Degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) by fungi originating from Vietnam.

Three different fungi were tested for their ability to degrade 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid and for the role of laccases and cytochromes P450-type in this process. We studied a white-rot fungus Rigidoporus sp. FMD21, which has a high laccase activity, for its efficiency to degrade these herbicides. A positive correlation was found between its laccase activity and the corresponding herbicide degradation rate. Even more, the doubling of the enzyme activity in this phase corresponded with a doubling of the herbicide degradation rate. It is, therefore, tempting to speculate that laccase is the most dominant enzyme in the degradation of 2,4-D and 2,4,5-T under these conditions. In addition, it was shown that Rigidoporus sp. FMD21 partly relies on cytochromes P450-type for the breakdown of the herbicides as well. Two filamentous fungi were isolated from soil contaminated with herbicides and dioxins located at Bien Hoa airbase. They belong to genera Fusarium and Verticillium of the phylum Ascomycota as judged by their 18S rRNA gene sequences. Both isolated fungi were able to degrade the herbicides but with different rates. Their laccase activity, however, was very low and did not correlate with the rate of breakdown of the herbicides. These data indicate that the white-rot fungus most likely synthesizes laccase and cytochromes P450-type for the breakdown of the herbicides, while the types of enzyme used for the breakdown of the herbicides by the two Ascomycota remain unclear.

In vitro human cell-based TTR-TRß CALUX assay indicates thyroid hormone transport disruption of short-chain, medium-chain, and long-chain chlorinated paraffins.

Over the last decades, short-chain chlorinated paraffins (SCCPs), medium-chain chlorinated paraffins (MCCPs), and long-chain chlorinated paraffins (LCCPs) have become the most heavily produced monomeric organohalogen compound class of environmental concern. However, knowledge about their toxicology is still scarce, although SCCPs were shown to have effects on the thyroid hormone system. The lack of data in the case of MCCPs and LCCPs and the structural similarity with perfluoroalkyl substances (PFAS) prompted us to test CPs in the novel TTR-TR CALUX assay for their thyroid hormone transport disrupting potential. Four self-synthesized and additionally purified single chain length CP mixtures (C10-CPs, C11-CPs, C14-CPs and C16-CPs) and two each of industrial MCCP and LCCP products were tested in parallel with PFOA. All CP mixtures influenced the TTR binding of T4, giving activities of 1,300 to 17,000 µg/g PFOA equivalents and lowest observable effect concentrations (LOELs) of 0.95 to 0.029 mM/L incubate. Highest activities and lowest LOELs were observed for C16-CPs (48.3% Cl content, activity 17,000, LOEL 0.047 mM/L) and a LCCP mixture (71.7% Cl content; activity 10,000; LOEL 0.029 mM/L). A trend of higher activities and lower LOELs towards longer chains and higher chlorination degrees was implied, but could not be statistically confirmed. Irrespectively, the less well examined and current-use LCCPs showed the highest response in the TTR-TRß CALUX assay.

Evolutionary ecology of beta-lactam gene clusters in animals.

Beta-lactam biosynthesis was thought to occur only in fungi and bacteria, but we recently reported the presence of isopenicillin N synthase in a soil-dwelling animal, Folsomia candida. However, it has remained unclear whether this gene is part of a larger beta-lactam biosynthesis pathway and how widespread the occurrence of penicillin biosynthesis is among animals. Here, we analysed the distribution of beta-lactam biosynthesis genes throughout the animal kingdom and identified a beta-lactam gene cluster in the genome of F. candida (Collembola), consisting of isopenicillin N synthase (IPNS), δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine synthetase (ACVS), and two cephamycin C genes (cmcI and cmcJ) on a genomic scaffold of 0.76 Mb. All genes are transcriptionally active and are inducible by stress (heat shock). A beta-lactam compound was detected in vivo using an ELISA beta-lactam assay. The gene cluster also contains an ABC transporter which is coregulated with IPNS and ACVS after heat shock. Furthermore, we show that different combinations of beta-lactam biosynthesis genes are present in over 60% of springtail families, but they are absent from genome- and transcript libraries of other animals including close relatives of springtails (Protura, Diplura and insects). The presence of beta-lactam genes is strongly correlated with an euedaphic (soil-living) lifestyle. Beta-lactam genes IPNS and ACVS each form a phylogenetic clade in between bacteria and fungi, while cmcI and cmcJ genes cluster within bacteria. This suggests a single horizontal gene transfer event most probably from a bacterial host, followed by differential loss in more recently evolving species.

Screening of molecular cell targets for carcinogenic heterocyclic aromatic amines by using CALUX® reporter gene assays.

Heterocyclic aromatic amines (HCAs) are compounds formed when meat or fish are cooked at high temperatures for a long time or over an open fire. To determine which pathways of toxicity are activated by HCAs, nine out of the ten HCAs known to be carcinogenic in rodents (2-amino-9H-pyrido[2,3-b]indole (AαC), 2-aminodipyrido[1,2-a:3',2-d]imidazole (Glu-P-2), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2)) were tested in the estrogen receptor α (ERα), androgen receptor (AR), glucocorticoid receptor (GR), peroxisome proliferator-activated receptor γ2 (PPARγ2), polycyclic aromatic hydrocarbons (PAH), Nrf2, and p53 CALUX® reporter gene assays. Trp-P-1 was the only HCA that led to a positive response in the ERα, PPARγ2, and Nrf2 CALUX® assays. In the PAH CALUX® assay, Trp-P-2, MeAαC, and AαC induced luciferase activity to a greater extent than MeIQ and PhIP. In the p53 CALUX® assay without a coupled metabolic activation, only Trp-P-1 and Trp-P-2 enhanced luciferase expression; when a metabolic activation step was coupled to the p53 CALUX® assay, Trp-P-1, Glu-P-2, MeIQ, MeIQx, and PhIP induced a positive response. No HCA was positive in the AR and GR CALUX® assays. Taken together, the results obtained show that the battery of CALUX® assays performed in the present study can successfully be used to screen for molecular cell targets of carcinogenic compounds such as HCAs.

Screening for PCDD/Fs and dl-PCBs in local and imported food and feed products available across the State of Kuwait and assessment of dietary intake.

A total of 318 local and imported meat, milk, eggs, fish, and animal feed samples collected in Kuwait were analyzed by cell-based reporter gene assay (Dioxin-Responsive Chemical Activated LUciferase gene eXpression DR-CALUX) for PCDD/Fs and dl-PCBs. The bioanalytical equivalents (BEQs) obtained by DR-CALUX bioassay were compared with the official maximum limits according to the European Commission (EC) regulations. Suspected and randomly chosen negative samples were analyzed by gas chromatography-high resolution mass spectrometry (GC-HRMS). The results showed that among suspected samples, one sample was confirmed to be non-compliant. The positive sample was of imported origin. The correlation coefficient of 0.98 between DR-CALUX and GC-HRMS was found. Moreover, the average daily intakes of PCDD/Fs and dl-PCBs for the Kuwaiti population were estimated. Results obtained in this study were discussed and compared with other published data.

Results of an international interlaboratory study on dioxin-like activities in drinking-, river surface- and wastewater using DR CALUX bioassay.

Aquatic animals and consumers of aquatic animals are exposed to increasingly complex mixtures of known and as-yet-unknown chemicals with dioxin-like toxicities in the water cycle. Effect- and cell-based bioanalysis can cover known and yet unknown dioxin and dioxin-like compounds as well as complex mixtures thereof but need to be standardized and integrated into international guidelines for environmental testing. In an international laboratory testing (ILT) following ISO/CD 24295 as standard procedure for rat cell-based DR CALUX un-spiked and spiked extracts of drinking-, surface-, and wastewater were validated to generate precision data for the development of the full ISO-standard. We found acceptable repeatability and reproducibility ranges below 36 % by DR CALUX bioassay for the tested un-spiked and spiked water of different origins. The presence of 17 PCDD/Fs and 12 dioxin-like PCBs was also confirmed by congener-specific GC-HRMS analysis. We compared the sum of dioxin-like activity levels measured by DR CALUX bioassay (expressed in 2,3,7,8-TCDD Bioanalytical Equivalents, BEQ; ISO 23196, 2022) with the obtained GC-HRMS chemical analysis results converted to toxic equivalents (TEQ; van den Berg et al., 2013).

Similarities in the endocrine-disrupting potencies of indoor dust and flame retardants by using human osteosarcoma (U2OS) cell-based reporter gene assays.

Indoor dust is a sink for many kinds of pollutants, including flame retardants (FRs), plasticizers, and their contaminants and degradation products. These pollutants can be migrated to indoor dust from household items such as televisions and computers. To reveal high-priority end points of and contaminant candidates in indoor dust, using CALUX reporter gene assays based on human osteosarcoma (U2OS) cell lines, we evaluated and characterized the endocrine-disrupting potencies of crude extracts of indoor dust collected from Japan (n = 8), the United States (n = 21), Vietnam (n = 10), the Philippines (n = 17), and Indonesia (n = 10) and for 23 selected FRs. The CALUX reporter gene assays used were specific for compounds interacting with the human androgen receptor (AR), estrogen receptor α (ERα), progesterone receptor (PR), glucocorticoid receptor (GR), and peroxisome proliferator-activated receptor γ2 (PPARγ2). Indoor dust extracts were agonistic to ERα, GR, and PPARγ2 and antagonistic against AR, PR, GR, and PPARγ2. In comparison, a majority of FRs was agonistic to ERα and PPARγ2 only, and some FRs demonstrated receptor-specific antagonism against all tested nuclear receptors. Hierarchical clustering clearly indicated that agonism of ERα and antagonism of AR and PR were common, frequently detected end points for indoor dust and tested FRs. Given our previous results regarding the concentrations of FRs in indoor dust and in light of our current results, candidate contributors to these effects include not only internationally controlled brominated FRs but also alternatives such as some phosphorus-containing FRs. In the context of indoor pollution, high-frequency effects of FRs such as agonism of ERα and antagonism of AR and PR are candidate high-priority end points for further investigation.

Nonribosomal Peptide Synthetases in Animals.

Nonribosomal peptide synthetases (NRPSs) are a class of cytosolic enzymes that synthesize a range of bio-active secondary metabolites including antibiotics and siderophores. They are widespread among both prokaryotes and eukaryotes but are considered rare among animals. Recently, several novel NRPS genes have been described in nematodes, schistosomes, and arthropods, which led us to investigate how prevalent NRPS genes are in the animal kingdom. We screened 1059 sequenced animal genomes and showed that NRPSs were present in 7 out of the 19 phyla analyzed. A phylogenetic analysis showed that the identified NRPSs form clades distinct from other adenylate-forming enzymes that contain similar domains such as fatty acid synthases. NRPSs show a remarkably scattered distribution over the animal kingdom. They are especially abundant in rotifers and nematodes. In rotifers, we found a large variety of domain architectures and predicted substrates. In the nematode Plectus sambesii, we identified the beta-lactam biosynthesis genes L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine synthetase, isopenicillin N synthase, and deacetoxycephalosporin C synthase that catalyze the formation of beta-lactam antibiotics in fungi and bacteria. These genes are also present in several species of Collembola, but not in other hexapods analyzed so far. In conclusion, our survey showed that NRPS genes are more abundant and widespread in animals than previously known.

Hormone receptor activities of complex mixtures of known and suspect chemicals in personal silicone wristband samplers worn in office buildings.

Humans are exposed to increasingly complex mixtures of hormone-disrupting chemicals from a variety of sources, yet, traditional research methods only evaluate a small number of chemicals at a time. We aimed to advance novel methods to investigate exposures to complex chemical mixtures. Silicone wristbands were worn by 243 office workers in the USA, UK, China, and India during four work shifts. We analyzed extracts of the wristbands for: 1) 99 known (targeted) chemicals; 2) 1000+ unknown chemical features, tentatively identified through suspect screening; and 3) total hormonal activities towards estrogen (ER), androgen (AR), and thyroid hormone (TR) receptors in human cell assays. We evaluated associations of chemicals with hormonal activities using Bayesian kernel machine regression models, separately for targeted versus suspect chemicals (with detection ≥50%). Every wristband exhibited hormonal activity towards at least one receptor: 99% antagonized TR, 96% antagonized AR, and 58% agonized ER. Compared to men, women were exposed to mixtures that were more estrogenic (180% higher, adjusted for country, age, and skin oil abundance in wristband), anti-androgenic (110% higher), and complex (median 836 detected chemical features versus 780). Adjusted models showed strong associations of jointly increasing chemical concentrations with higher hormonal activities. Several targeted and suspect chemicals were important co-drivers of overall mixture effects, including chemicals used as plasticizers, fragrance, sunscreen, pesticides, and from other or unknown sources. This study highlights the role of personal care products and building microenvironments in hormone-disrupting exposures, and the substantial contribution of chemicals not often identifiable or well-understood to those exposures.

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.

Maternal diet and dioxin-like activity, bulky DNA adducts and micronuclei in mother-newborns.

Maternal diet can contribute to carcinogenic exposures and also modify effects of environmental exposures on maternal and fetal genetic stability. In this study, associations between maternal diet and the levels of dioxin-like plasma activity, bulky DNA adducts in white blood cells and micronuclei (MN) in lymphocytes from mother to newborns were examined. From 98 pregnant women living in the greater area of Copenhagen, Denmark in 2006-2007, maternal peripheral blood and umbilical cord blood were collected, together with information on health, environmental exposure and lifestyle. Maternal diet was estimated on the basis of maternal food frequency questionnaire (FFQ) completed by the end of pregnancy. Biomarkers were detected in paired blood samples through the dioxin-responsive chemical-activated luciferase expression (CALUX)(®) bioassay, (32)P-postlabelling technique and cytokinesis-block MN assay. Maternal preference for meats with dark surface were significantly associated with higher bulky DNA adducts in both maternal (ß 95%CI; 0.46 (0.08, 0.84)) and cord blood (ß 95%CI; 0.46 (0.05, 0.86)) before and after adjustment for potential confounders. No other significant associations between the 18 dietary variables and the biomarkers measured in maternal and fetal samples were identified. The present study suggests that maternal intake of meats with dark surface contributes to the bulky DNA adduct levels in maternal and umbilical cord blood. Relationship between food preparation and bulky DNA adducts appear to be captured by a FFQ while potential associations for other biomarkers might be more complex or need larger sample size.

A Functional Carbohydrate Degrading Enzyme Potentially Acquired by Horizontal Gene Transfer in the Genome of the Soil Invertebrate Folsomia candida.

Horizontal gene transfer (HGT) is defined as the acquisition by an organism of hereditary material from a phylogenetically unrelated organism. This process is mostly observed among bacteria and archaea, and considered less likely between microbes and multicellular eukaryotes. However, recent studies provide compelling evidence of the evolutionary importance of HGT in eukaryotes, driving functional innovation. Here, we study an HGT event in Folsomia candida (Collembola, Hexapoda) of a carbohydrate-active enzyme homologous to glycosyl hydrase group 43 (GH43). The gene encodes an N-terminal signal peptide, targeting the product for excretion, which suggests that it contributes to the diversity of digestive capacities of the detritivore host. The predicted α-L-arabinofuranosidase shows high similarity to genes in two other Collembola, an insect and a tardigrade. The gene was cloned and expressed in Escherichia coli using a cell-free protein expression system. The expressed protein showed activity against p-nitrophenyl-α-L-arabinofuranoside. Our work provides evidence for functional activity of an HGT gene in a soil-living detritivore, most likely from a bacterial donor, with genuine eukaryotic properties, such as a signal peptide. Co-evolution of metazoan GH43 genes with the Panarthropoda phylogeny suggests the HGT event took place early in the evolution of this ecdysozoan lineage.

Anti-androgenic compounds in breast milk and cryptorchidism among Norwegian boys in the HUMIS birth cohort.

The prevalence of cryptorchidism has increased over the past decades, yet its origins remain poorly understood. Testis descent is dependent on androgens and likely affected by endocrine disrupting compounds (EDCs), targeting the androgen receptor (AR). We investigated the association between anti-androgenic activity, not derived from natural hormones, in maternal breast milk and impaired testis descent among boys. We performed a case-control study based on 199 breast milk samples from 94 mothers of cryptorchid boys and 105 random non-cryptorchid boys participating in the Norwegian HUMIS (Human Milk Study) cohort. For each participant, apolar, and polar fractions were extracted, and combined to reconstitute a mixture. Anti-androgenic activity was measured in all three fractions using the human cell-based in vitro anti-AR CALUX® assay and expressed in µg of flutamide equivalent, a well-known antiandrogen. Results from fraction analyses were compared among boys with cryptorchidism and controls using multiple logistic regression, controlling for appropriate confounders identified using a directed acyclic graph. Children's daily exposure to anti-androgenic EDCs through breastfeeding was estimated to 78 µg flutamide eq./kg of body weigh/day. The activity was higher in the polar fraction (1.48 ± 1.37 µg flutamide eq./g of milk) mainly representing non-persistent chemicals, in contrast to other fractions. However, the activity in the polar extracts was decreased when in mixtures with the apolar fraction, indicating synergistic interactions. No significant difference in the activity was observed according to cryptorchid status for polar, apolar or mixed breast milk fractions. The study showed anti-androgenic activity in nearly all human milk samples, and at levels higher than the advisory threshold. However, no significant association was observed between cryptorchidism and antiandrogenic activity measured in either polar, apolar, or mixture fractions derived from breast milk.

Paleotoxicity of petrogenic and pyrogenic hydrocarbon mixtures in sediment cores from the Athabasca oil sands region, Alberta (Canada).

Despite the economic benefits of the oil and gas industry in Northern Alberta, significant concerns exist regarding the impacts of increased oil production on the environment and human health. Several studies have highlighted increases in the concentrations of polycyclic aromatic compounds (PACs) and other hydrocarbons in the atmosphere, water, soil and sediments, plants, wildlife and fish in the Athabasca Oil Sands Region (AOSR) as a result of oil sands industrial activity. Sediment cores can provide information on the temporal trends of contaminants to the environment and provide important baseline information when monitoring data are absent. Here we combined analytical chemistry and a mammalian cell-based bioassay in dated lake sediment cores to assess paleotoxicity in freshwater systems in the AOSR. Sediment intervals were radiometrically dated and subsequently analysed for PACs. PAC extracts from select dated intervals were used in cell-based bioassays to evaluate their endocrine disrupting properties. We demonstrated spatial and temporal variability in the PAC composition of sediment cores around the AOSR with some of the highest concentrations of PACs detected near oil sands industrial activity north of Fort McMurray (AB) in La Saline Natural Area. Recent sediment had positive enrichment factors across most PAC analytes at this site with heavier pyrogenic compounds such as benz(a)anthracene/chrysene and benzofluoranthene/benzopyrene dominating. Our study is the first to link chemical analysis of sediment cores with biological effect assessments of endocrine activity showing feasibility of extending the usefulness of sediment cores in monitoring programs interested in complex mixture assessments. While we observed no spatial or temporal differences in ERα mediated signaling, AhR CALUX results mirrored those of the chemical analysis, demonstrating the utility of coupling biological effects assessments to historical reconstructions of contaminant inputs to the natural environment.

Correlating biodegradation kinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin to the dynamics of microbial communities originating from soil in Vietnam contaminated with herbicides and dioxins.

We studied the succession of bacterial communities during the biodegradation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). The communities originated from a mesocosm with soil from Bien Hoa airbase in Vietnam heavily contaminated with herbicides and dioxins. They were grown in defined media with different carbon and Gibbs energy sources and 2,3,7,8-TCDD. Cultures with dimethyl sulfoxide (DMSO) as the sole carbon and energy source degraded about 95% of 2,3,7,8-TCDD within 60 days of cultivation. Those with an additional 1 mM of vanillin did that in roughly 90 days. Further 16S rRNA gene amplicon sequencing showed that the increase in relative abundance of members belonging to the genera Bordetella, Sphingomonas, Proteiniphilum, and Rhizobium correlated to increased biodegradation of 2,3,7,8-TCDD in these cultures. A higher concentration of vanillin slowed down the biodegradation rate. Addition of alternative carbon and Gibbs energy sources, such as amino acids, sodium lactate and sodium acetate, even stopped the degradation of 2,3,7,8-TCDD completely. Bacteria from the genera Bordetella, Achromobacter, Sphingomonas and Pseudomonas dominated most of the cultures, but the microbial profiles also significantly differed between cultures as judged by non-metric multidimensional scaling (NMDS) analyses. Our study indicates that 2,3,7,8-TCDD degradation may be stimulated by bacterial communities preadapted to a certain degree of starvation with respect to the carbon and energy source. It also reveals the succession and abundance of defined bacterial genera in the degradation process.

Identification of major dioxin-like compounds and androgen receptor antagonist in acid-treated tissue extracts of high trophic-level animals.

We evaluated the applicability of combining in vitro bioassays with instrument analyses to identify potential endocrine disrupting pollutants in sulfuric acid-treated extracts of liver and/or blubber of high trophic-level animals. Dioxin-like and androgen receptor (AR) antagonistic activities were observed in Baikal seals, common cormorants, raccoon dogs, and finless porpoises by using a panel of rat and human cell-based chemical-activated luciferase gene expression (CALUX) reporter gene bioassays. On the other hand, no activity was detected in estrogen receptor α (ERα)-, glucocorticoid receptor (GR)-, progesterone receptor (PR)-, and peroxisome proliferator-activated receptor γ2 (PPARγ2)-CALUX assays with the sample amount applied. All individual samples (n = 66) showed dioxin-like activity, with values ranging from 21 to 5500 pg CALUX-2,3,7,8-tetrachlorodibenzo-p-dioxin equivalent (TEQ)/g-lipid. Because dioxins are expected to be strong contributors to CALUX-TEQs, the median theoretical contribution of dioxins calculated from the result of chemical analysis to the experimental CALUX-TEQs was estimated to explain up to 130% for all the tested samples (n = 54). Baikal seal extracts (n = 31), but not other extracts, induced AR antagonistic activities that were 8-150 µg CALUX-flutamide equivalent (FluEQ)/g-lipid. p,p'-DDE was identified as an important causative compound for the activity, and its median theoretical contribution to the experimental CALUX-FluEQs was 59% for the tested Baikal seal tissues (n = 25). Our results demonstrate that combining in vitro CALUX assays with instrument analysis is useful for identifying persistent organic pollutant-like compounds in the tissue of wild animals on the basis of in vitro endocrine disruption toxicity.

Developing potency factors for thyroid hormone disruption by PFASs using TTR-TRß CALUX® bioassay and assessment of PFASs mixtures in technical products.

BACKGROUND: Over the last decade, per- and polyfluoroalkyl substances (PFASs) have become one of the most heavily investigated persistent organohalogen compound class of environmental concern. However, knowledge about their toxicology is still scarce, although PFASs as individual compounds and their industrial mixtures were shown to exert effects on the thyroid hormone system. METHODS: In vitro toxicity potency factors were established for thyroid hormone transport disruption potential using the novel TTR-TRß CALUX® bioassay for major PFASs. We assessed technical PFASs mixtures, including aqueous film-forming foam (AFFF) surfactants and chromium mist suppressants (CMS) applications with and without total oxidizable precursor (TOP) by TTR-TRß CALUX® assay for their thyroid hormone transport disrupting potential. RESULTS: All PFASs listed in the German guideline for drinking water (German Environment Agency, 2017) affected the T4 binding to TTR, an important plasma thyroid hormone transport protein. For all tested PFASs, potency factors based on PC80 values relative to PFOA could be obtained and ranged between PFBA (0.0018) and PFOS (2.0). Applying in vitro potency factors obtained from the present in vitro TTR-TRß CALUX® assay study and recently reported in vivo potency factors (Zeilmaker et al., 2018; Bil et al., 2021) on the above-mentioned German guideline for PFAS in drinking water, showed that the cumulative effect-based trigger values (in vivo and in vitro) are comparable (3.0 vs. 2.9 to 4.6 µg PFOA-EQ/l). Additionally, AFFF surfactants and CMS with and without TOP assay were tested. Highest activities were found in the older AFFF surfactants (2013/2014) due to higher PFOS/PFOA levels, which were already substituted with 6:2 FTS in 2019, resulting in much lower PFOA-EQ levels. As expected also the PFOA-EQ levels increased in the samples with TOP treatment compared to the original AFFF surfactants and CMS as confirmed here by biological and chemical PFOA-equivalents (PFOA-EQ) analysis. Additionally, CMS (which have been used in the electroplating chromium industry since the 1950s) as well as PFOS-free, but not PFAS-free fume suppressants (such as Fumetrol® 21) have been tested in the TTR-TRß CALUX® assay and showed much lower activity levels then the AFFFs, confirmed by the similar potency determination based on chemical PFASs analysis followed by transformation to PFOA-EQ for comparison. The potency factor of 6:2 FTS, which is the main substitute for PFOS in CMS, indicates that it is approximately 100-times less potent as a thyroid hormone disruptor as compared to PFOS. CONCLUSION: Potency factors based on PC80 values from TTR-TRß CALUX® relative to PFOA have been developed for major PFASs. In AFFF surfactants and CMS a trend of higher activities with higher amounts of PFOS and PFOA have been found. PFOA and PFOS showed high responses in the TTR-TRß CALUX® assay and had the largest contributions to the PFOA-EQs in the AFFF surfactants and CMS applications. Using potency factors as determined in the TTR-TRß CALUX® to convert PFASs assessed by chemical analysis to PFOA-EQ led to comparable results as compared to the results from PFASs measured directly by the TTR-TRß CALUX® assay. This study supports the claim that semiquantitative effect- and group-based in vitro CALUX bioanalysis tools can be applied effectively to assess industrial products containing complex mixtures with PFAS compounds for which no instrumental analysis are established, and for many compounds where in vitro toxicity data are not yet available.

Elucidating fungal Rigidoporus species FMD21 lignin-modifying enzyme genes and 2,3,7,8-tetrachlorodibenzo-p-dioxin degradation by laccase isozymes.

White-rot fungus Rigidoporus sp. FMD21 is a lignin-modifying enzyme producing fungus that can degrade dioxin. Extracellular enzymes from FMD21 include laccase and manganese peroxidase which are promising enzymes for myco-remediation because of their wide substrate specificity and mild catalysis conditions. The FMD21 genome was sequenced using Ion Torrent technology and consists of 38.98 Mbps with a GC content of 47.4 %. Gene prediction using Augustus with Basidiomycota reference setting resulted in 8245 genes. Functional gene annotations were carried out by using several programs and databases. We focused on laccase and ligninolytic peroxidase genes, which are most likely involved in the degradation of aromatic pollutants. The genome of FMD21 contains 12 predicted laccase genes (10 out of 12 predicted as full length) and 13 putative ligninolytic peroxidases which were annotated as MnP or versatile peroxidases. Four predicted laccases showed a higher than 65 % binding chance to 2,3,7,8-TCDD with the highest at 72 % in in silico docking analysis. Heterologous expressed laccases showed activity towards three tested substrates included ABTS, guaiacol and 2,6-DMP. ABTS displayed two-stage oxidation which differed from natural FMD21 laccases. 2,3,7,8-TCDD was degraded by 50 % after two weeks of enzymatic treatment by three out of five laccase isozymes which were natural laccases secreted by FMD21. In this study, we provide direct evidence for the 2,3,7,8-TCDD biodegradation capability of fungal laccases.

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.

Characterization of 2,3,7,8-tetrachlorodibenzo-p-dioxin biodegradation by extracellular lignin-modifying enzymes from ligninolytic fungus.

Ligninolytic fungi secrete extracellular lignin-modifying enzymes (LME) that degrade plant polymers for fungal nutrition but that are, because of their broad substrate specificity, also applicable for the degradation of many hazardous pollutants. Laccase is one of the most well characterized LME and is involved in the removal and degradation of recalcitrant aromatic compounds with or without the assistance of laccase-mediators. The Ligninolytic fungus Rigidoporus sp. FMD21 can degrade 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) with a half-life of 6.2 days. Using Rigidoporus sp. FMD21 crude extracellular enzyme extract (ExE) that mainly consisted of laccase, 77.4% of 2,3,7,8-TCDD was degraded within 36 days. The degradation rate did not depend on the 2,3,7,8-TCDD concentration in the tested range between 0.005 and 0.5 pgTEQ/µL. 2,3,7,8-TCDD was analysed by DR-CALUX® bioassay and the degradation was confirmed by GC-HRMS. In this study, we found evidence for cleavage of the diaryl ether bond in the 2,3,7,8-TCDD molecule and here we propose a new degradation mechanism in which 3,4-dichlorophenol is the main metabolite of 2,3,7,8-TCDD degradation by FMD21's ExE. Six laccase-mediators were tested. Three of them 1-hydroxybenzotriazole (HBT), syringaldehyde (Syr) and violuric acid (Vio) showed an equipotent added effect on 2,3,7,8-TCDD degradation by ExE, however only in case of Vio a level of significance was reached. The others showed no effect or negatively impacted degradation. In conclusion, we have shown that Rigidoporus sp. FMD21 produces extracellular enzymes, mainly laccases that apparently are able to degrade the highly recalcitrant and most toxic 2,3,7,8-congener of TCDD via diaryl bond cleavage into 3,4-dichlorophenol.