (RGN) is a calcium-binding protein, which has been shown to be underexpressed in cancer cases. This study aimed to determine the association of RGN expression with clinicopathological parameters of human breast cancer. In addition, the role of RGN in malignancy of mammary gland using transgenic rats overexpressing the protein (Tg-RGN) was investigated. Wild-type (Wt) and Tg-RGN rats were treated with 7,12-dimethylbenz[α]anthracene (DMBA). Carcinogen-induced tumors were histologically classified and the Ki67 proliferation index was estimated. Immunohistochemistry analysis showed that RGN immunoreactivity was negatively correlated with the histological grade of breast infiltrating ductal carcinoma suggesting that progression of breast cancer is associated with loss of RGN. Tg-RGN rats displayed lower incidence of carcinogen-induced mammary gland tumors, as well as lower incidence of invasive forms. Moreover, higher proliferation was observed in non-invasive tumors of Wt animals comparatively with Tg-RGN. Overexpression of RGN was associated with diminished expression of cell-cycle inhibitors and increased expression of apoptosis inducers. Augmented activity of apoptosis effector caspase-3 was found in the mammary gland of Tg-RGN. RGN overexpression protected from carcinogen-induced mammary gland tumor development and was linked with reduced proliferation and increased apoptosis. These findings indicated the protective role of RGN in the carcinogenesis of mammary gland.

Evidence That a Peptide-Drug/p53 Gene Complex Promotes Cognate Gene Expression and Inhibits the Viability of Glioblastoma Cells.

Glioblastoma multiform (GBM) is considered the deadliest brain cancer. Conventional therapies are followed by poor patient survival outcomes, so novel and more efficacious therapeutic strategies are imperative to tackle this scourge. Gene therapy has emerged as an exciting and innovative tool in cancer therapy. Its combination with chemotherapy has significantly improved therapeutic outcomes. In line with this, our team has developed temozolomide-transferrin (Tf) peptide (WRAP5)/p53 gene nanometric complexes that were revealed to be biocompatible with non-cancerous cells and in a zebrafish model and were able to efficiently target and internalize into SNB19 and U373 glioma cell lines. The transfection of these cells, mediated by the formulated peptide-drug/gene complexes, resulted in p53 expression. The combined action of the anticancer drug with p53 supplementation in cancer cells enhances cytotoxicity, which was correlated to apoptosis activation through quantification of caspase-3 activity. In addition, increased caspase-9 levels revealed that the intrinsic or mitochondrial pathway of apoptosis was implicated. This assumption was further evidenced by the presence, in glioma cells, of Bax protein overexpression-a core regulator of this apoptotic pathway. Our findings demonstrated the great potential of peptide TMZ/p53 co-delivery complexes for cellular transfection, p53 expression, and apoptosis induction, holding promising therapeutic value toward glioblastoma.

Six transmembrane epithelial antigen of the prostate 1 is down-regulated by sex hormones in prostate cells.

BACKGROUND: STEAP1 is over-expressed in several types of tumors, especially prostate cancer, where it is localized in the plasma membrane of epithelial cells, at cell-cell junctions. Its role in prostate carcinogenesis and its regulation in prostate cells remain unknown. Therefore, we propose to study the effect of sex hormones in the regulation of STEAP1 expression in prostate cells in vitro and in vivo. METHODS: LNCaP prostate cells were incubated with fetal bovine serum (FBS), charcoal-stripped FBS (CS-FBS), 5α-dihydrotestosterone (DHT), and 17ß-estradiol (E2 ) for different periods of stimulation. In addition, adult male Wistar rats were castrated and treated with DHT and E2 . The levels of STEAP1 in response to treatments were analyzed by real-time PCR, Western blot, and immunohistochemistry. RESULTS: The treatment of LNCaP cells with DHT or E2 induces a down-regulation of STEAP1 expression, while incubation with CS-FBS has the opposite effect. Experiments using inhibitors of androgen and estrogen receptor (AR and ER) showed that down-regulation of STEAP1 is AR-dependent, but ER-independent. However, the mediation of six transmembrane epithelial antigen of the prostate 1 (STEAP1) expression by AR seems to be dependent of de novo protein synthesis. In vivo studies showed that castrated rats express higher levels of STEAP1 protein when compared to intact rats, an effect reversed by DHT or E2 replacement. CONCLUSIONS: STEAP1 is down-regulated by DHT and E2 in LNCaP cells and in rat prostate.

Bitter taste signaling in cancer.

Pharmacoresistance of cancer cells to many drugs used in chemotherapy remains a major challenge for the treatment of cancer. Multidrug resistance transporters, especially ATP-binding cassette (ABC) transporters, are a major cause of cancer drug resistance since they translocate a broad range of drug compounds across the cell membrane, extruding them out of the cells. The regulation of ABC transporters by bitter taste receptors (TAS2Rs), which might be activated by specific bitter tasting compounds, was described in several types of cells/organs, becoming a potential target for cancer therapy. TAS2Rs expression has been reported in many organs and several types of cancer, like breast, ovarian, prostate, and colorectal cancers, where their activation was shown to be involved in various biological actions (cell survival, apoptosis, molecular transport, among others). Moreover, many TAS2Rs' ligands, such as flavonoids and alkaloids, with well-recognized beneficial properties, including several anticancer effects, have been reported as potential adjuvants in cancer therapies. In this review, we discuss the potential therapeutic role of TAS2Rs and bitter tasting compounds in different types of cancer as a possible way to circumvent chemoresistance.

The impact of biological clock and sex hormones on the risk of disease.

Molecular clocks are responsible for defining 24-h cycles of behaviour and physiology that are called circadian rhythms. Several structures and tissues are responsible for generating these circadian rhythms and are named circadian clocks. The suprachiasmatic nucleus of the hypothalamus is believed to be the master circadian clock receiving light input via the optic nerve and aligning internal rhythms with environmental cues. Studies using both in vivo and in vitro methodologies have reported the relationship between the molecular clock and sex hormones. The circadian system is directly responsible for controlling the synthesis of sex hormones and this synthesis varies according to the time of day and phase of the estrous cycle. Sex hormones also directly interact with the circadian system to regulate circadian gene expression, adjust biological processes, and even adjust their own synthesis. Several diseases have been linked with alterations in either the sex hormone background or the molecular clock. So, in this chapter we aim to summarize the current understanding of the relationship between the circadian system and sex hormones and their combined role in the onset of several related diseases.

Neuroprotective and neuroregenerative properties of metallothioneins.

Metallothioneins (MTs) are low-molecular weight cysteine- and metal-rich proteins with unquestionable metal binding capacity, antioxidant and anti-inflammatory properties, and a clear involvement in diverse physiological actions as inhibition of proapoptotic mechanisms, enhancement of cell survival, and tissue regeneration. Concurrent with this wide array of functions, MT-1/2 have been implicated in neuroprotection and neuroregeneration. The zinc binding capacity and antioxidant properties of MTs may account for most of their physiological features in the brain. However, some receptor-mediated actions of MT-1/2 have also been reported recently, a subject to be fully elucidated. This review analyses and updates the current knowledge on the actions of MTs related to neuroprotection and neuroregeneration in an effort to distinguish receptor-mediated actions of MTs from those arising from its zinc binding capacity and its antioxidant properties.

The brain as a source and a target of prolactin in mammals.

Prolactin is a polypeptide hormone associated with an extensive variety of biological functions. Among the roles of prolactin in vertebrates, some were preserved throughout evolution. This is the case of its function in the brain, where prolactin receptors, are expressed in different structures of the central nervous system. In the brain, prolactin actions are principally associated with reproduction and parental behavior, and involves the modulation of adult neurogenesis, neuroprotection, and neuroplasticity, especially during pregnancy, thereby preparing the brain to parenthood. Prolactin is mainly produced by specialized cells in the anterior pituitary gland. However, during vertebrate evolution many other extrapituitary tissues do also produce prolactin, like the immune system, endothelial cells, reproductive structures and in several regions of the brain. This review summarizes the relevance of prolactin for brain function, the sources of prolactin in the central nervous system, as well as its local production and secretion. A highlight on the impact of prolactin in human neurological diseases is also provided.

The druggability of bitter taste receptors for the treatment of neurodegenerative disorders.

The delivery of therapeutic drugs to the brain remains a major pharmacology challenge. A complex system of chemical surveillance to protect the brain from endogenous and exogenous toxicants at brain barriers hinders the uptake of many compounds with significant in vitro and ex vivo therapeutic properties. Despite the advances in the field in recent years, the components of this system are not completely understood. Recently, a large group of chemo-sensing receptors, have been identified in the blood-cerebrospinal fluid barrier. Among these chemo-sensing receptors, bitter taste receptors (TAS2R) hold promise as potential drug targets, as many TAS2R bind compounds with recognized neuroprotective activity (quercetin, resveratrol, among others). Whether activation of TAS2R by their ligands contributes to their diverse biological actions described in other cells and tissues is still debatable. In this review, we discuss the potential role of TAS2R gene family as the mediators of the biological activity of their ligands for the treatment of central nervous system disorders and discuss their potential to counteract drug resistance by improving drug delivery to the brain.

The role of circadian rhythm in choroid plexus functions.

For several years, a great effort has been devoted to understand how circadian oscillations in physiological processes are determined by the circadian clock system. This system is composed by the master clock at the suprachiasmatic nucleus which sets the pace and tunes peripheral clocks in several organs. It was recently demonstrated that the choroid plexus epithelial cells that compose the blood-cerebrospinal fluid barrier hold a circadian clock which might control their multiple functions with implications for the maintenance of brain homeostasis. However, the choroid plexus activities regulated by its inner clock are still largely unknown. In this review, we propose that several choroid plexus functions might be regulated by the circadian clock, alike in other tissues. We provide evidences that the timing of cerebrospinal fluid secretion, clearance of amyloid-beta peptides and xenobiotics, and the barrier function of the blood-cerebrospinal fluid barrier are regulated by the circadian clock. These data, highlight that the circadian regulation of the blood-cerebrospinal fluid barrier must be taken into consideration for enhancing drug delivery to central nervous system disorders.

Adenosine Inhibits Cell Proliferation Differently in Human Astrocytes and in Glioblastoma Cell Lines.

Glioblastoma (GBM) is the most common brain primary tumour. Hypoxic regions in GBM are associated to tumour growth. Adenosine accumulates in hypoxic regions and can affect cell proliferation and survival. However, how proliferating GBM cells respond/adapt to increased adenosine levels compared to human astrocytes (HA) is not clarified and was addressed in the present work. GBM cell lines and HA were treated for 3 days with test drugs. Thirty Adenosine (30 µM) caused a 43% ± 5% (P < 0.05) reduction of cell proliferation/viability in HA, through an adenosine receptor-independent mechanism, but had no effect in GBM cell lines U87MG, U373MG and SNB19. Contrastingly, inhibition of adenosine phosphorylation (using the adenosine kinase (ADK) inhibitor 5-iodotubercidin (ITU) (25 µM)), produced a strong and similar decrease on cell proliferation in both HA and GBM cells. The effect of adenosine on HA proliferation/viability was potentiated by 100 µM-homocysteine. Combined application of 30 µM-adenosine and 100 µM-homocysteine reduced the cell proliferation/viability in all three GBM cell lines, but this reduction was much lower than that observed in HA. Adenosine alone did not induce cell death, assessed by lactate dehydrogenase (LDH) release, both in HA and GBM cells, but potentiated the cytotoxic effect of homocysteine in HA and in U87MG and U373MG cells. Results show a strong attenuation of adenosine anti-proliferative effect in GBM cells compared to HA, probably resulting from increased adenosine elimination by ADK, suggesting a proliferative-prone adaptation of tumour cells to increased adenosine levels.

Promoter Demethylation Upregulates STEAP1 Gene Expression in Human Prostate Cancer: In Vitro and In Silico Analysis.

The Six Transmembrane Epithelial Antigen of the Prostate (STEAP1) is an oncogene overexpressed in several human tumors, particularly in prostate cancer (PCa). However, the mechanisms involved in its overexpression remain unknown. It is well known that epigenetic modifications may result in abnormal gene expression patterns, contributing to tumor initiation and progression. Therefore, this study aimed to analyze the methylation pattern of the STEAP1 gene in PCa versus non-neoplastic cells. Bisulfite amplicon sequencing of the CpG island at the STEAP1 gene promoter showed a higher methylation level in non-neoplastic PNT1A prostate cells than in human PCa samples. Bioinformatic analysis of the GEO datasets also showed the STEAP1 gene promoter as being demethylated in human PCa, and a negative association with STEAP1 mRNA expression was observed. These results are supported by the treatment of non-neoplastic PNT1A cells with DNMT and HDAC inhibitors, which induced a significant increase in STEAP1 mRNA expression. In addition, the involvement of HDAC in the regulation of STEAP1 mRNA expression was corroborated by a negative association between STEAP1 mRNA expression and HDAC4,5,7 and 9 in human PCa. In conclusion, our work indicates that STEAP1 overexpression in PCa can be driven by the hypomethylation of STEAP1 gene promoter.

Cadaverine and Spermine Elicit Ca2+ Uptake in Human CP Cells via a Trace Amine-Associated Receptor 1 Dependent Pathway.

The choroid plexus (CP) constitutes a barrier between the blood and the cerebrospinal fluid (CSF) which regulates the exchange of substances between these two fluids through mechanisms that are not completely understood. Polyamines as spermine, spermidine and putrescine are produced by all cells and are present in the CSF. Interestingly, their levels are altered in some neuronal disorders as Alzheimer's and Parkinson's diseases, thus increasing the interest in their signalling in the central nervous system (CNS). Cadaverine, on the other hand, is synthetized by the intestinal microbiome, suggesting that the presence of this bacterial metabolite in the CSF requires that it is up taken to the CNS across brain barriers. We knew that polyamines are detected by the olfactory signalling cascade operating at the CP, but the receptor involved had not been identified. The zebrafish TAAR13c was the only receptor known to bind a polyamine-cadaverine. Thus, we searched for a human receptor with homology to TAAR13c and found that some human TAARs including TAAR1 showed great homology. Then, we confirmed the expression of TAAR1 mRNA and protein in a human cell line of the CP, and in human CP samples. Calcium imaging assays after TAAR1 knockdown in these cells with a specific siRNA against TAAR1 showed a consistent reduction in the responses of these cells to cadaverine and spermidine, but not to spermine, suggesting that TAAR1 is activated by cadaverine and spermidine, but not spermine.