Conflicts of Interest in the Assessment of Chemicals, Waste, and Pollution.

Pollution by chemicals and waste impacts human and ecosystem health on regional, national, and global scales, resulting, together with climate change and biodiversity loss, in a triple planetary crisis. Consequently, in 2022, countries agreed to establish an intergovernmental science-policy panel (SPP) on chemicals, waste, and pollution prevention, complementary to the existing intergovernmental science-policy bodies on climate change and biodiversity. To ensure the SPP's success, it is imperative to protect it from conflicts of interest (COI). Here, we (i) define and review the implications of COI, and its relevance for the management of chemicals, waste, and pollution; (ii) summarize established tactics to manufacture doubt in favor of vested interests, i.e., to counter scientific evidence and/or to promote misleading narratives favorable to financial interests; and (iii) illustrate these with selected examples. This analysis leads to a review of arguments for and against chemical industry representation in the SPP's work. We further (iv) rebut an assertion voiced by some that the chemical industry should be directly involved in the panel's work because it possesses data on chemicals essential for the panel's activities. Finally, (v) we present steps that should be taken to prevent the detrimental impacts of COI in the work of the SPP. In particular, we propose to include an independent auditor's role in the SPP to ensure that participation and processes follow clear COI rules. Among others, the auditor should evaluate the content of the assessments produced to ensure unbiased representation of information that underpins the SPP's activities.

An evaluation of DNA extraction methods on historical and roadkill mammalian specimen.

Guidelines identifying appropriate DNA extraction methods for both museum and modern biological samples are scarce or non-existent for mammalian species. Yet, obtaining large-scale genetic material collections are vital for conservation and management purposes. In this study, we evaluated five protocols making use of either spin-column, organic solvents, or magnetic bead-based methods for DNA extraction on skin samples from both modern, traffic-killed (n = 10) and museum (n = 10) samples of European hedgehogs, Ericaneus europaeus. We showed that phenol-chloroform or silica column (NucleoSpin Tissue) protocols yielded the highest amount of DNA with satisfactory purity compared with magnetic bead-based protocols, especially for museum samples. Furthermore, extractions using the silica column protocol appeared to produce longer DNA fragments on average than the other methods tested. Our investigation demonstrates that both commercial extraction kits and phenol-chloroform protocol retrieve acceptable DNA concentrations for downstream processes, from degraded remnants of traffic-killed and museum samples of mammalian specimens. Although all the tested methods could be applied depending on the research questions and laboratory conditions, commercial extraction kits may be preferred due to their effectiveness, safety and the higher quality of the DNA extractions.

Estrogen receptor genes in gastropods: phylogenetic divergence and gene expression responses to a synthetic estrogen.

Endocrine disrupting chemicals (EDCs) have the potential to affect development and reproduction in gastropods. However, one is today lacking basic understanding of the Molluscan endocrine system and one can therefore not fully explain these EDC-induced affects. Furthermore, only a few genes that potentially may be connected to the endocrine system have been sequenced in gastropods. An example is the estrogen receptor gene (er) that have been identified in a restricted number of freshwater and marine gastropods. Here, we have identified a new partial coding sequence of an estrogen receptor gene (er) in the European common heterobranch Radix balthica. The following phylogenetic analysis divided the ers of heterobranchs and ceanogastropods in two branches. Furthermore, exposure to the synthetic estrogen 17α-ethinylestradiol (EE2) showed that exposure could significantly affect er expression level in the heterobranch R. balthica. This paper is the first that phylogenetically compares gastropods' er, basal er expression profiles, and transcriptional estrogenic responses in gastropods from two different evolutionary groups.

17α-Ethinylestradiol (EE2) treatment of wild roach (Rutilus rutilus) during early life development disrupts expression of genes directly involved in the feedback cycle of estrogen.

Fish are more sensitive to introduced disturbances from synthetic endocrine disrupting compounds during early life phases compared with mature stages. 17α-Ethinylestradiol (EE2), which is the active compound in human oral contraceptives and hormone replacement therapies, is today ever present in the effluents from sewage treatment plants. EE2 targets and interacts with the endogenous biological systems of exposed vertebrates resulting in to large extents unknown short- and long-term effects. We investigated how EE2 exposure affects expression profiles of a large number of target genes during early life of roach (Rutilus rutilus). We exposed fertilized roach eggs collected from a lake in Southern Sweden to EE2 for 12weeks together with 1+-year-old roach in aquaria. We measured the gene expression of the estrogen receptor (esr)1/2a/2b, androgen receptor (ar), vitellogenin, cytochrome P450 (cyp)19a1a/1b in fertilized eggs; newly hatched larvae; 12-week-old fry; and juvenile wild roach (1+-year-old). Results shows that an EE2 concentration as low as 0.5ng/L significantly affects gene expression during early development. Gene expression responses vary both among life stages and molecular receptors. We also show that the gene profile of the estrogen feedback cycle to a large extent depends on the relationship between the three esr genes and the two cyp19a1 genes, which are all up-regulated with age. Results indicate that a disruption of the natural activity of the dominant esr gene could lead to detrimental biological effects if EE2 exposure occurs during development, even if this exposure occurred for only a short period.

Unraveling the estrogen receptor (er) genes in Atlantic salmon (Salmo salar) reveals expression differences between the two adult life stages but little impact from polychlorinated biphenyl (PCB) load.

Estrogen receptors (ers) not only are activated by hormones but also interact with many human-derived environmental contaminants. Here, we present evidence for four expressed er genes in Atlantic salmon cDNA - two more ers (erα2 and erß2) than previously published. To determine if er gene expression differs between two adult life-stages we sampled 20 adult salmon from the feeding phase in the Baltic Sea and during migration in the River Mörrum, Sweden. Results show that all four er genes are present in the investigated tissues, except for erα2 not appearing in the spleen. Overall, a profile analysis reveals the erα1 gene to be the most highly expressed er gene in both female and male Baltic Sea salmon tissues, and also in female River Mörrum salmon. In contrast, this gene has the lowest gene expression level of the four er genes in male salmon from the River Mörrum. The erα2 gene is expressed at the lowest levels in both female/male Baltic Sea salmon and in female River Mörrum salmon. Statistical analyses indicate a significant and complex interaction where both sex and adult life stage can impact er gene expression. Regression analyses did not demonstrate any significant relationship between polychlorinated biphenyl (PCB) body burden and er gene expression level, suggesting that accumulated pollutants from the Baltic Sea may be deactivated inside the salmon's lipid tissues and have limited impact on er activity. This study is the first comprehensive analysis of four er gene expression levels in two wild salmon populations from two different adult life stages where information about PCB load is also available.

Identification of an estrogen receptor gene in the natural freshwater snail Bithynia tentaculata.

Mollusks have received increasing interest in ecotoxicological studies but so far the available scientific analyses of how their genes are affected by anthropogenic pollutants are scarce. The focus of this study is to identify an estrogen receptor (er) gene in the common prosobranch snail Bithynia tentaculata and to test a hypothesis that 17α-Ethinylestradiol (EE2) will modulate er gene expression after short-term exposure. We set up exposure experiments with a total of 144 snails, which were collected from a natural population in southern Sweden. Snails were exposed to either 10ng/L or 100ng/L EE2 during 24h and/or 72h. From the isolated B. tentaculata RNA we successfully identified and characterized a novel er gene and phylogenetic analyses strongly indicate that the Bithynia er gene is an ortholog to the human ERα (ESR1, NR3A1). We found a significant interaction between EE2-dose and exposure duration on the er's gene expression (Two-way ANOVA; p=0.04). We also found a significant difference in the gene expression of the er when comparing the control and 100ng/L treatment groups after 72h in female snails (One-way ANOVA; p=0.047). The results from this study should be useful for future field-related studies of estrogen receptors in natural populations of mollusks.

Polychlorinated biphenyl (PCB) load, lipid reserves and biotransformation activity in migrating Atlantic salmon from River Mörrum, Sweden.

Atlantic salmon accumulate high levels of contaminants such as polychlorinated biphenyls (PCBs) in their lipids during the adult growth phase spent at sea. The lipids are later utilized during migration for swimming and biological adaptations. We hypothesize that migrating salmons' biotransformation processes are affected by the high levels of built-up PCBs compared to salmon that in a pre-migrational stage. For these analyses we sampled adult Atlantic salmon during migration in the Swedish River Mörrum and measured the 21 most common PCB congeners ( summation operatorPCB) and lipid levels in muscle tissue, aryl hydrocarbon receptor (AHR2) and cytochrome P4501A1 (CYP1A1) transcript levels as well as ethoxyresorufin-O-deethylase activity (EROD) in liver. We also determined which AHR2 genotypes the salmon carried. We show that EROD activity is correlated to CYP1A1 level but not to summation operatorPCB concentration. summation operatorPCB concentration does not predict levels of neither the AHR2 nor CYP1A1 genes. We find no associations between specific AHR2 transcription levels and AHR2 genotypes or a correlation between AHR2 and CYP1A1 transcription levels, which is in direct contrast to pre-migrational adult salmon from the Baltic Sea. When we compare River Mörrum to salmon we have previously sampled in the Baltic Sea we show that migrating salmon have significantly lower lipid levels in their muscles; higher muscle concentrations of summation operatorPCB on a lipid basis; and significantly lower CYP1A1 and EROD levels compared to salmon from the Baltic Sea. Also, transcript levels of three out of four AHR2 genes are significantly different. In conclusion, migrating Swedish Atlantic salmon carry higher concentrations of PCBs in their lipids compared to salmon in the Baltic Sea, but have lower activation of biotransformation genes and enzymes. Our results indicate that accumulated pollutants from the Baltic Sea are deactivated inside the migrating salmon's lipid tissues and increase in concentration when migration is initiated thereby limiting their impact on biotransformation processes.

Functional properties of the four Atlantic salmon (Salmo salar) aryl hydrocarbon receptor type 2 (AHR2) isoforms.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which organochlorine contaminants including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and some polycyclic aromatic hydrocarbons induce toxicity and altered gene expression. Atlantic salmon has multiple AHR genes, of which two belong to the AHR1 clade and four belong to the AHR2 clade. The four AHR2 forms (alpha, beta, gamma, delta) are more highly expressed than the AHR1 (alpha, beta,) forms and all six AHRs are highly similar in pairs, likely originating from a whole-genome duplication in the salmonid ancestor. It has been speculated that having multiple AHRs contributes to the very high sensitivity of salmonid species to TCDD and related chemicals. To test the hypothesis that all four salmon AHR2 proteins are expressed and functional, we measured mRNA transcription for each AHR2 in several tissues, cloned the cDNAs and evaluated the functional properties of the expressed proteins. Analysis by real-time PCR revealed that the receptors showed differences in transcript levels among salmon tissues and that in general AHR2alpha was transcribed at higher levels than the other three AHR2s. Velocity sedimentation analysis showed that all four in vitro-expressed AHR2 proteins exhibit specific, high-affinity binding of [(3)H]TCDD. When expressed in COS-7 cells, all four AHR2 proteins were able to drive the expression of a reporter gene under control of murine CYP1A1 enhancer elements. From EC(50) values determined in TCDD concentration-response experiments, all four salmon AHR2s show similar sensitivity to TCDD. In summary, all four Atlantic salmon AHR2 appear to function in AHR-mediated signaling, suggesting that all four proteins are involved in TCDD-mediated toxicity.

Polychlorinated biphenyl load, aryl hydrocarbon receptor, and cytochrome P4501A1 induction in a wild population of Atlantic salmon (Salmo salar) from the Baltic Sea.

The toxicity induced by several environmental pollutants is mediated by the aryl hydrocarbon receptor (AHR), which controls the expression of many biotransformation genes, such as cytochrome P4501A1 (CYP1A1). Previous studies have indicated that fish populations can evolve tolerance to persistent chlorinated pollutants by down-regulating the AHR pathway. Here, we measure to what extent tissue loads of polychlorinated biphenyl (PCB) congeners and AHR genotypes contribute to biotransformation capacity in wild, foraging Atlantic salmon (Salmo salar L.) from the Baltic Sea. In muscle, the sum of the 21 most common PCB congeners (ZPCB) was correlated with three extracted AHR agonists (PCBs 77/110, 118/123/149, and 105/132/153). Both the AHR agonists as well as sigmaPCB were correlated with lipid content. The sigmaPCB, controlled for the effects of sex and lipid content in muscle tissue, did not predict mRNA transcript levels of the measured AHRs (AHR2alpha, AHR2gamma, and AHR2delta) or CYP1A1 in liver. However, all AHR2 mRNA transcript levels were positively correlated with CYP1A1 level. In turn, the CYP1A1 level was negatively correlated with concentration of the muscle-tissue antioxidant astaxanthin, suggesting that astaxanthin is depleted when biotransformation processes (CYP1A1) are activated. No correlation was found between ethoxyresorufin-O-deethylase activity and sigmaPCB, CYP1A1, or antioxidant levels. In 5'-flanking regions of the AHR2 genes, we identified multiple allelic variants that were used for genotyping. The mRNA transcript level of AHR2alpha was significantly associated with the AHR2alpha 5'-flanking region genotype and with the interaction of the genotype and individual PCB level. These results suggest that in wild Atlantic salmon from the Baltic Sea, active production of AHR2 mRNA by means of PCB exposure may be affected by genetic polymorphisms at the AHR2 loci.

Effect of P availability on temporal dynamics of carbon allocation and glomus intraradices high-affinity P transporter gene induction in arbuscular mycorrhiza.

Arbuscular mycorrhizal (AM) fungi depend on a C supply from the plant host and simultaneously provide phosphorus to the colonized plant. We therefore evaluated the influence of external P on C allocation in monoxenic Daucus carota-Glomus intraradices cultures in an AM symbiosis. Fungal hyphae proliferated from a solid minimal medium containing colonized roots into a C-free liquid minimal medium with high or low P availability. Roots and hyphae were harvested periodically, and the flow of C from roots to fungus was measured by isotope labeling. We also measured induction of a G. intraradices high-affinity P transporter to estimate fungal P demand. The prevailing hypothesis is that high P availability reduces mycorrhizal fungal growth, but we found that C flow to the fungus was initially highest at the high P level. Only at later harvests, after 100 days of in vitro culture, were C flow and fungal growth limited at high P availability. Thus, AM fungi can benefit initially from P-enriched environments in terms of plant C allocation. As expected, the P transporter induction was significantly greater at low P availability and greatest in very young mycelia. We found no direct link between C flow to the fungus and the P transporter transcription level, which indicates that a good C supply is not essential for induction of the high-affinity P transporter. We describe a mechanism by which P regulates symbiotic C allocation, and we discuss how this mechanism may have evolved in a competitive environment.

Unprecedented genomic diversity of AhR1 and AhR2 genes in Atlantic salmon (Salmo salar L.).

Aryl hydrocarbon receptor (AhR) genes encode proteins involved in mediating the toxic responses induced by several environmental pollutants. Here, we describe the identification of the first two AhR1 (alpha and beta) genes and two additional AhR2 (alpha and beta) genes in the tetraploid species Atlantic salmon (Salmo salar L.) from a cosmid library screening. Cosmid clones containing genomic salmon AhR sequences were isolated using a cDNA clone containing the coding region of the Atlantic salmon AhR2gamma as a probe. Screening revealed 14 positive clones, from which four were chosen for further analyses. One of the cosmids contained genomic AhR sequences that were highly similar to the rainbow trout (Oncorhynchus mykiss) AhR2alpha and beta genes. SMART RACE amplified two complete, highly similar but not identical AhR type 2 sequences from salmon cDNA, which from phylogenetic analyses were determined as the rainbow trout AhR2alpha and beta orthologs. The salmon AhR2alpha and beta encode proteins of 1071 and 1058 residues, respectively, and encompass characteristic AhR sequence elements like a basic-helix-loop-helix (bHLH) and two PER-ARNT-SIM (PAS) domains. Both genes are transcribed in liver, spleen and muscle tissues of adult salmon. A second cosmid contained partial sequences, which were identical to the previously characterized AhR2gamma gene. The last two cosmids contained partial genomic AhR sequences, which were more similar to other AhR type 1 fish genes than the four characterized salmon AhR2 genes. However, attempts to amplify the corresponding complete cDNA sequences of the inserts proved very difficult, suggesting that these genes are non-functional or very weakly transcribed in the examined tissues. Phylogenetic analyses of the conserved regions did, however, clearly indicate that these two AhRs belong to the AhR type 1 clade and have been assigned as the Atlantic salmon AhR1alpha and AhR1beta genes. Taken together, these findings demonstrate that multiple AhR genes are present in Atlantic salmon genome, which likely is a consequence of previous genome duplications in the evolutionary past of salmonids. Plausible explanations for the high incidence of AhR genes in fish and more specifically in salmonids, like rapid divergences in specialized functions, are discussed.

Characterization of two distinct aryl hydrocarbon receptor (AhR2) genes in Atlantic salmon (Salmo salar) and evidence for multiple AhR2 gene lineages in salmonid fish.

The aryl hydrocarbon receptor (AhR) mediates the toxicity of several environmental contaminants, e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin, and other halogenated hydrocarbons in vertebrates. This receptor initiates the transcription of several biotransformation enzymes, which in turn are responsible for causing severe harm to biological tissue. Here we describe the isolation and complete characterization of the first two AhR genes from the teleost fish Atlantic salmon (Salmo salar). The predicted amino acid sequences contain regions characteristic of other vertebrate AhRs including basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) domains but show little similarity to other vertebrate AhRs across the C-terminal half. Furthermore, they do not contain distinct Q-rich domains as found in the mammalian AhR, which is in line with previously described fish AhR genes. The salmon cDNAs encode 1106 and 1107 putative residues, respectively, approximately 50 amino acids longer than previously characterized AhR genes. Phylogenetic analyses demonstrated that the two salmon AhR sequences cluster within the AhR subfamily of the bHLH-PAS family, in a clade containing fish AhR2 genes. Although the two AhR2 forms are 92% identical at the amino acid level, the distribution of sequence differences and the presence of both forms in 30 tested individuals suggest that they are not allelic but derived from separate loci. Interestingly, they are not orthologs of the rainbow trout (Oncorhynchus mykiss) AhR2 alpha and beta genes and the new salmon loci are therefore here designated AhR2 gamma and AhR2 delta. In line with this, PCR with DNA from rainbow trout revealed a new trout AhR locus that was more similar to the two salmon genes than to the trout AhR2 alpha and beta genes, suggesting that the rainbow trout possesses at least three distinct AhR2 genes. The presence of multiple AhR genes in these species is probably a consequence of the genome duplications that occurred in the early evolution of fish and later also specifically in the salmonid lineage. Reverse transcription-PCR analyses revealed that both AhR2 gamma and AhR2 delta are transcribed in the liver, spleen and muscles of adult salmon.