Stabilization of myoglobin from different species (produced by cellular agriculture) using food-grade natural and synthetic antioxidants.

Cellular agriculture products, like myoglobin, are increasingly used by the food industry to provide desirable sensory properties to plant-based meat substitutes. This study elucidated the physicochemical properties and redox stability of myoglobin from both natural (equine) and cellular agriculture (bovine, sperm whale, and leopard) sources. The electrical characteristics and water-solubility of the different myoglobin samples were measured from pH 2.5 to 8.5. The isoelectric point of the myoglobin samples depended on the species, being pH 5.5 for equine, pH 4.5 for leopard and bovine, and pH 6.5 for sperm whale. All myoglobin samples had a solubility greater than 80% across the entire pH range studied. All myoglobin solutions appeared red and had two peaks in their UV-visible absorbance spectra after one day, which is consistent with oxymyoglobin formation. Equine myoglobin at pH 8 was selected to study its redox and color stability over time, where the oxymyoglobin oxidative status closely paralleled with the redness of the solutions. The effects of antioxidants (ascorbic acid, caffeic acid, catechin, gallic acid, quercetin, taxifolin, Trolox, and 4-methylcatechol) on the redox and color stability (redness) of the equine myoglobin (pH 8.0) was also studied. Antioxidants with low reduction potential values (ascorbic acid and quercetin) were particularly effective at enhancing the color stability of oxymyoglobin. The computational modeling study showed that amino acids on the myoglobin interacted with antioxidants through hydrogen bonds. The insights obtained may have important implications for the use of cellular agriculture to produce myoglobin for food applications.

Influence of the heme distal pocket on nitrite binding orientation and reactivity in Sperm Whale myoglobin.

Nitrite binding to recombinant wild-type Sperm Whale myoglobin (SWMb) was studied using a combination of spectroscopic methods including room-temperature magnetic circular dichroism. These revealed that the reactive species is free nitrous acid and the product of the reaction contains a nitrite ion bound to the ferric heme iron in the nitrito- (O-bound) orientation. This exists in a thermal equilibrium with a low-spin ground state and a high-spin excited state and is spectroscopically distinct from the purely low-spin nitro- (N-bound) species observed in the H64V SWMb variant. Substitution of the proximal heme ligand, histidine-93, with lysine yields a novel form of myoglobin (H93K) with enhanced reactivity towards nitrite. The nitrito-mode of binding to the ferric heme iron is retained in the H93K variant again as a thermal equilibrium of spin-states. This proximal substitution influences the heme distal pocket causing the pKa of the alkaline transition to be lowered relative to wild-type SWMb. This change in the environment of the distal pocket coupled with nitrito-binding is the most likely explanation for the 8-fold increase in the rate of nitrite reduction by H93K relative to WT SWMb.

Biosynthesis of ambrein in ambergris: evidence from isotopic data and identification of possible intermediates.

Ambrein is found in ambergris, a coprolith occurring in the rectum of the sperm whale. In vitro, ambrein is produced by enzymatic cyclisation of squalene, via a monocyclic intermediate. However, little is known of the in vivo process. In order to find evidence for the reaction in vivo, a comparison was made of the δ13C relative isotopic ratios of ambrein in ambergris with those of co-occurring sterols. A statistically significant difference was noted. This suggests that ambrein originates via a different biosynthetic mechanism from that of the sterols. Examination of the minor constituents of a hydrogenolysed extract of ambergris revealed compounds with a bicyclic polypodane nucleus, rather than those with monocyclic structures. It is hypothesised that in vivo biosynthesis of ambrein proceeds, at least in some cases, via bacterial production of bicyclic polypodenols. The latter are known products of non-concerted squalene (or squalene oxide) cyclisations in other organisms.

Construction of an artificial system for ambrein biosynthesis and investigation of some biological activities of ambrein.

Ambergris, a sperm whale metabolite, has long been used as a fragrance and traditional medication, but it is now rarely available. The odor components of ambergris result from the photooxidative degradation of the major component, ambrein. The pharmacological activities of ambergris have also been attributed to ambrein. However, efficient production of ambrein and odor compounds has not been achieved. Here, we constructed a system for the synthesis of ambrein and odor components. First, we created a new triterpene synthase, "ambrein synthase," for mass production of ambrein by redesigning a bacterial enzyme. The ambrein yields were approximately 20 times greater than those reported previously. Next, an efficient photooxidative conversion system from ambrein to a range of volatiles of ambergris was established. The yield of volatiles was 8-15%. Finally, two biological activities, promotion of osteoclast differentiation and prevention of amyloid ß-induced apoptosis, were discovered using the synthesized ambrein.

Stable isotope ratios of carbon, nitrogen and sulphur and mercury concentrations as descriptors of trophic ecology and contamination sources of Mediterranean whales.

The Mediterranean Sea remains a complex system for mercury (Hg) cycling and accumulation in marine vertebrates. The extremely high levels these animals present demand for an urgent understanding of such processes and the development of new analytical techniques that go beyond the simple contamination monitoring. It was often proposed that prey selection or habitat use may affect Hg contamination in animals; however, it was never possible to measure which factor influences more rates and pathways of contamination. In this paper, we directly integrate toxicological information (Hg levels) and ecological tracers (stable isotopes of C, N and S) into a common data analysis framework (isotopic niches), with the aim of quantifying the influence of species' trophic behaviour on Hg contamination. The analysis was conducted on skin biopsies of fin whales Balaenoptera physalus, long-finned pilot whales Globicephala melas and sperm whales Physeter microcephalus. Their different trophic modes and residency in the area make them model species for the analysis of Hg accumulation along NWMS food webs. We measured Total Hg (T-Hg) concentrations through absorbance spectrometry with the DMA80 Milestone. Carbon, nitrogen and sulphur isotope compositions were measured via mass spectrometry in an IRMS coupled to an Elemental Analyser (EA) Isoprime. Comparison of ecological and contamination niches allowed to explain Hg accumulation in Mediterranean marine predators. Factors such as food web complexity, trophic position, hunting distribution or habitat use (e.g., foraging depth) did not influence Hg exposure. It is rather the selection of prey type, which determines the range of potential Hg sources and as a consequence the rates of accumulation in whales' tissues. A generalist piscivorous species such as the pilot whales will bioaccumulate more Hg than specialised sperm whales feeding mostly on cephalopods.

Ingestion of macroplastics by odontocetes of the Greek Seas, Eastern Mediterranean: Often deadly!

Plastic pollution is an omnipresent problem that threatens marine animals through ingestion and entanglement. Marine mammals are no exception to this rule but their interaction with plastic remains understudied in the Mediterranean Sea. Here we highlight this problem by analyzing the stomach contents of 34 individuals from seven odontocete species stranded in Greece. Macroplastic (>5 mm) was found in the stomachs of nine individuals from four species (harbour porpoise Phocoena phocoena, Risso's dolphin Grampus griseus, Cuvier's beaked whale Ziphius cavirostris and sperm whale Physeter macrocephalus) with the highest frequency of occurrence in sperm whales (60%). Gastric blockage from plastic was presumably lethal in three cases, with plastic bags being the most common finding (46%). Plastic ingestion is of particular conservation concern for the endangered Mediterranean sperm whales. A regular examination of stranded cetaceans with a standardised protocol is critical for allowing spatiotemporal comparisons within and across species.

Tissue distribution of organic contaminants in stranded pregnant sperm whale (Physeter microcephalus) from the Huizhou coast of the South China Sea.

Twelve persistent organic pollutants (POPs) were measured in 11 tissue samples from a pregnant sperm whale stranded on the Huizhou coast of the South China Sea, China, in March 2017. POPs were found to be more concentrated in the irrigated tissues such as placenta, ovary, mammary gland, and liver than the less irrigated tissues such as epidermis. High POP levels detected in the placenta might result in abnormal hormone secretion in the placenta, which would affect the unborn offspring. We hypothesized that ovary is potentially vulnerable to the exposure of higher contaminant levels. The PAH concentrations were higher in the lung than in other tissues, which suggest that PAH levels in the lung were breath-dependent in the sperm whale. The concentrations of POPs except PAHs in the sperm whale blubber were lower than those in the same species in the Northern Hemisphere and were comparable to or lower than those in the same species in the Southern Hemisphere.

A whale of a tale: A One Environmental Health approach to study metal pollution in the Sea of Cortez.

Marine metal pollution is an emerging concern for human, animal, and ecosystem health. We considered metal pollution in the Sea of Cortez, which is a relatively isolated sea rich in biodiversity. Here there are potentially significant anthropogenic inputs of pollution from agriculture and metal mining. We considered the levels of 23 heavy metals and selenium in seven distinct cetacean species found in the area. Our efforts considered two different periods of time: 1999 and 2016/17. We considered the metal levels in relation to (1) all species together across years, (2) differences between suborders Odontoceti and Mysticeti, (3) each species individually across years, and (4) gender differences for each of these comparisons. We further compared metal levels found in sperm whale skin samples collected during these voyages to a previous voyage in 1999, to assess changes in metal levels over a longer timescale. The metals Mg, Fe, Al, and Zn were found at the highest concentrations across all species and all years. For sperm whales, we observed decreased metal levels from 1999 to 2016/2017, except for iron (Fe), nickel (Ni), and chromium (Cr), which either increased or did not change during this time period. These results indicate a recent change in the metal input to the Sea of Cortez, which may indicate a decreased concern for human, animal, and ecosystem health for some metals, but raises concern for the genotoxic metals Cr and Ni. This work was supported by NIEHS grant ES016893 (J.P.W.) and numerous donors to the Wise Laboratory.

From banana fields to the deep blue: Assessment of chlordecone contamination of oceanic cetaceans in the eastern Caribbean.

In the French West Indies (Caribbean), the insecticide Chlordecone (CLD) has been extensively used to reduce banana weevil (Cosmopolites sordidus) infestations in banana plantations. Previous studies have shown high CLD concentrations in freshwater and coastal communities of the region. CLD concentrations, however, have not yet been assessed in marine top predators. We investigated CLD concentrations in cetacean blubber tissues from Guadeloupe, including Physeter macrocephalus, Lagenodelphis hosei, Stenella attenuata and Pseudorca crassidens. Chlordecone was detected in all blubber samples analysed, with the exception of four P. macrocephalus. Concentrations (range: 1 to 329 ng·g-1 of lipid weight) were, however, lower than those found in species from fresh and brackish water. Ecological factors (open ocean habitat), CLD kinetics, and cetacean metabolism (high or specific enzymatic activity) might explain low concentrations found in cetacean blubber. Future analyses that include internal organ sampling would help to confirm CLD levels observed in this study.

Halogenated flame retardants in stranded sperm whales (Physeter macrocephalus) from the Mediterranean Sea.

In recent years, decline of marine mammals' populations and increased frequency of strandings have arised the interest on the role that pollution may have in these events. The present work aimed at quantifying levels of brominated flame retardants (BFRs) and dechloranes (DECs) in tissues of 3 adult females and one foetus of sperm whales stranded in the Southern Adriatic Sea coasts (Italy). Results proved the presence of different flame retardants (FRs) in tissues of sperm whales, including various polybrominated diphenyl ethers (PBDE) congeners (47, 99, 100, 154, entering the composition of PentaBDE mixture), hexabromocyclodecanes (HBCDs), Dec 602 and methoxylated polibrominated diphenyl ethers (MeO-BDEs). In blubber, a target tissue for contaminant accumulation, ΣPBDEs reached values of 160, 158 and 183 ng/g lw, α-HBCD of 5.75 ng/g lw, Dec 602 of 1632 ng/g lw and MeO-BDEs of 563 ng/g lw. The availability of foetal tissues allowed evaluating the potential maternal transfer on many of these compounds, and to discuss the potential adverse effects on foetal health. To the best of our knowledge, obtained data are the first reporting placental transfer of FRs in sperm whales. PBDE levels detected in foetus suggested a potentially long-term exposure to BFRs, which could cause severe damages to the developing organism, likely at the cerebral, endocrine and immunologic levels. Dec 602, which was detected at the highest concentrations among all FRs considered, could potentially cause dysfunctional effects on the immune system of adult females.

A three year study of metal levels in skin biopsies of whales in the Gulf of Mexico after the Deepwater Horizon oil crisis.

In response to the explosion of the Deepwater Horizon and the massive release of oil that followed, we conducted three annual research voyages to investigate how the oil spill would impact the marine offshore environment. Most investigations into the ecological and toxicological impacts of the Deepwater Horizon Oil crisis have mainly focused on the fate of the oil and dispersants, but few have considered the release of metals into the environment. From studies of previous oil spills, other marine oil industries, and analyses of oil compositions, it is evident that metals are frequently encountered. Several metals have been reported in the MC252 oil from the Deepwater Horizon oil spill, including the nonessential metals aluminum, arsenic, chromium, nickel, and lead; genotoxic metals, such as these are able to damage DNA and can bioaccumulate in organisms resulting in persistent exposure. In the Gulf of Mexico, whales are the apex species; hence we collected skin biopsies from sperm whales (Physeter macrocephalus), short-finned pilot whales (Globicephala macrorhynchus), and Bryde's whales (Balaenoptera edeni). The results from our three-year study of monitoring metal levels in whale skin show (1) genotoxic metals at concentrations higher than global averages previously reported and (2) patterns for MC252-relevant metal concentrations decreasing with time from the oil spill.

Large amounts of marine debris found in sperm whales stranded along the North Sea coast in early 2016.

30 sperm whales (Physeter macrocephalus) stranded along the coasts of the North Sea between January and February 2016. The gastro-intestinal tracts of 22 of the carcasses were investigated. Marine debris including netting, ropes, foils, packaging material and a part of a car were found in nine of the 22 individuals. Here we provide details about the findings and consequences for the animals. While none of the items was responsible for the death of the animal, the findings demonstrate the high level of exposure to marine debris and associated risks for large predators, such as the sperm whale.

Benchmarking Rapid TLES Simulations of Gas Diffusion in Proteins: Mapping O2 Migration and Escape in Myoglobin as a Case Study.

Standard molecular dynamics (MD) simulations of gas diffusion consume considerable computational time and resources even for small proteins. To combat this, temperature-controlled locally enhanced sampling (TLES) examines multiple diffusion trajectories per simulation by accommodating multiple noninteracting copies of a gas molecule that diffuse independently, while the protein and water molecules experience an average interaction from all copies. Furthermore, gas migration within a protein matrix can be accelerated without altering protein dynamics by increasing the effective temperature of the TLES copies. These features of TLES enable rapid simulations of gas diffusion within a protein matrix at significantly reduced (∼98%) computational cost. However, the results of TLES and standard MD simulations have not been systematically compared, which limits the adoption of the TLES approach. We address this drawback here by benchmarking TLES against standard MD in the simulation of O2 diffusion in myoglobin (Mb) as a case study since this model system has been extensively characterized. We find that 2 ns TLES and 108 ns standard simulations map the same network of diffusion tunnels in Mb and uncover the same docking sites, barriers, and escape portals. We further discuss the influence of simulation time as well as the number of independent simulations on the O2 population density within the diffusion tunnels and on the sampling of Mb's conformational space as revealed by principal component analysis. Overall, our comprehensive benchmarking reveals that TLES is an appropriate and robust tool for the rapid mapping of gas diffusion in proteins when the kinetic data provided by standard MD are not required. Furthermore, TLES provides explicit ligand diffusion pathways, unlike most rapid methods.

Global assessment of cadmium concentrations in the skin of free-ranging sperm whales (Physeter macrocephalus).

Cadmium is a non-essential, toxic metal found accumulated in the organs of stranded cetaceans. Currently, there is no baseline cadmium concentration reported in a free-ranging, pelagic cetacean. The aim was to determine cadmium concentrations in the skin of free-ranging sperm whales (n=340) collected from 16 regions around the world during the voyage of the Odyssey (2000-2005) considering region, gender, and age in males. Cadmium was detected in 81% of skin biopsies with a mean of 0.3±0.04µg/g ww (0.02 to 12.4µg/g ww). These concentrations were higher than reported in literature in toothed whale skin (0.002-0.1µg/g ww). Concentrations by region were significantly different (p<0.0001) with the highest mean in Maldives and the Sea of Cortez (0.8 and 0.6µg/g ww, respectively). There was no significant difference in cadmium concentration by gender (p=0.42). Cadmium is known to have a long biological half-life, and cadmium concentrations in males were significantly higher in adults with a mean of 0.3µg/g ww compared to subadults with 0.2µg/g ww (p=0.03). Selenium, an element that binds to cadmium inhibiting its toxicity, had a moderately positive correlation with cadmium (r=0.41). Mercury, a toxic metal that positively correlates with cadmium in cetacean tissue, had a weakly positive relationship (r=0.20). The regional baselines reported in this study may be used to develop residue criteria for prediction of toxicological risk in sperm whale skin. Additionally, this study shows the extent of cadmium exposure in a pelagic cetacean that has global distribution.

Analysis of mercury and methylmercury concentrations, and selenium:mercury molar ratios for a toxicological assessment of sperm whales (Physeter macrocephalus) in the most recent stranding event along the Adriatic coast (Southern Italy, Mediterranean Sea).

Mass stranding of sperm whales (Physeter macrocephalus) is a rare event in the Mediterranean Sea. In September 2014, a pod of seven sperm whales became stranded along the Adriatic coast of Southern Italy. This is the seventh occurrence of this type since 1555 in this sea basin. Total concentrations of mercury (T-Hg), methylmercury (MeHg) and selenium (Se) were measured from brain, muscle, liver and kidney of three female sperm whales, which died in this event. Analyses showed considerable, age-dependent variations in Hg and Se concentrations in the different organs. The contamination levels of T-Hg in the liver (up to 200 mg kg(-1)) and brain (up to 21 mg kg(-1)) samples were markedly higher than those in the kidney and muscle samples. The liver and brain also showed the highest Se levels. Se:Hg molar ratios ⩾1 were observed in all the organs of the three sperm whales, suggesting that Se could protect the animals from Hg toxicity. The risk of Hg-associated neurotoxicity was assessed by comparing our values to thresholds set for neurotoxicity in mammals, and the role of Se in the detoxification process of T-Hg/MeHg is discussed herein.

Structures of K42N and K42Y sperm whale myoglobins point to an inhibitory role of distal water in peroxidase activity.

Sperm whale myoglobin (Mb) functions as an oxygen-storage protein, but in the ferric state it possesses a weak peroxidase activity which enables it to carry out H2O2-dependent dehalogenation reactions. Hemoglobin/dehaloperoxidase from Amphitrite ornata (DHP) is a dual-function protein represented by two isoproteins DHP A and DHP B; its peroxidase activity is at least ten times stronger than that of Mb and plays a physiological role. The `DHP A-like' K42Y Mb mutant (K42Y) and the `DHP B-like' K42N mutant (K42N) were engineered in sperm whale Mb to mimic the extended heme environments of DHP A and DHP B, respectively. The peroxidase reaction rates increased ∼3.5-fold and ∼5.5-fold in K42Y and K42N versus Mb, respectively. The crystal structures of the K42Y and K42N mutants revealed that the substitutions at position 42 slightly elongate not only the distances between the distal His55 and the heme iron but also the hydrogen-bonding distances between His55 and the Fe-coordinated water. The enhanced peroxidase activity of K42Y and K42N thus might be attributed in part to the weaker binding of the axial water molecule that competes with hydrogen peroxide for the binding site at the heme in the ferric state. This is likely to be the mechanism by which the relationship `longer distal histidine to Fe distance - better peroxidase activity', which was previously proposed for heme proteins by Matsui et al. (1999) (J. Biol. Chem. 274, 2838-2844), works. Furthermore, positive cooperativity in K42N was observed when its dehaloperoxidase activity was measured as a function of the concentration of the substrate trichlorophenol. This serendipitously engineered cooperativity was rationalized by K42N dimerization through the formation of a dityrosine bond induced by excess H2O2.

Carbon and nitrogen isotopes from top predator amino acids reveal rapidly shifting ocean biochemistry in the outer California Current.

Climatic variation alters biochemical and ecological processes, but it is difficult both to quantify the magnitude of such changes, and to differentiate long-term shifts from inter-annual variability. Here, we simultaneously quantify decade-scale isotopic variability at the lowest and highest trophic positions in the offshore California Current System (CCS) by measuring δ15N and δ13C values of amino acids in a top predator, the sperm whale (Physeter macrocephalus). Using a time series of skin tissue samples as a biological archive, isotopic records from individual amino acids (AAs) can reveal the proximate factors driving a temporal decline we observed in bulk isotope values (a decline of ≥1 ‰) by decoupling changes in primary producer isotope values from those linked to the trophic position of this toothed whale. A continuous decline in baseline (i.e., primary producer) δ15N and δ13C values was observed from 1993 to 2005 (a decrease of ∼4‰ for δ15N source-AAs and 3‰ for δ13C essential-AAs), while the trophic position of whales was variable over time and it did not exhibit directional trends. The baseline δ15N and δ13C shifts suggest rapid ongoing changes in the carbon and nitrogen biogeochemical cycling in the offshore CCS, potentially occurring at faster rates than long-term shifts observed elsewhere in the Pacific. While the mechanisms forcing these biogeochemical shifts remain to be determined, our data suggest possible links to natural climate variability, and also corresponding shifts in surface nutrient availability. Our study demonstrates that isotopic analysis of individual amino acids from a top marine mammal predator can be a powerful new approach to reconstructing temporal variation in both biochemical cycling and trophic structure.

Involvement of ferryl in the reaction between nitrite and the oxy forms of globins.

The reaction between nitrite and the oxy forms of globins has complex autocatalytic kinetics with several branching steps and evolves through chain reactions mediated by reactive species (including radicals) such as hydrogen peroxide, ferryl and nitrogen dioxide, starting with a lag phase, after which it proceeds onto an autocatalytic phase. Reported here are UV-Vis spectra collected upon stopped-flow mixing of myoglobin with a supraphysiological excess of nitrite. The best fit to the experimental data follows an A â†’ B â†’ C reaction scheme involving the formation of a short-lived intermediate identified as ferryl. This is consistent with a mechanism where nitrite binds to oxy myoglobin to generate an undetectable ferrous-peroxynitrate intermediate, whose decay leads to nitrate and ferryl. The ferryl is then reduced to met by the excess nitrite. DFT calculations reveal an essentially barrierless reaction between nitrite and the oxy heme, with a notable outer-sphere component; the resulting metastable ferrous-peroxynitrate adduct is found to feature a very low barrier towards nitrate liberation, with ferryl as a final product-in good agreement with experiment.

Spectroscopic and computational study of a nonheme iron nitrosyl center in a biosynthetic model of nitric oxide reductase.

A major barrier to understanding the mechanism of nitric oxide reductases (NORs) is the lack of a selective probe of NO binding to the nonheme FeB center. By replacing the heme in a biosynthetic model of NORs, which structurally and functionally mimics NORs, with isostructural ZnPP, the electronic structure and functional properties of the FeB nitrosyl complex was probed. This approach allowed observation of the first S=3/2 nonheme {FeNO}(7) complex in a protein-based model system of NOR. Detailed spectroscopic and computational studies show that the electronic state of the {FeNO}(7) complex is best described as a high spin ferrous iron (S=2) antiferromagnetically coupled to an NO radical (S=1/2) [Fe(2+)-NO(.)]. The radical nature of the FeB -bound NO would facilitate N-N bond formation by radical coupling with the heme-bound NO. This finding, therefore, supports the proposed trans mechanism of NO reduction by NORs.

Global assessment of arsenic pollution using sperm whales (Physeter macrocephalus) as an emerging aquatic model organism.

Arsenic is an oceanic pollutant of global concern due to its toxicity, ability to bioaccumulate and continued input into the environment by anthropogenic activities. The sperm whale (Physeter macrocephalus) is an emerging aquatic model for both human disease and ocean health having global distribution and high trophic level. The aim of this study was to establish global and regional baselines of total arsenic concentrations using free-ranging sperm whales. Skin biopsies (n=342) were collected during the voyage of the Odyssey (2000-2005) from 17 regions considering gender and age in males. Arsenic was detectable in 99% of samples with a global mean of 1.9µg/g ww ranging from 0.1 to 15.6µg/g ww. Previous work in toothed whale skin found mean concentrations 3 fold lower with 0.6µg/g ww. A significant gender-related effect was found with males having higher mean arsenic concentrations than females. There was no significant age-related effect between adult and subadult males. Arsenic concentrations in sloughed skin samples were similar to levels in skin biopsies indicating that arsenic excretion can occur by skin sloughing. Regional mean concentrations were highest in the Maldives, Seychelles and Sri Lanka with 3.5, 2.5, and 2.4µg/g ww, respectively, raising concern for arsenic pollution in the Indian Ocean. Literature suggests that arsenic exposure is emitted from natural sources and the heavy use of arsenic-containing pesticides and herbicides in this region. These data suggest that research is needed in determining the extent and source of arsenic pollution in the Indian Ocean.