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Background Alpha-chloralose (AC) is a compound known to be toxic to various animal species and humans. In 2018 and 2019 an increase in suspected cases of AC poisoning in cats related to the use of AC as a rodenticide was reported to national veterinary and chemical authorities in Finland, Norway and Sweden by veterinarians working in clinical practices in respective country. The aims of this study were to prospectively investigate AC poisoning in cats, including possible secondary poisoning by consuming poisoned mice, and to study metabolism and excretion of AC in cats through analysis of feline urine. Methods Data on signalment, history and clinical findings were prospectively collected in Finland, Norway and Sweden from July 2020 until March of 2021 using a questionnaire which the attending veterinarian completed and submitted together with a serum sample collected from suspected feline cases of AC-poisoning. The diagnosis was confirmed by quantification of AC in serum samples. Content of AC was studied in four feline urine samples, including screening for AC metabolites by UHPLC-HRMS/MS. Bait intake and amount of AC consumed by mice was observed in wild mice during an extermination of a rodent infestation. Results In total, 59 of 70 collected questionnaires and accompanying serum samples were included, with 127 to 70 100 ng/mL AC detected in the serum. Several tentative AC-metabolites were detected in the analysed feline urine samples, including dechlorinated and oxidated AC, several sulfate conjugates, and one glucuronic acid conjugate of AC. The calculated amount of AC ingested by each mouse was 33 to 106 mg with a mean of 61 mg. Conclusions Clinical recognition of symptoms of AC poisoning in otherwise healthy cats roaming free outdoors and known to be rodent hunters strongly correlated with confirmation of the diagnosis through toxicological analyses of serum samples. The collected feline exposure data regarding AC show together with the calculation of the intake of bait and subsequent AC concentrations in mice that secondary poisoning from ingestion of mice is possible. The results of the screening for AC metabolites in feline urine confirm that cats excrete AC both unchanged and metabolized through dechlorination, oxidation, glucuronidation and sulfatation pathways.

Microcystins are potent cyclic heptapeptide toxins found in many freshwater cyanobacteria. Most microcystins contain an α,β-unsaturated amide that can react with thiol-containing amino acids, peptides, and proteins in vivo and in vitro. While soluble conjugates formed from small peptides can be extracted and analyzed directly by LC-MS, microcystins conjugated to proteins are analyzed after oxidative cleavage of their Adda side chains, but information on which microcystin analogues were present is lost. Observations during the development of thiol-derivatization-based LC-MS methods for microcystin analysis indicated that the reaction of thiols with microcystins was reversible. The kinetics of deconjugation was investigated with mercaptoethanol as a model thiol to identify suitable reaction conditions. A range of microcystins conjugated to mercaptoethanol, methanethiol, cysteine, and glutathione were then successfully deconjugated, demonstrating the feasibility of releasing conjugated forms of microcystins for chemical analysis. Reagents for removing the released thiols or for trapping the released microcystins increased the reaction rate. Optimization of methodologies based on this reaction should increase the method's utility for measuring free and conjugated microcystins. The results also indicate that thiol-conjugated microcystins slowly release free microcystins, even at neutral pH, with consequences for assessment of toxin exposure, metabolism, and trophic transfer. A range of other common natural and environmental toxins, such as deoxynivalenol and acrylamide, also contain α,β-unsaturated carbonyl groups and can be expected to behave in a similar manner.

Flounder (Platichthys flesus) is among the most common fish-species in Norwegian and European estuaries. It lives in or on sediments from which it also finds most of its food. The aim of the present work was to evaluate biomarkers in flounder for possible future use in monitoring programmes. There were clear biomarker responses in flounder following injection of model contaminants benzo[a]pyrene (B[a]P), PCB #156 and Cd, singly or in sequence. Cytochrome P4501A responded following injection of the organic contaminants and metallothionein (MT) following Cd injection. All groups receiving B[a]P, either singly or in combination with other contaminants, accumulated high levels of B[a]P-metabolites in bile. There was little change in glutathione-S-transferase activity (measured using CDNB as substrate) following the treatments. Starvation appeared to affect the response of hepatic MT to Cd, but none of the other biomarkers. PAH in sediments elicited strong biomarker responses in caged flounder, whereas sediment-associated metals appeared to be largely unavailable to flounder in this study.

The objective of this study was to investigate the response of acetylcholinesterase (AChE) activities in Clarias gariepinus in response to Organophosphates (Ops) and carbamate exposure. The AChE activities were determined in plasma, and eye and brain homogenates of unexposed and exposed fish using Ellman's method and 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) chromophore. The baseline AChE activities in plasma, eyes and brain tissues in unexposed fish were comparable between males and females (P > 0.05). Concentrations of pesticides that inhibited 50% (IC(50)) of AChE activities in brain homogenates following in vitro exposures were 0.003, 0.03, 0.15, 190, 0.2, 0.003 and 0.002 microM for carbaryl, chlorfenvinphos, diazinon, dimethoate, fenitrothion, pirimiphosmethyl and profenofos, respectively. The in vivo dose-effect relationships were assessed using chlorfenvinphos and carbaryl at different concentrations that ranged from 0.0003 to 0.06 microM and 0.0005 to 0.05 microM, respectively. Acetylcholinesterase activities were comparable in plasma, and eye and brain homogenates from control and carbaryl-exposed fish. Following exposure of fish to chlorfenvinphos at concentrations above 0.03 microM, a significant inhibition of AChE activities in plasma (84%) and eye homogenate (50%) was observed. The AChE activities in brain homogenate were comparable between chlorfenvinphos-exposed fish and controls. Because carbaryl cause reversible inhibition of AChE activities was found to be more potent than chlorfenvinphos that cause irreversible inhibition following in vitro exposure. Contrary, carbaryl was less potent than chlorfenvinphos after in vivo exposure possibly due to more rapid biotransformation of carbaryl than chlorfenvinphos. Findings from this study have demonstrated that inhibition of AChE activity in C. gariepinus is a useful biomarker in assessing aquatic environment contaminated by anticholinesterases.

Many persistent organic pollutants are known to have endocrine-disrupting effects in several aquatic and terrestrial species. In this regard, hydroxylated metabolites of polychlorinated biphenyls (OH-PCBs) represent serious health and environmental concern because they are shown to act agonistic or antagonistic at hormone receptors (HRs) or to cause hormone-receptor-mediated responses. In the present study, salmon primary hepatocytes were used to study alterations in an estrogen signaling pathway resulting from exposure to four hydroxylated (4OH-CB 107, 4OH-CB146, 4OH-CB187, and 3OH-CB138) metabolites of PCB at different concentrations using quantitative real-time polymerase chain reaction. The effects of the PCB metabolites were compared to the mRNA expression in 17alpha-ethynylestradiol (EE2)-treated cells. Concentration-specific increase of vitellogenin (Vtg) mRNA transcription after exposure to OH-PCBs was observed. Decreased mRNA transcription was observed for zona radiata protein (Zr-protein) and cytochrome P450 side-chain cleavage (P450scc) enzyme. For estrogen receptor beta (ERbeta), the mRNA expression pattern was OH-PCB-metabolite congener-specific. A novel aspect of this study is that OH-PCBs produced effects on hepatic steroidogenic pathways by targeting the StAR protein and P450scc genes. Given that endocrine toxicology research mainly has focused on estrogenicity involving direct ER-mediated effects and that steroidogenic enzyme and proteins are highly tissue- and cell-type-specific and controlled by different promoters and second-messenger pathways, the present findings provide potential new targets for interaction with xenobiotics such as hydroxylated congeners of certain chemicals. The quantitative expression patterns of hepatic and extrahepatic steroidogenic genes and proteins after exposure to environmental contaminants are the subject of systematic investigations in our laboratory.

Hydroxylated metabolites of PCBs [OH-PCBs] represent new health and environmental concern because they have been shown to have agonist or antagonist interactions with hormone receptors (HRs) or hormone-receptor mediated responses. The present study was designed to investigate the estrogenic potency based on anti-AhR signalling effect of three 4-OH substituted PCB congeners (#107, #146 and #187), one 3-OH substituted congener (#138), and the pharmaceutical synthetic estrogen, ethynylestradiol (EE2) in fish in vitro system using primary culture of Atlantic salmon hepatocytes. The effects were studied by quantifying changes in transcripts with gene-sequence primer pairs for a suite of gene responses (AhRalpha, ARNT, CYP1A1, CYP3A, UGT and GST) belonging to the xenobiotic biotransformation system. Our data show that OH-PCB congeners and EE2, decreased AhRalpha and ARNT transcript levels, and CYP1A1, UGT and GST gene expressions, together with CYP3A gene expression. The decreased expression of transcripts for xenobiotic biotransformation system is related to the concentration of individual OH-PCB congener and these responses are typical of reported estrogenic and estrogen-like effects on the CYP system. Modulation of biotransformation pathways by OH-PCBs may alter xenobiotic metabolism leading to the production of toxic reactive molecules, altering pharmacokinetics and diminishing the clearance rate of individual chemicals from the organism.

Dose dependent effects of Benzo[a]pyrene (BaP) on cytochrome P4501A (CYP1A), glutathione-S-transferase (GST) and fluorescent aromatic compounds (FACs) metabolites biomarker responses were studied in African sharptooth catfish (Clarias gariepinus) following 24 h of waterborne exposures. Based on biomass of C. gariepinus in different tanks, BaP concentrations of 1.60, 3.44, and 18.21 microg/L that corresponded to 0.5, 1.0, and 5.0 mg/kg body weight were used. Significant induction of EROD activities in gill filaments was observed at all doses and the accumulation of FACs metabolites in bile was significantly different between groups. Accumulation of FACs metabolites in bile strongly correlated (r (2) = 0.99) with BaP doses. Hepatic EROD activities were undetectable and no effect on GST activities was observed. The highest dose of BaP from the dose dependent study was further studied to assess the interactive and temporal responses of C. gariepinus on CYP1A, GST, and FACs metabolites biomarkers following exposure to either BaP alone, 17alpha-ethynylestradiol (EE(2)) alone or a combination of both compounds at concentrations of 54.17 microg/L for BaP, 51.38 microg/L for EE(2) and 54.44 microg/L for each of both compounds. Based on biomass in each tank, these concentrations corresponded to 5 mg/kg body weight. While a group of six fish was sacrificed on day 0 from the control tank only, other groups of six fish were sacrificed after 1, 3, and 6 days of exposure from the control and exposed groups. Maximum induction of gill filament and hepatic EROD activities was observed after 1 day of exposure. Both EROD activities in gill filaments and liver were significantly induced by exposure to BaP alone or co-administration with EE(2). Gill filament EROD induction was significantly inhibited (50%) by co-administration of BaP and EE(2) compared to administration of BaP alone. Levels of FACs in bile for BaP and BaP + EE(2) exposed groups were significantly different from the control at all doses and time points. A significant induction of GST activities was observed in fish exposed to BaP and BaP + EE(2) after 3 days. Exposure to EE(2) alone caused significant induction of this enzyme after day 6. This study reports for the first time the significant antagonistic influence of EE(2) on BaP in gills of fish following waterborne exposures. The results also indicate that chemical mixtures may affect biomarker responses differently from compounds administered alone and that the sensitivity of CYP1A to interactive chemicals is different in gills and liver.

The ability of African sharptooth catfish (Clarias gariepinus) in inducing cytochrome P-450 class 1A (CYP1A) and glutathione S-transferase (GST) biomarkers was determined in liver and gill filaments after 4 days of waterborne exposure to the polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P). Male (n=6) and female (n=6) fish were exposed to B[a]P added to the water (30 microg/l) corresponding to 5 mg/kg total body weight. Five female and six male fish, exposed to acetone alone added to the water served as controls. The 7-ethoxyresurufin-O-deethylase (EROD) activity was measured in hepatic microsomes and gill filaments. In addition, GST activity was determined in the hepatic cytosolic fraction and fluorescent aromatic compounds (FACs) in bile and biliverdin. Benzo[a]pyrene strongly induced EROD activities in gill filaments of both sex. Levels of FACs per ml of bile were 17-fold higher in exposed fish compared to the controls. Correlations between induction of EROD activities in gill filaments and liver and between induction of EROD activities in gill filaments and levels of biliary FACs metabolites were strong. GST activities in the hepatic cytosolic fraction were similar amongst the treated and control groups. This is the first report on studies determining EROD activities in gill filaments and hepatic tissue, FACs in bile and GST in hepatic tissues of C. gariepinus after waterborne exposure to B[a]P. The findings suggest that the gill filament-based EROD assay in C. gariepinus can be used to monitor the pollution of AhR agonists in aquatic ecosystems in eastern and southern African countries.

Two novel pectenotoxins (PTXs) were detected by LC-MS in solid phase extracts of net hauls taken at Flodevigen, Norway, in June 2002 that were dominated by Dinophysis acuminata and Dinophysis norvegica. The new compounds were isolated as minor components from a large collection of a Dinophysis acuta-dominated bloom obtained from Skjer, Sognefjorden, Norway, in October 2002. LC-MS and NMR analyses revealed that the new components, 36S-PTX-12 and 36R-PTX-12, occurred as a pair of equilibrating diastereoisomers differing from PTX-2 in that they contained an exocylic olefinic methylene rather than a methyl group at C-38. Analyses of shellfish extracts revealed that PTX-12 accumulated in Norwegian blue mussels (Mytilus edulis) and cockles (Cerastoderma edule), along with PTX-12 seco acids occurring as a complex mixture of diastereoisomers. LC-MS analysis of algal cells picked from the net haul from Flodevigen revealed that PTX-12 predominated in D. acuta and D. norvegica, whereas PTX-2 was the predominant pectenotoxin in D. acuminata. Preliminary observations indicate that the relative contents of PTX-2 and PTX-12 vary between sites and years in Norway, even within a single species of Dinophysis. Our data also suggest that heterotrophic dinoflagellates may accumulate toxins from their prey.

Thirty-nine leaf samples of Narthecium ossifragum collected from eight sites in More og Romsdal County, Norway, during June-September 1997 and 41 leaf samples collected at five sites in the same county during June-August 1998 were analysed for the concentrations of steroidal sapogenins using GC-MS. The 1998 samples were also examined for fungal elements (conidia and hyphae) after incubation in a moist chamber for 10-14 days. The highest 1997 and 1998 leaf sapogenin concentrations (4881 and 7115 mg/kg dry matter, respectively) were 13-14 times greater than the lowest sapogenin concentrations found (344 and 531 mg/kg dry matter, respectively). The results did not reveal systematic differences in sapogenin concentrations between the two seasons, or between samples harvested early or late in the same seasons, or between sapogenin concentrations in plants harvested at different sites. Cladosporium magnusianum was the predominant fungus found in the samples. The degree of fungal infection on the samples was in generally low, but the number of C. magnusianum colonies in the moist chamber preparations and fungal elements (conidia and hyphae) in leaf washings and on leaves tended to increase with time. Factor analysis and multiple regression analysis performed on the chemical and fungal results suggest that sporulation may have occurred in the fungi in response to increase in sapogenin concentrations.

Okadaic acid analogues are well known as protein phosphatase inhibitors and occur naturally in marine shellfish feeding on dinoflagellates of the genus Dinophysis, leading to diarrhetic shellfish poisoning of shellfish consumers. Knowledge of the correct structures for these toxins is important in understanding their toxicology, biochemistry, and biosynthesis. We have performed extensive NMR analyses on okadaic acid (1), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2) obtained from natural sources. Consequently, we were able to unambiguously deduce the stereochemistries at C-35 for DTX-1 and DTX-2 based on analysis of NMR coupling constants and NOE interactions. Our results revealed that DTX-2 (3) has a stereochemistry opposite to that of DTX-1 (2) at C-35. Molecular modeling of the docking of 1-3 with protein phosphatase-1 and protein phosphatase 2A (PP2A) suggested that the reduced affinity of DTX-2 for PP2A may be due to the newly defined stereochemistry at the 35-methyl group. The implications of these findings for biosynthesis and toxicology are discussed.