Oligomeric α-Synuclein induces skin degeneration in reconstructed human epidermis.

Aged and photoaged skin exhibit fine wrinkles that are signs of epidermal inflammation and degeneration. It has been shown that healthy elderly skin expresses amyloidogenic proteins, including α-Synuclein, which are known to oligomerize and trigger inflammation and neurodegeneration. However, little is known about their putative role in skin physiology and sensitivity. To unravel this possible role, we investigated the impact of oligomeric α-Synuclein (Oα-Syn) in 2D and 3D keratinocyte human models. Exogenous Oα-Syn caused degeneration of reconstructed human epidermis (RHE) by diminishing proliferation and thickness of the stratum basale. Oα-Syn also increased NF-kB nuclear translocation in keratinocytes and triggered inflammation in the RHE, by increasing expression of interleukin-1ß and tumor necrosis factor-alpha, and the release of tumor necrosis factor-alpha in a time-dependent manner. Dexamethasone and an IL-1ß inhibitor partially diminished RHE degeneration caused by Oα-Syn. These findings suggest that Oα-Syn induces epidermal inflammation and decreases keratinocyte proliferation, and therefore might contribute to epidermal degeneration observed in human skin aging.

Short term changes in the proteome of human cerebral organoids induced by 5-MeO-DMT.

Dimethyltryptamines are entheogenic serotonin-like molecules present in traditional Amerindian medicine recently associated with cognitive gains, antidepressant effects, and changes in brain areas related to attention. Legal restrictions and the lack of adequate experimental models have limited the understanding of how such substances impact human brain metabolism. Here we used shotgun mass spectrometry to explore proteomic differences induced by 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) on human cerebral organoids. Out of the 6,728 identified proteins, 934 were found differentially expressed in 5-MeO-DMT-treated cerebral organoids. In silico analysis reinforced previously reported anti-inflammatory actions of 5-MeO-DMT and revealed modulatory effects on proteins associated with long-term potentiation, the formation of dendritic spines, including those involved in cellular protrusion formation, microtubule dynamics, and cytoskeletal reorganization. Our data offer the first insight about molecular alterations caused by 5-MeO-DMT in human cerebral organoids.

Harmine stimulates proliferation of human neural progenitors.

Harmine is the ß-carboline alkaloid with the highest concentration in the psychotropic plant decoction Ayahuasca. In rodents, classical antidepressants reverse the symptoms of depression by stimulating neuronal proliferation. It has been shown that Ayahuasca presents antidepressant effects in patients with depressive disorder. In the present study, we investigated the effects of harmine in cell cultures containing human neural progenitor cells (hNPCs, 97% nestin-positive) derived from pluripotent stem cells. After 4 days of treatment, the pool of proliferating hNPCs increased by 71.5%. Harmine has been reported as a potent inhibitor of the dual specificity tyrosine-phosphorylation-regulated kinase (DYRK1A), which regulates cell proliferation and brain development. We tested the effect of analogs of harmine, an inhibitor of DYRK1A (INDY), and an irreversible selective inhibitor of monoamine oxidase (MAO) but not DYRK1A (pargyline). INDY but not pargyline induced proliferation of hNPCs similarly to harmine, suggesting that inhibition of DYRK1A is a possible mechanism to explain harmine effects upon the proliferation of hNPCs. Our findings show that harmine enhances proliferation of hNPCs and suggest that inhibition of DYRK1A may explain its effects upon proliferation in vitro and antidepressant effects in vivo.

Differential expression of CYP1A1 and CYP1A2 genes in H4IIE rat hepatoma cells exposed to TCDD and PAHs.

Rat hepatoma cells H4IIE were treated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons (PAHs) (dibenz(a,h)anthracene, benzo(a)pyrene, benz(a)anthracene, chrysene), low-concentration mixtures of PAHs and TCDD, and environmental mixtures contaminated by PAHs and their derivatives. Expression of the gene battery comprising cytochrome P450 Cyp1a1, Cyp1a2, Cyp1b1, and glutathione-s-transferase Gsta2 and Gstp was investigated using quantitative real time polymerase chain reaction (qRT-PCR) analysis. The results revealed that TCDD induce Cyp1a1>Cyp1a2>Cyp1b1, while PAHs and PAH-containing environmental mixtures induce Cyp1a2>Cyp1a1>Cyp1b1 gene expression pattern. While low-concentration mixtures elicited a more pronounced response in comparison to single treatments, the typical gene expression patterns were not observed. In all samples, Gsta2 was predominantly expressed relative to Gstp. These findings indicate that differential Cyp1a1 and Cyp1a2 expression in the H4IIE cells might be used for detection of PAHs in highly contaminated environmental mixtures, but not in low-concentration mixtures of these compounds.

Acute effects of hexabromocyclododecane on Leydig cell cyclic nucleotide signaling and steroidogenesis in vitro.

Hexabromocyclododecane (HBCDD), an additive brominated flame retardant routinely added to various consumer products, was reported to have toxic effects upon biota, including endocrine disruption. In this study, the potential toxicity of HBCDD was tested in peripubertal rat Leydig cells in vitro during 6h exposure. HBCDD inhibited human chorionic gonadotropin- and forskolin-supported cAMP accumulation and steroidogenesis. It also inhibited basal cAMP production, but elevated basal steroidogenesis. The expression of several cAMP-dependent genes, including steroidogenic acute regulatory protein, cholesterol side chain cleavage enzyme, and 3ß-hydroxysteroid dehydrogenase, was also inhibited by HBCDD treatment. Nevertheless, this was not accompanied by a decrease in steroidogenic acute regulatory protein expression, as documented by western blot analysis, and activity of steroidogenic enzymes, as documented by unaffected steroidogenesis in the presence of permeable 22(R)-hydroxycholesterol. However, HBCDD caused significant decrease in mitochondrial membrane potential in untreated and human chorionic gonadotropin-treated cells. This indicates that HBCDD acute toxicity in Leydig cells reflects changes in mitochondrial membrane potential-dependent cAMP production and basal and cAMP-regulated cholesterol transport. This in turn facilitates basal but inhibits cAMP-dependent steroidogenesis. Acute effects of HBCDD treatment on transcription are also indicative of its sustained effects on Leydig cell function.

Atrazine effects on antioxidant status and xenobiotic metabolizing enzymes after oral administration in peripubertal male rat.

Previously, we reported that in vivo applied atrazine from postnatal day 23 to 50 induced strong inhibition of testicular steroidogenesis. Therefore, the aim of the present study was to investigate, in the same experimental model, the oxidative status in androgen-producing testicular interstitial compartment characterized by diminished steroidogenesis. In parallel, we determined activities of antioxidative and cytochrome P450 (CYP) xenobiotic-metabolizing enzymes in liver. To confirm the results on atrazine induced-inhibition of testicular androgenesis, we measured ex vivo production of androgen in Leydig cells. The results revealed decreased activity of antioxidant enzymes, especially glutathione S-transferase (GST), but also glutathione peroxidase (GSH-Px) and catalase (CAT) in testicular interstitial cells, in parallel with strongly diminished ex vivo basal and agonist-stimulated Leydig cell androgenesis. In liver, atrazine increased the activity of GSH-Px, GST, and CYP1A1/2 enzyme, but not lipid peroxidation. These results indicate that atrazine markedly affects both antioxidant status and androgenesis in peripubertal rats.

Transcriptional responses in neonate and adult Daphnia magna in relation to relative susceptibility to genotoxicants.

Little information is available on the responses of lower animals to genotoxic chemicals or on their sensitivity for detecting genotoxic chemicals, especially at different life-stages, despite the established use of the water flea Daphnia magna in ecotoxicity testing. Comet assay methodology was developed and applied to daphnid cells but only limited, non-statistically significant responses to the genotoxicants sodium dichromate (0.2-1 µM), chrysoidine (0.1-2 µM), and mixtures of benzo-a-pyrene (BaP) and sodium dichromate were found (from 0.01 µM BaP & 0.1 µM sodium dichromate to 0.25 µM BaP & 0.75 µM sodium dichromate). Transcriptomic analyses using Agilent D. magna oligonucleotide microarrays were undertaken to assess the effect of a mixture of sodium dichromate and BaP (designed to produce both adducted and oxidised DNA) on gene transcription. Neonates (<24h) and adults (day 7) were exposed for 6h and 24h at two combination concentration levels (0.02 µM BaP & 0.15 µM sodium dichromate and 0.1 µM BaP & 0.75 µM sodium dichromate). The greatest differences in transcriptional profile occurred between adults and neonates. Subsets of the transcriptional profiles distinguished genotoxicant-exposed animals from controls, both for neonates and adults. Higher transcript levels of DNA repair genes were found in adults and adults also displayed significant induction of DNA repair gene transcripts in response to exposure whereas neonates did not. Transcriptional changes in response to genotoxicant exposure proved more sensitive than measurement of DNA strand breaks by the Comet assay and the extensive differences in transcription between adults and neonates emphasized the importance of life stage in toxicant testing with Daphnia.

Upregulation of peripubertal rat Leydig cell steroidogenesis following 24 h in vitro and in vivo exposure to atrazine.

Atrazine is currently one of the most widely used herbicides in the United States and elsewhere. Here we examined 24 h in vitro and in vivo effects of atrazine on androgen production and on expression and activity of steroidogenic enzymes and regulatory proteins involved in cyclic adenosine monophosphate (cAMP)-signaling pathway in peripubertal rat Leydig cells. When in vitro added, 1-50 µM atrazine increased basal and human chorion gonadotropin-stimulated testosterone production and accumulation of cAMP in the medium of treated cells. The stimulatory action of atrazine on androgen production but not on cAMP accumulation was abolished in cells with inhibited protein kinase A. Atrazine also stimulated the expression of mRNA transcripts for steroidogenic factor-1, steroidogenic acute regulatory protein, cytochrome P450 (CYP)17A1, and 17ß-hydroxysteroid dehydrogenase (HSD), as well as the activity of CYP17A1 and 17ßHSD. The stimulatory effects of atrazine on cAMP accumulation and androgen production were also observed during the first 3 days of in vivo treatment (200 mg/kg body weight, by gavage) followed by a decline during further treatment. These results indicate that atrazine has a transient stimulatory action on cAMP signaling pathway in Leydig cells, leading to facilitated androgenesis.

Atrazine oral exposure of peripubertal male rats downregulates steroidogenesis gene expression in Leydig cells.

In the present study, we investigated the effects of oral dosing of atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) to peripubertal male rats (50 and 200 mg/kg body weight daily from postnatal days 23-50) on ex vivo Leydig cell steroidogenesis. Leydig cells from treated rats were characterised by significant decline in mRNA transcripts of several genes responsible for steroidogenesis: luteinizing hormone receptor (LHR), scavenger receptor-B1, steroidogenic acute regulatory protein, translocator protein, steroidogenic factor-1, phosphodiesterase 4B, 3beta-hydroxysteroid dehydrogenase (HSD), CYP17A1, and 17betaHSD. In the presence of human chorion gonadotropin, the dose-dependent decrease in extracellular cAMP level and accordingly strong inhibition of androgenesis were obtained. The transcription of LHR gene in Leydig cells of atrazine-treated rats was downregulated in a dose-dependent manner, which could be the reason for reduction in cAMP level and expression of cAMP-dependent genes. To clarify the activity of the steroidogenic enzymes responsible for androgenesis, purified Leydig cells were challenged with different steroid substrates (22OH-cholesterol, pregnenolone, progesterone, and Delta(4)-androstenedione), and the obtained results indicated inhibition of androgen production in Leydig cells isolated from atrazine-treated animals in the presence of all those substrates. However, when Leydig cells were challenged with 22OH-cholesterol, the progesterone level in the incubation medium was unchanged, indicating that decrease in cholesterol transport and/or CYP17A1 and 17betaHSD activity are most probably responsible for inhibition of androgen production after the addition of different substrates. Our results demonstrated that in vivo exposure to atrazine affects Leydig cell steroidogenesis via the inhibition of steroidogenesis gene expression, which is accompanied by decreased androgenesis.