Proteomic examination of polyester-polyurethane degradation by Streptomyces sp. PU10: Diverting polyurethane intermediates to secondary metabolite production.

Global plastic waste accumulation has become omnipresent in public discourse and the focus of scientific research. Ranking as the sixth most produced polymer globally, polyurethanes (PU) significantly contribute to plastic waste and environmental pollution due to the toxicity of their building blocks, such as diisocyanates. In this study, the effects of PU on soil microbial communities over 18 months were monitored revealing that it had marginal effects on microbial diversity. However, Streptomyces sp. PU10, isolated from this PU-contaminated soil, proved exceptional in the degradation of a soluble polyester-PU (Impranil) across a range of temperatures with over 96% degradation of 10 g/L in 48 h. Proteins involved in PU degradation and metabolic changes occurring in this strain with Impranil as the sole carbon source were further investigated employing quantitative proteomics. The proposed degradation mechanism implicated the action of three enzymes: a polyester-degrading esterase, a urethane bond-degrading amidase and an oxidoreductase. Furthermore, proteome data revealed that PU degradation intermediates were incorporated into Streptomyces sp. PU10 metabolism via the fatty acid degradation pathway and subsequently channelled to polyketide biosynthesis. Most notably, the production of the tri-pyrrole undecylprodigiosin was confirmed paving the way for establishing PU upcycling strategies to bioactive metabolites using Streptomyces strains.

Polyurethane-Degrading Potential of Alkaline Groundwater Bacteria.

Plastic waste is a global environmental burden and long-lasting plastic polymers, including ubiquitous and toxic polyurethanes (PUs), rapidly accumulate in the water environments. In this study, samples were collected from the three alkaline groundwater occurrences in the geotectonic regions of the Pannonian basin of northern Serbia (Torda and Slankamen Banja) and Inner Dinarides of western Serbia (Mokra Gora) with aim to isolate and identify bacteria with plastic- and lignocellulose-degrading potential, that could be applied to reduce the burden of environmental plastic pollution. The investigated occurrences belong to cold, mildly alkaline (pH: 7.6-7.9) brackish and hyperalkaline (pH: 11.5) fresh groundwaters of the SO4 - Na + K, Cl - Na + K and OH, Cl - Ca, Na + K genetic type. Full-length 16S rDNA sequencing, using Oxford Nanopore sequencing device, was performed with DNA extracted from colonies obtained by cultivation of all groundwater samples, as well as with DNA extracted directly from one groundwater sample. The most abundant genera belong to Pseudomonas, Acidovorax, Kocuria and Methylotenera. All screened isolates (100%) had the ability to grow on at least 3 of the tested plastic and lignocellulosic substrates, with 53.9% isolates degrading plastic substrate Impranil® DLN-SD (SD), a model compound for PUs degradation. Isolates degrading SD that were identified by partial 16S rDNA sequencing belong to the Stenotrophomonas, Pseudomonas, Paraburkholderia, Aeromonas, Vibrio and Acidovorax genera. Taking into account that plastics, including commonly produced PUs, are widespread in groundwater, identification of PUs-degrading bacteria may have potential applications in bioremediation of groundwater polluted with this polymer.

Brackish Groundwaters Contain Plastic- and Cellulose-Degrading Bacteria.

The selected brackish groundwater occurrences in the geotectonic regions of Inner Dinarides of western Serbia (Obrenovacka Banja) and Serbian crystalline core (Lomnicki Kiseljak and Velika Vrbnica) were sampled for isolation and identification of plastic- and lignocellulose-degrading bacteria, as well as for the assessment of their enzymatic potential. The examined occurrences belong to the cold and warm (subthermal), weakly alkaline, neutral, and weakly acidic groundwater, and their genetic types are HCO3-Na + K and HCO3-Ca, Mg. The most abundant genera identified by next-generation 16S sequencing of cultivated groundwater samples belong to Aeromonas and Exiguobacterium. Of isolates screened on plastic and lignocellulosic substrates, 85.3% demonstrated growth and/or degrading activity on at least one tested substrate, with 27.8% isolates degrading plastic substrate Impranil® DLN-SD (SD), 1.9% plastic substrate bis(2-hydroxyethyl)terephthalate, and 5.6% carboxymethyl cellulose (CMC). Isolates degrading SD that were identified by 16S rDNA sequencing belonged to genera Stenotrophomonas, Flavobacterium, Pantoea, Enterobacter, Pseudomonas, Serratia, Acinetobacter, and Proteus, while isolates degrading CMC belonged to genera Rhizobium and Shewanella. All investigated brackish groundwaters harbor bacteria with potential in degradation of plastics or cellulose. Taking into account that microplastics contamination of groundwater resources is becoming a significant problem, the finding of plastic-degrading bacteria may have potential in bioremediation treatments of polluted groundwater. Subterranean ecosystems, which are largely untapped resources of biotechnologically relevant enzymes, are not traditionally considered the environment of choice for screening for plastic- and cellulose-degrading bacteria and therefore deserve a special attention from this aspect.

Global survey of dioxin- and thyroid hormone-like activities in consumer products and toys.

BACKGROUND: Children and consumers are exposed to increasingly complex mixtures of known and as-yet-unknown toxic chemicals from toys and products. However traditional chemical analysis methods only evaluate a small number of chemicals at a time thereby restricting consumer awareness of the full range of potentially harmful chemicals in products. METHODS: We used high-throughput effect-based non-animal methods to investigate exposures to complex chemical mixtures of several kinds of brominated flame retardants (BFRs) for their dioxin- and thyroid hormone-like activities in various kinds of consumer products and toys from 26 different countries, on four continents (Africa, America, Asia and Europe) in combination with chemical analysis of various polybrominated flame retardants (BFRs) and their impurities (such as polyhalogenated PCDD/Fs and PBDD/Fs). RESULTS: We found high levels of polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs) in toys and now, for the first time, also in consumer products that are manufactured from black plastics containing certain brominated flame retardants (BFRs). The presence of PBDD/PBDFs as well as other BFRs in various black plastic materials from additional countries as well as additional kinds of consumer products as confirmed by effect-based in vitro reporter gene DR CALUX and TTR-TRß CALUX assays as well as congener-specific chemical analysis. We compared total Toxicity Equivalent (TEQ) levels of PBDD/F-TEQs analysed by chemical analysis to by CALUX bioassay measured Biological equivalence (BEQ) concentrations (for further info see at ISO 23196, ISO, 2022). In the case of TBBPA, both chemical and TTR-TRß CALUX analysis measure direct the amount of TBBPA. Finally, the daily ingestion of 2,3,7,8-TCDD equivalents from PBDD/Fs-contaminated plastic toys by child mouthing habits have been related to our earlier study (Budin et al., 2020). CONCLUSIONS: Interaction of children with such contaminated plastics may significantly contribute to the daily uptake of dioxin- and thyroid hormone transport disrupting-like compounds. Effect-based bioassays for dioxin- and thyroid hormone-like activities are relevant to pick-out such complex mixtures of known and yet unknown (and therefore not regulated) substances for safer and more sustainable plastics. Low POPs Content Levels and other mechanisms set under the Basel and Stockholm Conventions are set far too high to prevent a significant flow of BFRs and PBDD/Fs into consumer products.

Versicolorin A enhances the genotoxicity of aflatoxin B1 in human liver cells by inducing the transactivation of the Ah-receptor.

Aflatoxins are a group of mycotoxins that have major adverse effects on human health. Aflatoxin B1 (AFB1) is the most important aflatoxin and a potent carcinogen once converted into a DNA-reactive form by cytochrome P450 enzymes (CYP450). AFB1 biosynthesis involves the formation of Versicolorin A (VerA) which shares structural similarities with AFB1 and can be found in contaminated commodities, often co-occurring with AFB1. This study investigated and compared the toxicity of VerA and AFB1, alone or in combination, in HepG2 human liver cells. Our results show that both toxins have similar cytotoxic effects and are genotoxic although, unlike AFB1, the main genotoxic mechanism of VerA does not involve the formation of DNA double-strand breaks. Additionally, we show that VerA activates the aryl hydrocarbon receptor (AhR) and significantly induce the expression of the CYP450-1A1 (CYP1A1) while AFB1 did not induce AhR-dependent CYP1A1 activation. Combination of VerA with AFB1 resulted in enhanced genotoxic effects, suggesting that AhR-activation by VerA influences AFB1 genotoxicity by promoting its bioactivation by CYP450s to a highly DNA-reactive metabolite. Our results emphasize the need for expanding the toxicological knowledge regarding mycotoxin biosynthetic precursors to identify those who may pose, directly or indirectly, a threat to human health.

Induction of AhR transactivation by PBDD/Fs and PCDD/Fs using a novel human-relevant, high-throughput DRhuman CALUX reporter gene assay.

Polychlorinated dioxins and dibenzofurans (PCDD/Fs) are highly toxic contaminants that are strictly regulated and monitored in the environment and food to reduce human exposure. Recently, the increasing occurrence of polybrominated dioxins and dibenzofurans (PBDD/Fs) in the environment is raising concerns about the impact on human health by the combined exposure to chlorinated and brominated analogues of dioxins. Toxicological properties of PBDD/Fs relative to PCDD/Fs have not been firmly established, and brominated dioxins are not included in routine monitoring programs. In this study, we set out to determine human-relevant congener-specific potency values for a range of brominated and chlorinated dioxin congeners, based on their aryl hydrocarbon receptor (AhR)-mediated mode of toxic action. Transactivation of the AhR was measured using dioxin-responsive (DR) CALUX reporter gene assays. Because of known species-differences in dioxin-mediated toxicity, we developed and used a HepG2 human liver cell-based DR human CALUX assay that is a variant of the rodent-based DR CALUX. The assay was found to be highly inducible and stable, with low variations between independent measurements. Using both DR CALUX assays in an automated high-throughput mode we found that overall PBDD/Fs were as potent as PCDD/Fs in inducing AhR transactivation, but congener-specific differences were observed. We also observed species-specific differences in sensitivity and potency when comparing DR human REP values to those obtained in the rat-based DR CALUX. Finally, we observed significant differences between WHO-TEF values and DR human REP values, suggesting that actual WHO-TEF values may underestimate the hazards associated with exposure of humans to dioxins.

Detection of high PBDD/Fs levels and dioxin-like activity in toys using a combination of GC-HRMS, rat-based and human-based DR CALUX® reporter gene assays.

Brominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs) are increasingly reported at significant levels in various matrices, including consumer goods that are manufactured from plastics containing certain brominated flame retardants. PBDD/Fs are known ligands for the aryl hydrocarbon receptor (AhR) but are not yet considered in the hazard assessment of dioxin mixtures. The aim of the present study was to determine if PBDD/Fs levels present in plastic constituents of toys could pose a threat to children's health. PBDD/Fs, unlike their chlorinated counterparts (PCDD/Fs), have not been officially assigned toxic equivalence factors (TEFs) by the WHO therefore, we determined their relative potency towards AhR activation in both human and rodent cell-based DR CALUX® bioassays. This allowed us to compare GC-HRMS PBDD/F congener levels, converted to total Toxic Equivalents (TEQ) by using the PCDD/F TEFs, to CALUX Bioanalytical Equivalents (BEQ) levels present in contaminated plastic constituents from children's toys. Finally, an estimate was made of the daily ingestion of TEQs from PBDD/Fs-contaminated plastic toys by child mouthing habits. It is observed that the daily ingestion of PBDD/Fs from contaminated plastic toys may significantly contribute to the total dioxin daily intake of young children.