Exposure to BaA inhibits trophoblast cell invasion and induces miscarriage by regulating the DEC1/ARHGAP5 axis and promoting ubiquitination-mediated degradation of MMP2.

Benz[a]anthracene (BaA), a hazardous polycyclic aromatic hydrocarbon classified by the EPA, is a probable reproductive toxicant. Epidemiological studies suggest that BaA exposure may be a risk factor for recurrent miscarriage (RM). However, the underlying mechanisms are not well understood. This study identified DEC1 as a key gene through RNA-seq and single-cell RNA sequencing analysis. DEC1 expression was found to be downregulated in villous tissues from women with RM and in primary extravillous trophoblasts (EVTs) exposed to BaA. BaA suppressed DEC1 expression by promoting abnormal methylation patterns. Further analysis revealed that ARHGAP5 is a direct target of DEC1 in EVTs, where DEC1 inhibits trophoblast invasion by directly regulating ARHGAP5 transcription. Additionally, BaA destabilized matrix metalloproteinase 2 (MMP2) by activating the aryl hydrocarbon receptor (AhR) and promoting E3 ubiquitin ligase MID1-mediated degradation. In a mouse model, BaA induced miscarriage by modulating the DEC1/ARHGAP5 and MID1/MMP2 axes. Notably, BaA-induced miscarriage in mice was prevented by DEC1 overexpression or MID1 knockdown. These findings indicate that BaA exposure leads to miscarriage by suppressing the DEC1/ARHGAP5 pathway and enhancing the MID1/MMP2 pathway in human EVTs.

Alternaria alternata Toxins Synergistically Activate the Aryl Hydrocarbon Receptor Pathway In Vitro.

Alternaria molds simultaneously produce a large variety of mycotoxins, of which several were previously reported to induce enzymes of phase I metabolism through aryl hydrocarbon receptor activation. Thus, we investigated the potential of naturally occurring Alternaria toxin mixtures to induce Cytochrome P450 (CYP) 1A1/1A2/1B1 activity. Two variants of an extract from cultured Alternaria alternata, as well as the toxins alternariol (AOH), alternariol monomethyl ether (AME), altertoxin I (ATX-I), and altertoxin II (ATX-II), were tested singularly and in binary mixtures applying the 7-ethoxy-resorufin-O-deethylase (EROD) assay in MCF-7 breast cancer cells. Sub-cytotoxic concentrations of the two toxin mixtures, as well as ATX-I, ATX-II and AOH, exhibited dose-dependent enhancements of CYP 1 activity. ATX-I and ATX-II interacted synergistically in this respect, demonstrating the two perylene quinones as major contributors to the extract's potential. Binary mixtures between AOH and the two altertoxins respectively exhibited concentration-dependent antagonistic as well as synergistic combinatory effects. Notably, AME showed no efficacy towards EROD enzyme activity or impact on other toxins' efficacy. Hence, this study provides insights into synergistic and other combinatory effects of Alternaria toxins in natural co-occurrence scenarios in the context of AhR signalling pathway activation in breast cancer cells.

Study of the Mechanism of the Antimicrobial Activity of Novel Water Soluble Ammonium Quaternary Benzanthrone on Model Membranes.

The increasing resistance of many pathogens to most of the common antimicrobials requires the development of new substances with more effective antimicrobial properties. In the present work, we investigated the mechanism of the antimicrobial activity of novel water soluble ammonium quaternary benzanthrone (Compound B) on model membranes, composed of dipalmitoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, 1-palmitoyl-2-oleoylphosphatidylglycerol, and dipalmitoylphosphatidylethanolamine (DPPE). The lipids were chosen to represent a model of a bacterial membrane. The changes in surface pressure of the model membranes, before and after the addition of Compound B, were studied by the Langmuir's monolayer method, and the compressional modulus for each monolayer was determined. In addition, the surface morphology of the lipid monolayers before and after injection of Compound B was monitored by Brewster Angle Microscopy. The results showed that Compound B penetrated all the monolayers studied. The most noticeable effects were found with the negatively charged phosphatidylglycerols and with DPPE leading to the conclusion that the electrostatic interactions between the compound and the lipid head groups and the possible formation of hydrogen bonds between the amino group of the ethanolamine and the keto groups in the structure of Compound B are of great importance. In addition, the penetration ability of the benzoquinone with all phospholipids studied was stable even at higher values of the surface pressure, i.e. thicker monolayers, due to the hydrophobic interaction, which plays also an important role for the antimicrobial activity of Compound B.

Structure-activity relationships and mechanism of action of tetragomycin derivatives as inhibitors of Staphylococcus aureus staphyloxanthin biosynthesis.

Despite the main strategy to overcome bacterial resistance has focused on the development of more potent antimicrobial agents, the evolutionary pressure caused by such drugs makes this strategy limited. Molecules that interfere with virulence factors appear as a promising alternative though, as they cause reduced selective pressure. As a matter of fact, staphyloxanthin biosynthesis inhibition (STXBI) has been pursued as promising strategy to reduce S. aureus virulence. Herein, we report the inhibitory profile of 27 tetrangomycin derivatives over staphyloxanthin production. The experimental result showed that naphthoquinone dehydro-α-lapachone (25 - EC50 = 57.29 ± 1.15 µM) and 2-Isopropylnaphtho[2,3-b]furan-4,9-dione (26 EC50 = 82.10 ± 1.09 µM) are the most potent compounds and suggest that hydrogen acceptor groups and lipophilic moieties decorating the naphthoquinone ring are crucial for STXBI. In addition, we present an in situ analysis, through RAMAN spectroscopy, that is inexpensive and might be employed to probe the mechanism of action of staphyloxanthin biosynthesis inhibitors. Therefore, our molecular simplification strategies afforded promising lead compounds for the development of drugs that modulate S. aureus staphyloxanthin biosynthesis.

Mycotoxin Altertoxin II Induces Lipid Peroxidation Connecting Mitochondrial Stress Response to NF-κB Inhibition in THP-1 Macrophages.

Prolonged exposure to mycotoxins, even in subtoxic concentrations, might contribute to modulate pro- or anti-inflammatory cascades and ultimately have long-term consequences on our health. In line, there is an increasing need to describe and comprehend the potential immunomodulatory effects of toxins that can be produced from fungi proliferating even in a domestic environment like, for instance, Alternaria alternata. Taking this as a starting point, we investigated the effects of one of the most potent genotoxic compounds produced by this fungi type, namely altertoxin II (ATXII) on THP-1 macrophages. In noncytotoxic concentrations (0.1-1 µM), ATXII inhibited the activation of the transcription factor NF-κB, and this event was accompanied by significant mitochondrial superoxide production (1 µM ATXII). Both responses seemed dependent on membrane structure and morphology since they were modulated by the coincubation with the cholesterol complexing agent methyl-ß-cyclodextrin (MßCD, 10-50 µM). Moreover, toxicity of ATXII was enhanced by cholesterol load (cholesterol-MßCD). The mycotoxin induced also lipid peroxidation (1-10 µM, ATXII) possibly streaming down at the mitochondrial level and suppressing NF-κB activation in THP-1 macrophages.

Dose-Dependent Response to 3-Nitrobenzanthrone Exposure in Human Urothelial Cancer Cells.

A product of incomplete combustion of diesel fuel, 3-nitrobenzanthrone (3-NBA), has been classified as a cancer-causing substance. It first gained attention as a potential urinary bladder carcinogen due to the presence of its metabolite in urine and formation of DNA adducts. The aim of the present study was to characterize the dose-response relationship of 3-NBA in human urothelial cancer cell line (RT4) exposed to concentrations ranging from 0.0003 µM (environmentally relevant) to 80 µM by utilizing toxicological and metabolomic approaches. We observed that the RT4 cells were capable of bioactivation of 3-NBA within 30 min of exposure. Activity measurements of various enzymes involved in the conversion of 3-NBA in RT4 cells demonstrated NAD(P)H:quinone oxidoreductase (NQO1) as the main contributor for its bioactivation. Moreover, cytotoxicity assessment exhibited an initiation of adaptive mechanisms at low dosages, which diminished at higher doses, indicating that the capacity of these mechanisms no longer suffices, resulting in increased levels of intracellular reactive oxygen species, reduced proliferation, and hyperpolarisation of the mitochondrial membrane. To characterize the underlying mechanisms of this cellular response, the metabolism of 3-NBA and metabolomic changes in the cells were analyzed. The metabolomic analysis of the cells (0.0003, 0.01, 0.08, 10, and 80 µM 3-NBA) showed elevated levels of various antioxidants at low concentrations of 3-NBA. However, at higher exposure concentrations, it appeared that the cells reprogrammed their metabolism to maintain the cell homeostasis via activation of pentose phosphate pathway (PPP).

Pre-steady-state kinetic investigation of bypass of a bulky guanine lesion by human Y-family DNA polymerases.

3-Nitrobenzanthrone (3-NBA), a byproduct of diesel exhaust, is highly present in the environment and poses a significant health risk. Exposure to 3-NBA results in formation of N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dGC8-N-ABA), a bulky DNA lesion that is of particular importance due to its mutagenic and carcinogenic potential. If not repaired or bypassed during genomic replication, dGC8-N-ABA can stall replication forks, leading to senescence and cell death. Here we used pre-steady-state kinetic methods to determine which of the four human Y-family DNA polymerases (hPolη, hPolκ, hPolι, or hRev1) are able to catalyze translesion synthesis of dGC8-N-ABAin vitro. Our studies demonstrated that hPolη and hPolκ most efficiently bypassed a site-specifically placed dGC8-N-ABA lesion, making them good candidates for catalyzing translesion synthesis (TLS) of this bulky lesion in vivo. Consistently, our publication (Biochemistry 53, 5323-31) in 2014 has shown that small interfering RNA-mediated knockdown of hPolη and hPolκ in HEK293T cells significantly reduces the efficiency of TLS of dGC8-N-ABA. In contrast, hPolι and hRev1 were severely stalled by dGC8-N-ABA and their potential role in vivo was discussed. Subsequently, we determined the kinetic parameters for correct and incorrect nucleotide incorporation catalyzed by hPolη at various positions upstream, opposite, and downstream from dGC8-N-ABA. Notably, nucleotide incorporation efficiency and fidelity both decreased significantly during dGC8-N-ABA bypass and the subsequent extension step, leading to polymerase pausing and error-prone DNA synthesis by hPolη. Furthermore, hPolη displayed nucleotide concentration-dependent biphasic kinetics at the two polymerase pause sites, suggesting that multiple enzyme•DNA complexes likely exist during nucleotide incorporation.

TP53 and lacZ mutagenesis induced by 3-nitrobenzanthrone in Xpa-deficient human TP53 knock-in mouse embryo fibroblasts.

3-Nitrobenzanthrone (3-NBA) is a highly mutagenic compound and possible human carcinogen found in diesel exhaust. 3-NBA forms bulky DNA adducts following metabolic activation and induces predominantly G:CT:A transversions in a variety of experimental systems. Here we investigated the influence of nucleotide excision repair (NER) on 3-NBA-induced mutagenesis of the human tumour suppressor gene TP53 and the reporter gene lacZ. To this end we utilised Xpa -knockout (Xpa-Null) human TP53 knock-in (Hupki) embryo fibroblasts (HUFs). As Xpa is essential for NER of bulky DNA adducts, we hypothesized that DNA adducts induced by 3-NBA would persist in the genomes of Xpa-Null cells and lead to an increased frequency of mutation. The HUF immortalisation assay was used to select for cells harbouring TP53 mutations following mutagen exposure. We found that Xpa-Null Hupki mice and HUFs were more sensitive to 3-NBA treatment than their wild-type (Xpa-WT) counterparts. However, following 3-NBA treatment and immortalisation, a similar frequency of TP53-mutant clones arose from Xpa-WT and Xpa-Null HUF cultures. In cells from both Xpa genotypes G:CT:A transversion was the predominant TP53 mutation type and mutations exhibited bias towards the non-transcribed strand. Thirty-two percent of 3-NBA-induced TP53 mutations occurred at CpG sites, all of which are hotspots for mutation in smokers' lung cancer (codons 157, 158, 175, 245, 248, 273, 282). We also examined 3-NBA-induced mutagenesis of an integrated lacZ reporter gene in HUFs, where we again observed a similar mutant frequency in Xpa-WT and Xpa-Null cells. Our findings suggest that 3-NBA-DNA adducts may evade removal by global genomic NER; the persistence of 3-NBA adducts in DNA may be an important factor in its mutagenicity.

Synthesis, photophysical and antimicrobial activity of new water soluble ammonium quaternary benzanthrone in solution and in polylactide film.

The synthesis of a new cationic water soluble fluorescent 1-[(7-oxo-7H-benzo[de]anthracen-3-ylcarbamoyl)-methyl]-triethylammonium chloride (B) has been described. Due to the presence of the quaternary amino group, the compound is soluble in water. Its photophysical characteristics in aqueous solution and organic solvents with different polarity have been determined using absorption and fluorescence spectroscopy. The photostability of compound B has been investigated in aqueous media. The newly synthesized compound has been tested in vitro for its antimicrobial activity against eight bacterial and two yeasts cultures. The results obtained suggest that the newly synthesized compound is effective in treating the relevant pathogens and is suitable in designing new effective antimicrobial preparations. The incorporation of the compound into thin polylactic acid film and its release into water solution has been also investigated. It was demonstrated that the compound released from the polymer polylactic acid matrix exhibited a prolonged good antibacterial activity.

Genome mining-directed activation of a silent angucycline biosynthetic gene cluster in Streptomyces chattanoogensis.

Genomic sequencing of actinomycetes has revealed the presence of numerous gene clusters seemingly capable of natural product biosynthesis, yet most clusters are cryptic under laboratory conditions. Bioinformatics analysis of the completely sequenced genome of Streptomyces chattanoogensis L10 (CGMCC 2644) revealed a silent angucycline biosynthetic gene cluster. The overexpression of a pathway-specific activator gene under the constitutive ermE* promoter successfully triggered the expression of the angucycline biosynthetic genes. Two novel members of the angucycline antibiotic family, chattamycins A and B, were further isolated and elucidated. Biological activity assays demonstrated that chattamycin B possesses good antitumor activities against human cancer cell lines and moderate antibacterial activities. The results presented here provide a feasible method to activate silent angucycline biosynthetic gene clusters to discover potential new drug leads.

Benzanthrone induced immunotoxicity via oxidative stress and inflammatory mediators in Balb/c mice.

Benzanthrone (BA) is an important dye intermediate which is used in the manufacturing of several polycyclic vat and disperse dyes in textile industries. Several studies have indicated that the general population is also exposed to BA owing to its release from furnace effluents and automobile exhausts in the environment. In several clinical studies, it has been shown that workers exposed to BA developed itching, burning sensation, erythema and hyperpigmentation of the skin, which could be an outcome of the dysregulated immune response. In this study, we have used female Balb/c mice as a model to study the immuno-inflammatory changes after systemic administration of BA (7.5mg/kgb.w. and 15mg/kgb.w.) for one week. BA exposed animals exhibited the signs of intense systemic inflammation as evident by enhanced DTH response, MPO activity, hyperplastic and dysplastic histopathological organization of spleen and lung tissue. Splenic evaluation revealed enhanced oxidative stress, upregulation of prominent inflammatory markers like iNOS and COX-2 and DNA damage. In coherence with the observed immuno-inflammatory alterations, the levels of several inflammatory and regulatory cytokines (IL-17, TNF-α, IFN-γ, IL-1, IL-10, IL-4) were significantly enhanced in serum as well as the spleen. In addition, BA administration significantly induced the activation of ERK1/2, p38, JNK MAPKs and their downstream transcription factors AP-1 (c-fos, c-jun), NF-κB and Nrf2 which comprise important mechanistic pathways involved in inflammatory manifestations. These results suggest the immunotoxic nature of the BA and have implications for the risk assessment and management of occupational workers, and even common masses considering its presence as an environmental contaminant.

Comparison of the metabolic activation of environmental carcinogens in mouse embryonic stem cells and mouse embryonic fibroblasts.

We compared mouse embryonic stem (ES) cells and fibroblasts (MEFs) for their ability to metabolically activate the environmental carcinogens benzo[a]pyrene (BaP), 3-nitrobenzanthrone (3-NBA) and aristolochic acid I (AAI), measuring DNA adduct formation by (32)P-postlabelling and expression of xenobiotic-metabolism genes by quantitative real-time PCR. At 2 µM, BaP induced Cyp1a1 expression in MEFs to a much greater extent than in ES cells and formed 45 times more adducts. Nqo1 mRNA expression was increased by 3-NBA in both cell types but induction was higher in MEFs, as was adduct formation. For AAI, DNA binding was over 450 times higher in MEFs than in ES cells, although Nqo1 and Cyp1a1 transcriptional levels did not explain this difference. We found higher global methylation of DNA in ES cells than in MEFs, which suggests higher chromatin density and lower accessibility of the DNA to DNA damaging agents in ES cells. However, AAI treatment did not alter DNA methylation. Thus mouse ES cells and MEFs have the metabolic competence to activate a number of environmental carcinogens, but MEFs have lower global DNA methylation and higher metabolic capacity than mouse ES cells.

Two new antioxidant actinosporin analogues from the calcium alginate beads culture of sponge-associated Actinokineospora sp. strain EG49.

Marine sponge-associated actinomycetes represent an exciting new resource for the identification of new and novel natural products . Previously, we have reported the isolation and structural elucidation of actinosporins A (1) and B (2) from Actinokineospora sp. strain EG49 isolated from the marine sponge Spheciospongia vagabunda. Herein, by employing different fermentation conditions on the same microorganism, we report on the isolation and antioxidant activity of structurally related metabolites, actinosporins C (3) and D (4). The antioxidant potential of actinosporins C and D was demonstrated using the ferric reducing antioxidant power (FRAP) assay. Additionally, at 1.25 µM, actinosporins C and D showed a significant antioxidant and protective capacity from the genomic damage induced by hydrogen peroxide in the human promyelocytic (HL-60) cell line.

Altertoxins with potent anti-HIV activity from Alternaria tenuissima QUE1Se, a fungal endophyte of Quercus emoryi.

Screening of a small library of natural product extracts derived from endophytic fungi of the Sonoran desert plants in a cell-based anti-HIV assay involving T-cells infected with the HIV-1 virus identified the EtOAc extract of a fermentation broth of Alternaria tenuissima QUE1Se inhabiting the stem tissue of Quercus emoryi as a promising candidate for further investigation. Bioactivity-guided fractionation of this extract led to the isolation and identification of two new metabolites, altertoxins V (1) and VI (2) together with the known compounds, altertoxins I (3), II (4), and III (5). The structures of 1 and 2 were determined by detailed spectroscopic analysis and those of 3-5 were established by comparison with reported data. When tested in our cell-based assay at concentrations insignificantly toxic to T-cells, altertoxins V (1), I (3), II (4), and III (5) completely inhibited replication of the HIV-1 virus at concentrations of 0.50, 2.20, 0.30, and 1.50 µM, respectively. Our findings suggest that the epoxyperylene structural scaffold in altertoxins may be manipulated to produce potent anti-HIV therapeutics.

A new compound from an endophytic fungus Alternaria tenuissima.

A new secondary metabolite, named altertoxin IV (1), together with altertoxin II (2), was isolated from the fermentation broth of Alternaria tenuissima, an endophytic fungal strain residing in the stem of Tribulus terrestris L. The structure of new compound 1 was established by HR-ESI-MS, multinuclear NMR spectroscopy, and single crystal X-ray diffraction method. In their in vitro bioassay, compound 2 exhibited moderate cytotoxic activity against PC-3 cell lines with an IC50 value of 14.28 µM.

Epoxide reduction to an alcohol: a novel metabolic pathway for perylene quinone-type alternaria mycotoxins in mammalian cells.

The group of perylene quinone-type Alternaria toxins contains several congeners with epoxide groups, for example, altertoxin II (ATX II) and stemphyltoxin III (STTX III). Recent studies in our laboratory have disclosed that the epoxide moieties of ATX II and STTX III are reduced to alcohols in human colon Caco-2 cells, thereby resulting in the formation of altertoxin I (ATX I) and alteichin, respectively. In the present study, this pathway was demonstrated for ATX II in three other mammalian cell lines. Furthermore, the chemical reaction of this toxin with monothiols like glutathione could be shown, and the structures of the reaction products were tentatively elucidated by UV and mass spectrometry. Chemical reaction of ATX II with dithiols capable of forming five- and six-membered rings gave rise to ATX I, thus providing a clue for the molecular mechanism of the epoxide reduction pathway of ATX II. Both epoxide reduction and glutathione conjugation appear to attenuate, but not completely abolish, the genotoxicity of ATX II.

Warkmycin, a novel angucycline antibiotic produced by Streptomyces sp. Acta 2930*.

A novel angucycline-type antibiotic, warkmycin, was isolated from the culture filtrate of Streptomyces strain Acta 2930. Its chemical structure was elucidated by HR-MS, one-dimensional and 2D NMR experiments. The compound inhibits the growth of Gram-positive bacteria and shows a strong antiproliferative activity against mouse fibroblast cell line NIH-3T3 and human cancer cell lines HepG2 and HT29.

Saccharosporones A, B and C, cytotoxic antimalarial angucyclinones from Saccharopolyspora sp. BCC 21906.

Three new angucyclinones, saccharosporones A, B and C, together with (+)-ochromycinone, (+)-rubiginone B2, tetrangulol methyl ether and fujianmycin A, were obtained from fermentation of the terrestrial actinomycete of the genus Saccharopolyspora BCC 21906 isolated from a soil collected in Chanthaburi Province, Thailand. Structures of the new compounds and their relative configurations were assigned by NMR spectral data interpretation. Saccharosporones A and B exhibited antimalarial activity against Plasmodium falciparum K1 with IC50 values of 4.1 and 3.9 µM. Both metabolites also possessed cytotoxic activities against cancer cell lines (KB, MCF-7 and NCI-H187) and nonmalignant Vero cell, while saccharosporone C only showed cytotoxic activity against NCI-H187.

Plasticity of hypothalamic dopamine neurons during lactation results in dissociation of electrical activity and release.

Tuberoinfundibular dopamine (TIDA) neurons are the central regulators of prolactin (PRL) secretion. Their extensive functional plasticity allows a change from low PRL secretion in the non-pregnant state to the condition of hyperprolactinemia that characterizes lactation. To allow this rise in PRL, TIDA neurons are thought to become unresponsive to PRL at lactation and functionally silenced. Here we show that, contrary to expectations, the electrical properties of the system were not modified during lactation and that the neurons remained electrically responsive to a PRL stimulus, with PRL inducing an acute increase in their firing rate during lactation that was identical to that seen in non-pregnant mice. Furthermore, we show a long-term organization of TIDA neuron electrical activity with an harmonization of their firing rates, which remains intact during lactation. However, PRL-induced secretion of dopamine (DA) at the median eminence was strongly blunted during lactation, at least in part attributable to lack of phosphorylation of tyrosine hydroxylase, the key enzyme involved in DA synthesis. We therefore conclude that lactation, rather than involving electrical silencing of TIDA neurons, represents a condition of decoupling between electrical activity at the cell body and DA secretion at the median eminence.

Organ-specific distribution of 7-chlorinated benz[a]anthracene and regulation of selected cytochrome P450 genes in rats.

We previously reported that 14-day exposure to 7-chlorinated benz[a]anthracene (7-Cl-BaA), a new environmental pollutant, selectively induced hepatic cytochrome P450 (CYP)1A2 in rats, although treatment with its parent, benz[a]anthracene (BaA), induced CYP1A1, CYP1A2, and CYP1B1. In this study, to better understand the relative contribution of chlorination to the toxicity of polycyclic aromatic hydrocarbons (PAHs), we investigated the organ-specific distributions of 7-Cl-BaA and BaA in F334 rats. After 14 days of oral administration of 7-Cl-BaA or BaA at a concentration of 1 or 10 mg/kg body weight/day, both chemicals were detected in their plasma, which was collected 24 hr after the last administration, even at the lower dosage. Dose-dependent accumulation patterns were observed in the liver, muscle, kidney, spleen, heart, and lung. The 7-Cl-BaA concentrations in the organs were higher than those of the BaA. Furthermore, at the end of the exposure, 7-Cl-BaA specifically regulated several CYP genes in the heart more so than in other organs, although these inductions were not significant in the BaA treatment. 7-Cl-BaA might also stimulate the metabolic pathways of chemicals other than AhR-mediated metabolism, which is specific to normal PAHs, because of the alterations of CYP2J4, CYP4B1, and CYP17A1 expression in rats. In conclusion, our results imply that the chlorination of PAHs may change their organ-specific distribution and consequently alter their toxicological impacts compared to their parent PAHs.