2023, Gori, Gio B., Aschner, Michael, Borgert, Christopher J., Cohen, Samuel M., Dietrich, Daniel R., Galli, Corrado L., Greim, Helmut, Heslop-Harrison, John S., Kacew, Sam, Kaminski, Norbert E.

The 1958 Delaney amendment to the Federal Food Drug and Cosmetics Act prohibited food additives causing cancer in animals by appropriate tests. Regulators responded by adopting chronic lifetime cancer tests in rodents, soon challenged as inappropriate, for they led to very inconsistent results depending on the subjective choice of animals, test design and conduct, and interpretive assumptions. Presently, decades of discussions and trials have come to conclude it is impossible to translate chronic animal data into verifiable prospects of cancer hazards and risks in humans. Such conclusion poses an existential crisis for official agencies in the US and abroad, which for some 65 years have used animal tests to justify massive regulations of alleged human cancer hazards, with aggregated costs of $trillions and without provable evidence of public health advantages. This article addresses suitable remedies for the US and potentially worldwide, by critically exploring the practices of regulatory agencies vis-á-vis essential criteria for validating scientific evidence. According to this analysis, regulations of alleged cancer hazards and risks have been and continue to be structured around arbitrary default assumptions at odds with basic scientific and legal tests of reliable evidence. Such practices raise a manifold ethical predicament for being incompatible with basic premises of the US Constitution, and with the ensuing public expectations of testable truth and transparency from government agencies. Potential remedies in the US include amendments to the US Administrative Procedures Act, preferably requiring agencies to justify regulations compliant with the Daubert opinion of the Daubert ruling of the US Supreme Court, which codifies the criteria defining reliable scientific evidence. International reverberations are bound to follow what remedial actions may be taken in the US, the origin of current world regulatory procedures to control alleged cancer causing agents.

2021-04-15, Monteleone, Adrian, Schary, Weronika, Wenzel, Folker, Langhals, Heinz, Dietrich, Daniel R.

As plastic pollution is becoming an increasing worldwide problem, a variety of different techniques for the detection and in-depth characterization of plastics, including spectroscopy and chromatography methods, were introduced to the public. Recently we presented fluorescence lifetime imaging microscopy (FLIM) a new approach for the identification and characterization of microplastics using their fluorescence lifetime (τ) for differentiation. A very powerful extension of the recently established FLIM could be phasor analysis, which allows data representation in an interactive 2D graphical phasor plot thereby enabling a global view of the fluorescence decay in each pixel of the measured image. Microplastic particles generated from six different types of plastics were subjected to excitation wavelengths of 440 nm, upon which specific fluorescence lifetimes as well as the photon yield were determined using FLIM and phasor analysis. We could show that phasor analysis for FLIM with a laser pulse repetition frequency of 40 MHz was able to generate specific locations in the phasor plot for the plastics for fast differentiation, e.g. resulting in well-defined phasor plot positions for ABS at 3.019 ns, PPE at 6.239 ns, PET bottle from Germany at 2.703 ns and PET bottle from USA at 2.711 ns. Phasor analysis for FLIM proves to be a fast, label-free, and sensitive method for the identification and differentiation of plastics also with the aid of visualization variation enabling techniques such as heat treatment of plastics.

2021, Monteleone, Adrian, Wenzel, Folker, Langhals, Heinz, Dietrich, Daniel R.

The problem of micro- and nanoplastic (short: plastics) pollution is an increasing global issue and therefore several detection methods for plastics, also investigating the chemical nature via spectroscopy and chromatography, have been developed over the years. A new approach for identification and characterization of plastics is fluorescence lifetime imaging microscopy (FLIM) – a microspectroscopic method to detect fluorescence lifetime (τ) of plastics. We tested whether FLIM can be employed for the identification and characterization of plastics. Six types of plastics (ABS, PA6, PET, PLA, PPE, PU), with and without prior heat treatment, were subjected to FLIM with excitation wavelengths of 470 nm and 440 nm. The results provided mean τ (intensity weighted) values of 3.850 (+- 0.033) ns for ABS, 8.143 (+- 0.060) ns for PPE, 3.519 (+-0.090) ns for PET of a bottle from Germany and 3.564 (+-0.126) ns for PET of a bottle from the USA. The combination of mean intensity weighted τ and mean amplitude weighted τ values allowed for the significant differentiation of 52 (94.55%) of the 55 possible plastic comparisons. Moreover, FLIM showed the potential for the sub-micrometer range plastic characterization, phasor analysis and allows for visual 3D-sectioning of samples that could be important for identification and characterization of plastics in tissue and environmental samples.

2020-08, Autrup, Herman, Barile, Frank A., Berry, Sir Colin, Blaauboer, Bas J., Boobis, Alan, Bolt, Herrmann, Hengstler, Jan, Borgert, Christopher J., Dietrich, Daniel R., Greim, Helmut

Theoretically, both synthetic endocrine disrupting chemicals (S-EDCs) and natural (exogenous and endogenous) endocrine disrupting chemicals (N-EDCs) can interact with endocrine receptors and disturb hormonal balance. However, compared to endogenous hormones, S-EDCs are only weak partial agonists with receptor affinities several orders of magnitude lower. Thus, to elicit observable effects, S-EDCs require considerably higher concentrations to attain sufficient receptor occupancy or to displace natural hormones and other endogenous ligands.

Significant exposures to exogenous N-EDCs may result from ingestion of foods such as soy-based diets, green tea and sweet mustard. While their potencies are lower as compared to natural endogenous hormones, they usually are considerably more potent than S-EDCs.

Effects of exogenous N-EDCs on the endocrine system were observed at high dietary intakes. A causal relation between their mechanism of action and these effects is established and biologically plausible. In contrast, the assumption that the much lower human exposures to S-EDCs may induce observable endocrine effects is not plausible. Hence, it is not surprising that epidemiological studies searching for an association between S-EDC exposure and health effects have failed.

Regarding testing for potential endocrine effects, a scientifically justified screen should use in vitro tests to compare potencies of S-EDCs with those of reference N-EDCs. When the potency of the S-EDC is similar or smaller than that of the N-EDC, further testing in laboratory animals and regulatory consequences are not warranted.

2022-05-06, Piossek, Felicitas, Beneke, Sascha, Schlichenmaier, Nadja, Mucic, Goran, Drewitz, Sabine, Dietrich, Daniel R.

Reliable prediction of compound mediated nephrotoxicity in humans is still unsatisfactory irrespective of the recent advancements in in silico, in vitro and in vivo models. Therefore, current in vitro approaches need refinement to better match the human in vivo situation, specifically with regard to the potential influence of other cell types (e.g. fibroblasts) and to the potential biases introduced by the excessive 21% O₂ (AtmOx) as employed in routine cell culturing. We used a transwell co-culture model combining human renal proximal tubule epithelial cells (RPTEC/TERT1) and human fibroblasts (fHDF/TERT166) to compare the functional properties and expression of selected marker proteins at 21% O₂ and at the physiologically normal 10% O₂ tension (PhysOx) commensurate with in vivo conditions. Culturing at PhysOx and co-culturing with fibroblasts significantly improved epithelial barrier integrity, expression of transporters (e.g. aquaporin 2; OCT-MATE; MRP-OAT) and metabolism. Moreover, beyond culturing these human cells in co-culture for up to 41 days, we were able to demonstrate increased functionality of cation transport, as shown via ASP⁺ (OCT-MATE axis), and anion transport, as shown via LY (MRP-OAT axis). Thus, adjusting the in vitro system to near physiological conditions had a major impact on functionality and provides the basis for the future development of true flow-through microfluidic renal testing systems with better predictability of human renal proximal toxicity.

2021, Dietrich, Daniel R.

2020-11, Weisbrod, Barbara, Riehle, Eva, Helmer, M., Martin-Creuzburg, Dominik, Dietrich, Daniel R.

Cyanobacterial blooms often consist of numerous co-existing cyanobacterial species, with predominant taxa dynamically varying intra-annually. Parasitism by fungi (chytrids) has come into focus as an important factor driving short-term bloom dynamics. Using microscopic analysis, Illumina sequencing and cyanobacterial toxin analyses, we monitored the seasonal succession of Dolichospermum blooms in a reservoir along with environmental parameters. We identified two consecutive Dolichospermum blooms that were characterized by a straight and a coiled morphotype, separated by a complete bloom collapse. Phylotyping provided evidence for three putative Dolichospermum amplicon sequence variants (ASVs); i.e. Dolichospermum1 & 2 in the first bloom (straight filaments) and Dolichospermum3 in the second bloom (coiled filaments). Morphotype succession as well as total filament concentration did not correlate with any of the measured environmental parameters. Fungal parasitism by the chytrid Rhizosiphon crassum occurred in straight Dolichospermum filaments only. Coiled filaments showed no infection despite ambient presence of chytrids, deduced from fungal ASVs, throughout the entire observation period. Toxin concentrations (microcystins (MCs) and anabaenopeptins) correlated significantly with the abundance of the straight Dolichospermum morphotype. Enhanced cyanotoxin biosynthesis in the straight Dolichospermum morphotype, interpreted as a defensive reaction to fungal parasitism, appeared to come at the expense of lowered competitiveness with the co-occurring coiled morphotype. Our findings support the hypothesis that selective parasitism by chytrids is an important factor driving short-term morphotype and toxin dynamics within cyanobacterial blooms.

2022-01-05, Jaeger, Sabrina, Nitschke, Jahn, Altaner, Stefan, Klein, Karsten, Dietrich, Daniel R., Schreiber, Falk

Microcystins (MC) are a group of structurally similar cyanotoxins with currently 279 described structural variants. Human exposure is frequent by consumption of contaminated water, food or food supplements. MC can result in serious intoxications, commensurate with ensuing pathology in various organs or in rare cases even mortality. The current WHO risk assessment primarily considers MC-LR, while all other structural variants are treated as equivalent to MC-LR, despite that current data strongly suggest that MC-LR is not the most toxic MC, and toxicity can be very different for MC congeners. To investigate and analyse binding and conformation of different MC congeners, we applied for the first time Molecular Dynamics (MD) simulation to four MC congeners (MC-LR, MC-LF, [Enantio-Adda5]MC-LF, [β-D-Asp3,Dhb7]MC-RR). We could show that ser/thr protein phosphatase 1 is stable in all MD simulations and that MC-LR backbone adopts to a second conformation in solvent MD simulation, which was previously unknown. We could also show that MC congeners can adopt to different backbone conformation when simulated in solvent or in complex with ser/thr protein phosphatase 1 and differ in their binding behaviour. Our findings suggest that MD Simulation of different MC congeners aid in understanding structural differences and binding of this group of structurally similar cyanotoxins.

2021, Kelly, Laura T., Puddick, Jonathan, Borges, Hugo, Dietrich, Daniel R., Hamilton, David P., Wood, Susanna A.

2020-09, Autrup, Herman, Barile, Frank A., Berry, Sir Colin, Blaauboer, Bas J., Boobis, Alan, Bolt, Herrmann, Borgert, Christopher J., Dekant, Wolfgang, Dietrich, Daniel R., Greim, Helmut

Theoretically, both synthetic endocrine disrupting chemicals (S-EDCs) and natural (exogenous and endogenous) endocrine disrupting chemicals (N-EDCs) can interact with endocrine receptors and disturb hormonal balance. However, compared to endogenous hormones, S-EDCs are only weak partial agonists with receptor affinities several orders of magnitude lower. Thus, to elicit observable effects, S-EDCs require considerably higher concentrations to attain sufficient receptor occupancy or to displace natural hormones and other endogenous ligands.

Significant exposures to exogenous N-EDCs may result from ingestion of foods such as soy-based diets, green tea and sweet mustard. While their potencies are lower as compared to natural endogenous hormones, they usually are considerably more potent than S-EDCs.

Effects of exogenous N-EDCs on the endocrine system were observed at high dietary intakes. A causal relation between their mechanism of action and these effects is established and biologically plausible. In contrast, the assumption that the much lower human exposures to S-EDCs may induce observable endocrine effects is not plausible. Hence, it is not surprising that epidemiological studies searching for an association between S-EDC exposure and health effects have failed.

Regarding testing for potential endocrine effects, a scientifically justified screen should use in vitro tests to compare potencies of S-EDCs with those of reference N-EDCs. When the potency of the S-EDC is similar or smaller than that of the N-EDC, further testing in laboratory animals and regulatory consequences are not warranted.