Correction: The Minderoo-Monaco Commission on Plastics and Human Health.

[This corrects the article DOI: 10.5334/aogh.4056.].

Structural modeling of cytochrome P450 51 from a deep-sea fish points to a novel structural feature in other CYP51s.

Cytochromes P450 (CYP), enzymes involved in the metabolism of endogenous and xenobiotic substrates, provide an excellent model system to study how membrane proteins with unique functions have catalytically adapted through evolution. Molecular adaptation of deep-sea proteins to high hydrostatic pressure remains poorly understood. Herein, we have characterized recombinant cytochrome P450 sterol 14α-demethylase (CYP51), an essential enzyme of cholesterol biosynthesis, from an abyssal fish species, Coryphaenoides armatus. C. armatus CYP51 was heterologously expressed in Escherichia coli following N-terminal truncation and purified to homogeneity. Recombinant C. armatus CYP51 bound its sterol substrate lanosterol giving a Type I binding spectra (KD 15 µM) and catalyzed lanosterol 14α-demethylation turnover at 5.8 nmol/min/nmol P450. C. armatus CYP51 also bound the azole antifungals ketoconazole (KD 0.12 µM) and propiconazole (KD 0.54 µM) as determined by Type II absorbance spectra. Comparison of C. armatus CYP51 primary sequence and modeled structures with other CYP51s identified amino acid substitutions that may confer an ability to function under pressures of the deep sea and revealed heretofore undescribed internal cavities in human and other non-deep sea CYP51s. The functional significance of these cavities is not known. PROLOGUE: This paper is dedicated in memory of Michael Waterman and Tsuneo Omura, who as good friends and colleagues enriched our lives. They continue to inspire us.

The Minderoo-Monaco Commission on Plastics and Human Health.

Background: Plastics have conveyed great benefits to humanity and made possible some of the most significant advances of modern civilization in fields as diverse as medicine, electronics, aerospace, construction, food packaging, and sports. It is now clear, however, that plastics are also responsible for significant harms to human health, the economy, and the earth's environment. These harms occur at every stage of the plastic life cycle, from extraction of the coal, oil, and gas that are its main feedstocks through to ultimate disposal into the environment. The extent of these harms not been systematically assessed, their magnitude not fully quantified, and their economic costs not comprehensively counted. Goals: The goals of this Minderoo-Monaco Commission on Plastics and Human Health are to comprehensively examine plastics' impacts across their life cycle on: (1) human health and well-being; (2) the global environment, especially the ocean; (3) the economy; and (4) vulnerable populations-the poor, minorities, and the world's children. On the basis of this examination, the Commission offers science-based recommendations designed to support development of a Global Plastics Treaty, protect human health, and save lives. Report Structure: This Commission report contains seven Sections. Following an Introduction, Section 2 presents a narrative review of the processes involved in plastic production, use, and disposal and notes the hazards to human health and the environment associated with each of these stages. Section 3 describes plastics' impacts on the ocean and notes the potential for plastic in the ocean to enter the marine food web and result in human exposure. Section 4 details plastics' impacts on human health. Section 5 presents a first-order estimate of plastics' health-related economic costs. Section 6 examines the intersection between plastic, social inequity, and environmental injustice. Section 7 presents the Commission's findings and recommendations. Plastics: Plastics are complex, highly heterogeneous, synthetic chemical materials. Over 98% of plastics are produced from fossil carbon- coal, oil and gas. Plastics are comprised of a carbon-based polymer backbone and thousands of additional chemicals that are incorporated into polymers to convey specific properties such as color, flexibility, stability, water repellence, flame retardation, and ultraviolet resistance. Many of these added chemicals are highly toxic. They include carcinogens, neurotoxicants and endocrine disruptors such as phthalates, bisphenols, per- and poly-fluoroalkyl substances (PFAS), brominated flame retardants, and organophosphate flame retardants. They are integral components of plastic and are responsible for many of plastics' harms to human health and the environment.Global plastic production has increased almost exponentially since World War II, and in this time more than 8,300 megatons (Mt) of plastic have been manufactured. Annual production volume has grown from under 2 Mt in 1950 to 460 Mt in 2019, a 230-fold increase, and is on track to triple by 2060. More than half of all plastic ever made has been produced since 2002. Single-use plastics account for 35-40% of current plastic production and represent the most rapidly growing segment of plastic manufacture.Explosive recent growth in plastics production reflects a deliberate pivot by the integrated multinational fossil-carbon corporations that produce coal, oil and gas and that also manufacture plastics. These corporations are reducing their production of fossil fuels and increasing plastics manufacture. The two principal factors responsible for this pivot are decreasing global demand for carbon-based fuels due to increases in 'green' energy, and massive expansion of oil and gas production due to fracking.Plastic manufacture is energy-intensive and contributes significantly to climate change. At present, plastic production is responsible for an estimated 3.7% of global greenhouse gas emissions, more than the contribution of Brazil. This fraction is projected to increase to 4.5% by 2060 if current trends continue unchecked. Plastic Life Cycle: The plastic life cycle has three phases: production, use, and disposal. In production, carbon feedstocks-coal, gas, and oil-are transformed through energy-intensive, catalytic processes into a vast array of products. Plastic use occurs in every aspect of modern life and results in widespread human exposure to the chemicals contained in plastic. Single-use plastics constitute the largest portion of current use, followed by synthetic fibers and construction.Plastic disposal is highly inefficient, with recovery and recycling rates below 10% globally. The result is that an estimated 22 Mt of plastic waste enters the environment each year, much of it single-use plastic and are added to the more than 6 gigatons of plastic waste that have accumulated since 1950. Strategies for disposal of plastic waste include controlled and uncontrolled landfilling, open burning, thermal conversion, and export. Vast quantities of plastic waste are exported each year from high-income to low-income countries, where it accumulates in landfills, pollutes air and water, degrades vital ecosystems, befouls beaches and estuaries, and harms human health-environmental injustice on a global scale. Plastic-laden e-waste is particularly problematic. Environmental Findings: Plastics and plastic-associated chemicals are responsible for widespread pollution. They contaminate aquatic (marine and freshwater), terrestrial, and atmospheric environments globally. The ocean is the ultimate destination for much plastic, and plastics are found throughout the ocean, including coastal regions, the sea surface, the deep sea, and polar sea ice. Many plastics appear to resist breakdown in the ocean and could persist in the global environment for decades. Macro- and micro-plastic particles have been identified in hundreds of marine species in all major taxa, including species consumed by humans. Trophic transfer of microplastic particles and the chemicals within them has been demonstrated. Although microplastic particles themselves (>10 µm) appear not to undergo biomagnification, hydrophobic plastic-associated chemicals bioaccumulate in marine animals and biomagnify in marine food webs. The amounts and fates of smaller microplastic and nanoplastic particles (MNPs <10 µm) in aquatic environments are poorly understood, but the potential for harm is worrying given their mobility in biological systems. Adverse environmental impacts of plastic pollution occur at multiple levels from molecular and biochemical to population and ecosystem. MNP contamination of seafood results in direct, though not well quantified, human exposure to plastics and plastic-associated chemicals. Marine plastic pollution endangers the ocean ecosystems upon which all humanity depends for food, oxygen, livelihood, and well-being. Human Health Findings: Coal miners, oil workers and gas field workers who extract fossil carbon feedstocks for plastic production suffer increased mortality from traumatic injury, coal workers' pneumoconiosis, silicosis, cardiovascular disease, chronic obstructive pulmonary disease, and lung cancer. Plastic production workers are at increased risk of leukemia, lymphoma, hepatic angiosarcoma, brain cancer, breast cancer, mesothelioma, neurotoxic injury, and decreased fertility. Workers producing plastic textiles die of bladder cancer, lung cancer, mesothelioma, and interstitial lung disease at increased rates. Plastic recycling workers have increased rates of cardiovascular disease, toxic metal poisoning, neuropathy, and lung cancer. Residents of "fenceline" communities adjacent to plastic production and waste disposal sites experience increased risks of premature birth, low birth weight, asthma, childhood leukemia, cardiovascular disease, chronic obstructive pulmonary disease, and lung cancer.During use and also in disposal, plastics release toxic chemicals including additives and residual monomers into the environment and into people. National biomonitoring surveys in the USA document population-wide exposures to these chemicals. Plastic additives disrupt endocrine function and increase risk for premature births, neurodevelopmental disorders, male reproductive birth defects, infertility, obesity, cardiovascular disease, renal disease, and cancers. Chemical-laden MNPs formed through the environmental degradation of plastic waste can enter living organisms, including humans. Emerging, albeit still incomplete evidence indicates that MNPs may cause toxicity due to their physical and toxicological effects as well as by acting as vectors that transport toxic chemicals and bacterial pathogens into tissues and cells.Infants in the womb and young children are two populations at particularly high risk of plastic-related health effects. Because of the exquisite sensitivity of early development to hazardous chemicals and children's unique patterns of exposure, plastic-associated exposures are linked to increased risks of prematurity, stillbirth, low birth weight, birth defects of the reproductive organs, neurodevelopmental impairment, impaired lung growth, and childhood cancer. Early-life exposures to plastic-associated chemicals also increase the risk of multiple non-communicable diseases later in life. Economic Findings: Plastic's harms to human health result in significant economic costs. We estimate that in 2015 the health-related costs of plastic production exceeded $250 billion (2015 Int$) globally, and that in the USA alone the health costs of disease and disability caused by the plastic-associated chemicals PBDE, BPA and DEHP exceeded $920 billion (2015 Int$). Plastic production results in greenhouse gas (GHG) emissions equivalent to 1.96 gigatons of carbon dioxide (CO2e) annually. Using the US Environmental Protection Agency's (EPA) social cost of carbonmetric, we estimate the annual costs of these GHG emissions to be $341 billion (2015 Int$).These costs, large as they are, almost certainly underestimate the full economic losses resulting from plastics' negative impacts on human health and the global environment. All of plastics' economic costs-and also its social costs-are externalized by the petrochemical and plastic manufacturing industry and are borne by citizens, taxpayers, and governments in countries around the world without compensation. Social Justice Findings: The adverse effects of plastics and plastic pollution on human health, the economy and the environment are not evenly distributed. They disproportionately affect poor, disempowered, and marginalized populations such as workers, racial and ethnic minorities, "fenceline" communities, Indigenous groups, women, and children, all of whom had little to do with creating the current plastics crisis and lack the political influence or the resources to address it. Plastics' harmful impacts across its life cycle are most keenly felt in the Global South, in small island states, and in disenfranchised areas in the Global North. Social and environmental justice (SEJ) principles require reversal of these inequitable burdens to ensure that no group bears a disproportionate share of plastics' negative impacts and that those who benefit economically from plastic bear their fair share of its currently externalized costs. Conclusions: It is now clear that current patterns of plastic production, use, and disposal are not sustainable and are responsible for significant harms to human health, the environment, and the economy as well as for deep societal injustices.The main driver of these worsening harms is an almost exponential and still accelerating increase in global plastic production. Plastics' harms are further magnified by low rates of recovery and recycling and by the long persistence of plastic waste in the environment.The thousands of chemicals in plastics-monomers, additives, processing agents, and non-intentionally added substances-include amongst their number known human carcinogens, endocrine disruptors, neurotoxicants, and persistent organic pollutants. These chemicals are responsible for many of plastics' known harms to human and planetary health. The chemicals leach out of plastics, enter the environment, cause pollution, and result in human exposure and disease. All efforts to reduce plastics' hazards must address the hazards of plastic-associated chemicals. Recommendations: To protect human and planetary health, especially the health of vulnerable and at-risk populations, and put the world on track to end plastic pollution by 2040, this Commission supports urgent adoption by the world's nations of a strong and comprehensive Global Plastics Treaty in accord with the mandate set forth in the March 2022 resolution of the United Nations Environment Assembly (UNEA).International measures such as a Global Plastics Treaty are needed to curb plastic production and pollution, because the harms to human health and the environment caused by plastics, plastic-associated chemicals and plastic waste transcend national boundaries, are planetary in their scale, and have disproportionate impacts on the health and well-being of people in the world's poorest nations. Effective implementation of the Global Plastics Treaty will require that international action be coordinated and complemented by interventions at the national, regional, and local levels.This Commission urges that a cap on global plastic production with targets, timetables, and national contributions be a central provision of the Global Plastics Treaty. We recommend inclusion of the following additional provisions:The Treaty needs to extend beyond microplastics and marine litter to include all of the many thousands of chemicals incorporated into plastics.The Treaty needs to include a provision banning or severely restricting manufacture and use of unnecessary, avoidable, and problematic plastic items, especially single-use items such as manufactured plastic microbeads.The Treaty needs to include requirements on extended producer responsibility (EPR) that make fossil carbon producers, plastic producers, and the manufacturers of plastic products legally and financially responsible for the safety and end-of-life management of all the materials they produce and sell.The Treaty needs to mandate reductions in the chemical complexity of plastic products; health-protective standards for plastics and plastic additives; a requirement for use of sustainable non-toxic materials; full disclosure of all components; and traceability of components. International cooperation will be essential to implementing and enforcing these standards.The Treaty needs to include SEJ remedies at each stage of the plastic life cycle designed to fill gaps in community knowledge and advance both distributional and procedural equity.This Commission encourages inclusion in the Global Plastic Treaty of a provision calling for exploration of listing at least some plastic polymers as persistent organic pollutants (POPs) under the Stockholm Convention.This Commission encourages a strong interface between the Global Plastics Treaty and the Basel and London Conventions to enhance management of hazardous plastic waste and slow current massive exports of plastic waste into the world's least-developed countries.This Commission recommends the creation of a Permanent Science Policy Advisory Body to guide the Treaty's implementation. The main priorities of this Body would be to guide Member States and other stakeholders in evaluating which solutions are most effective in reducing plastic consumption, enhancing plastic waste recovery and recycling, and curbing the generation of plastic waste. This Body could also assess trade-offs among these solutions and evaluate safer alternatives to current plastics. It could monitor the transnational export of plastic waste. It could coordinate robust oceanic-, land-, and air-based MNP monitoring programs.This Commission recommends urgent investment by national governments in research into solutions to the global plastic crisis. This research will need to determine which solutions are most effective and cost-effective in the context of particular countries and assess the risks and benefits of proposed solutions. Oceanographic and environmental research is needed to better measure concentrations and impacts of plastics <10 µm and understand their distribution and fate in the global environment. Biomedical research is needed to elucidate the human health impacts of plastics, especially MNPs. Summary: This Commission finds that plastics are both a boon to humanity and a stealth threat to human and planetary health. Plastics convey enormous benefits, but current linear patterns of plastic production, use, and disposal that pay little attention to sustainable design or safe materials and a near absence of recovery, reuse, and recycling are responsible for grave harms to health, widespread environmental damage, great economic costs, and deep societal injustices. These harms are rapidly worsening.While there remain gaps in knowledge about plastics' harms and uncertainties about their full magnitude, the evidence available today demonstrates unequivocally that these impacts are great and that they will increase in severity in the absence of urgent and effective intervention at global scale. Manufacture and use of essential plastics may continue. However, reckless increases in plastic production, and especially increases in the manufacture of an ever-increasing array of unnecessary single-use plastic products, need to be curbed.Global intervention against the plastic crisis is needed now because the costs of failure to act will be immense.

Characterization of a Virally Encoded Flavodoxin That Can Drive Bacterial Cytochrome P450 Monooxygenase Activity.

Flavodoxins are small electron transport proteins that are involved in a myriad of photosynthetic and non-photosynthetic metabolic pathways in Bacteria (including cyanobacteria), Archaea and some algae. The sequenced genome of 0305φ8-36, a large bacteriophage that infects the soil bacterium Bacillus thuringiensis, was predicted to encode a putative flavodoxin redox protein. Here we confirm that 0305φ8-36 phage encodes a FMN-containing flavodoxin polypeptide and we report the expression, purification and enzymatic characterization of the recombinant protein. Purified 0305φ8-36 flavodoxin has near-identical spectral properties to control, purified Escherichia coli flavodoxin. Using in vitro assays we show that 0305φ8-36 flavodoxin can be reconstituted with E. coli flavodoxin reductase and support regio- and stereospecific cytochrome P450 CYP170A1 allyl-oxidation of epi-isozizaene to the sesquiterpene antibiotic product albaflavenone, found in the soil bacterium Streptomyces coelicolor. In vivo, 0305φ8-36 flavodoxin is predicted to mediate the 2-electron reduction of the ß subunit of phage-encoded ribonucleotide reductase to catalyse the conversion of ribonucleotides to deoxyribonucleotides during viral replication. Our results demonstrate that this phage flavodoxin has the potential to manipulate and drive bacterial P450 cellular metabolism, which may affect both the host biological fitness and the communal microbiome. Such a scenario may also be applicable in other viral-host symbiotic/parasitic relationships.

Metabolic arsenal of giant viruses: Host hijack or self-use?

Viruses generally are defined as lacking the fundamental properties of living organisms in that they do not harbor an energy metabolism system or protein synthesis machinery. However, the discovery of giant viruses of amoeba has fundamentally challenged this view because of their exceptional genome properties, particle sizes and encoding of the enzyme machinery for some steps of protein synthesis. Although giant viruses are not able to replicate autonomously and still require a host for their multiplication, numerous metabolic genes involved in energy production have been recently detected in giant virus genomes from many environments. These findings have further blurred the boundaries that separate viruses and living organisms. Herein, we summarize information concerning genes and proteins involved in cellular metabolic pathways and their orthologues that have, surprisingly, been discovered in giant viruses. The remarkable diversity of metabolic genes described in giant viruses include genes encoding enzymes involved in glycolysis, gluconeogenesis, tricarboxylic acid cycle, photosynthesis, and ß-oxidation. These viral genes are thought to have been acquired from diverse biological sources through lateral gene transfer early in the evolution of Nucleo-Cytoplasmic Large DNA Viruses, or in some cases more recently. It was assumed that viruses are capable of hijacking host metabolic networks. But the giant virus auxiliary metabolic genes also may represent another form of host metabolism manipulation, by expanding the catalytic capabilities of the host cells especially in harsh environments, providing the infected host cells with a selective evolutionary advantage compared to non-infected cells and hence favoring the viral replication. However, the mechanism of these genes' functionality remains unclear to date.

Orphan cytochrome P450 20a1 CRISPR/Cas9 mutants and neurobehavioral phenotypes in zebrafish.

Orphan cytochrome P450 (CYP) enzymes are those for which biological substrates and function(s) are unknown. Cytochrome P450 20A1 (CYP20A1) is the last human orphan P450 enzyme, and orthologs occur as single genes in every vertebrate genome sequenced to date. The occurrence of high levels of CYP20A1 transcripts in human substantia nigra and hippocampus and abundant maternal transcripts in zebrafish eggs strongly suggest roles both in the brain and during early embryonic development. Patients with chromosome 2 microdeletions including CYP20A1 show hyperactivity and bouts of anxiety, among other conditions. Here, we created zebrafish cyp20a1 mutants using CRISPR/Cas9, providing vertebrate models with which to study the role of CYP20A1 in behavior and other neurodevelopmental functions. The homozygous cyp20a1 null mutants exhibited significant behavioral differences from wild-type zebrafish, both in larval and adult animals. Larval cyp20a1-/- mutants exhibited a strong increase in light-simulated movement (i.e., light-dark assay), which was interpreted as hyperactivity. Further, the larvae exhibited mild hypoactivity during the adaptation period of the optomotor assays. Adult cyp20a1 null fish showed a pronounced delay in adapting to new environments, which is consistent with an anxiety paradigm. Taken together with our earlier morpholino cyp20a1 knockdown results, the results described herein suggest that the orphan CYP20A1 has a neurophysiological role.

Developmental exposure to non-dioxin-like polychlorinated biphenyls promotes sensory deficits and disrupts dopaminergic and GABAergic signaling in zebrafish.

The most abundant polychlorinated biphenyl (PCB) congeners found in the environment and in humans are neurotoxic. This is of particular concern for early life stages because the exposure of the more vulnerable developing nervous system to neurotoxic chemicals can result in neurobehavioral disorders. In this study, we uncover currently unknown links between PCB target mechanisms and neurobehavioral deficits using zebrafish as a vertebrate model. We investigated the effects of the abundant non-dioxin-like (NDL) congener PCB153 on neuronal morphology and synaptic transmission linked to the proper execution of a sensorimotor response. Zebrafish that were exposed during development to concentrations similar to those found in human cord blood and PCB contaminated sites showed a delay in startle response. Morphological and biochemical data demonstrate that even though PCB153-induced swelling of afferent sensory neurons, the disruption of dopaminergic and GABAergic signaling appears to contribute to PCB-induced motor deficits. A similar delay was observed for other NDL congeners but not for the potent dioxin-like congener PCB126. The effects on important and broadly conserved signaling mechanisms in vertebrates suggest that NDL PCBs may contribute to neurodevelopmental abnormalities in humans and increased selection pressures in vertebrate wildlife.

Resistance to Cyp3a induction by polychlorinated biphenyls, including non-dioxin-like PCB153, in gills of killifish (Fundulus heteroclitus) from New Bedford Harbor.

Previous reports suggested that non-dioxin-like (NDL) PCB153 effects on cytochrome P450 3A (Cyp3a) expression in Atlantic killifish (Fundulus heteroclitus) gills differed between F0 generation fish from a PCB site (New Bedford Harbor; NBH) and a reference site (Scorton Creek; SC). Here, we examined effects of PCB153, dioxin-like (DL) PCB126, or a mixture of both, on Cyp3a56 mRNA in killifish generations removed from the wild, without environmental PCB exposures. PCB126 effects in liver and gills differed between populations, as expected. Gill Cyp3a56 was not affected by either congener in NBH F2 generation fish, but was induced by PCB153 in SC F1 fish, with females showing a greater response. PCB153 did not affect Cyp3a56 in liver of either population. Results suggest a heritable resistance to NDL-PCBs in killifish from NBH, in addition to that reported for DL PCBs. Induction of Cyp3a56 in gills may be a biomarker of exposure to NDL PCBs in fish populations that are not resistant to PCBs.

Concerning P450 Evolution: Structural Analyses Support Bacterial Origin of Sterol 14α-Demethylases.

Sterol biosynthesis, primarily associated with eukaryotic kingdoms of life, occurs as an abbreviated pathway in the bacterium Methylococcus capsulatus. Sterol 14α-demethylation is an essential step in this pathway and is catalyzed by cytochrome P450 51 (CYP51). In M. capsulatus, the enzyme consists of the P450 domain naturally fused to a ferredoxin domain at the C-terminus (CYP51fx). The structure of M. capsulatus CYP51fx was solved to 2.7 Å resolution and is the first structure of a bacterial sterol biosynthetic enzyme. The structure contained one P450 molecule per asymmetric unit with no electron density seen for ferredoxin. We connect this with the requirement of P450 substrate binding in order to activate productive ferredoxin binding. Further, the structure of the P450 domain with bound detergent (which replaced the substrate upon crystallization) was solved to 2.4 Å resolution. Comparison of these two structures to the CYP51s from human, fungi, and protozoa reveals strict conservation of the overall protein architecture. However, the structure of an "orphan" P450 from nonsterol-producing Mycobacterium tuberculosis that also has CYP51 activity reveals marked differences, suggesting that loss of function in vivo might have led to alterations in the structural constraints. Our results are consistent with the idea that eukaryotic and bacterial CYP51s evolved from a common cenancestor and that early eukaryotes may have recruited CYP51 from a bacterial source. The idea is supported by bioinformatic analysis, revealing the presence of CYP51 genes in >1,000 bacteria from nine different phyla, >50 of them being natural CYP51fx fusion proteins.

Human Health and Ocean Pollution.

Background: Pollution - unwanted waste released to air, water, and land by human activity - is the largest environmental cause of disease in the world today. It is responsible for an estimated nine million premature deaths per year, enormous economic losses, erosion of human capital, and degradation of ecosystems. Ocean pollution is an important, but insufficiently recognized and inadequately controlled component of global pollution. It poses serious threats to human health and well-being. The nature and magnitude of these impacts are only beginning to be understood. Goals: (1) Broadly examine the known and potential impacts of ocean pollution on human health. (2) Inform policy makers, government leaders, international organizations, civil society, and the global public of these threats. (3) Propose priorities for interventions to control and prevent pollution of the seas and safeguard human health. Methods: Topic-focused reviews that examine the effects of ocean pollution on human health, identify gaps in knowledge, project future trends, and offer evidence-based guidance for effective intervention. Environmental Findings: Pollution of the oceans is widespread, worsening, and in most countries poorly controlled. It is a complex mixture of toxic metals, plastics, manufactured chemicals, petroleum, urban and industrial wastes, pesticides, fertilizers, pharmaceutical chemicals, agricultural runoff, and sewage. More than 80% arises from land-based sources. It reaches the oceans through rivers, runoff, atmospheric deposition and direct discharges. It is often heaviest near the coasts and most highly concentrated along the coasts of low- and middle-income countries. Plastic is a rapidly increasing and highly visible component of ocean pollution, and an estimated 10 million metric tons of plastic waste enter the seas each year. Mercury is the metal pollutant of greatest concern in the oceans; it is released from two main sources - coal combustion and small-scale gold mining. Global spread of industrialized agriculture with increasing use of chemical fertilizer leads to extension of Harmful Algal Blooms (HABs) to previously unaffected regions. Chemical pollutants are ubiquitous and contaminate seas and marine organisms from the high Arctic to the abyssal depths. Ecosystem Findings: Ocean pollution has multiple negative impacts on marine ecosystems, and these impacts are exacerbated by global climate change. Petroleum-based pollutants reduce photosynthesis in marine microorganisms that generate oxygen. Increasing absorption of carbon dioxide into the seas causes ocean acidification, which destroys coral reefs, impairs shellfish development, dissolves calcium-containing microorganisms at the base of the marine food web, and increases the toxicity of some pollutants. Plastic pollution threatens marine mammals, fish, and seabirds and accumulates in large mid-ocean gyres. It breaks down into microplastic and nanoplastic particles containing multiple manufactured chemicals that can enter the tissues of marine organisms, including species consumed by humans. Industrial releases, runoff, and sewage increase frequency and severity of HABs, bacterial pollution, and anti-microbial resistance. Pollution and sea surface warming are triggering poleward migration of dangerous pathogens such as the Vibrio species. Industrial discharges, pharmaceutical wastes, pesticides, and sewage contribute to global declines in fish stocks. Human Health Findings: Methylmercury and PCBs are the ocean pollutants whose human health effects are best understood. Exposures of infants in utero to these pollutants through maternal consumption of contaminated seafood can damage developing brains, reduce IQ and increase children's risks for autism, ADHD and learning disorders. Adult exposures to methylmercury increase risks for cardiovascular disease and dementia. Manufactured chemicals - phthalates, bisphenol A, flame retardants, and perfluorinated chemicals, many of them released into the seas from plastic waste - can disrupt endocrine signaling, reduce male fertility, damage the nervous system, and increase risk of cancer. HABs produce potent toxins that accumulate in fish and shellfish. When ingested, these toxins can cause severe neurological impairment and rapid death. HAB toxins can also become airborne and cause respiratory disease. Pathogenic marine bacteria cause gastrointestinal diseases and deep wound infections. With climate change and increasing pollution, risk is high that Vibrio infections, including cholera, will increase in frequency and extend to new areas. All of the health impacts of ocean pollution fall disproportionately on vulnerable populations in the Global South - environmental injustice on a planetary scale. Conclusions: Ocean pollution is a global problem. It arises from multiple sources and crosses national boundaries. It is the consequence of reckless, shortsighted, and unsustainable exploitation of the earth's resources. It endangers marine ecosystems. It impedes the production of atmospheric oxygen. Its threats to human health are great and growing, but still incompletely understood. Its economic costs are only beginning to be counted.Ocean pollution can be prevented. Like all forms of pollution, ocean pollution can be controlled by deploying data-driven strategies based on law, policy, technology, and enforcement that target priority pollution sources. Many countries have used these tools to control air and water pollution and are now applying them to ocean pollution. Successes achieved to date demonstrate that broader control is feasible. Heavily polluted harbors have been cleaned, estuaries rejuvenated, and coral reefs restored.Prevention of ocean pollution creates many benefits. It boosts economies, increases tourism, helps restore fisheries, and improves human health and well-being. It advances the Sustainable Development Goals (SDG). These benefits will last for centuries. Recommendations: World leaders who recognize the gravity of ocean pollution, acknowledge its growing dangers, engage civil society and the global public, and take bold, evidence-based action to stop pollution at source will be critical to preventing ocean pollution and safeguarding human health.Prevention of pollution from land-based sources is key. Eliminating coal combustion and banning all uses of mercury will reduce mercury pollution. Bans on single-use plastic and better management of plastic waste reduce plastic pollution. Bans on persistent organic pollutants (POPs) have reduced pollution by PCBs and DDT. Control of industrial discharges, treatment of sewage, and reduced applications of fertilizers have mitigated coastal pollution and are reducing frequency of HABs. National, regional and international marine pollution control programs that are adequately funded and backed by strong enforcement have been shown to be effective. Robust monitoring is essential to track progress.Further interventions that hold great promise include wide-scale transition to renewable fuels; transition to a circular economy that creates little waste and focuses on equity rather than on endless growth; embracing the principles of green chemistry; and building scientific capacity in all countries.Designation of Marine Protected Areas (MPAs) will safeguard critical ecosystems, protect vulnerable fish stocks, and enhance human health and well-being. Creation of MPAs is an important manifestation of national and international commitment to protecting the health of the seas.

CRISPR-Cas9-Mutated Pregnane X Receptor (pxr) Retains Pregnenolone-induced Expression of cyp3a65 in Zebrafish (Danio rerio) Larvae.

Pregnane X receptor (PXR; NR1I2) is a nuclear receptor that regulates transcriptional responses to drug or xenobiotic exposure, including induction of CYP3A transcription, in many vertebrate species. PXR is activated by a wide range of ligands that differ across species, making functional studies on its role in the chemical defensome most relevant when approached in a species-specific manner. Knockout studies in mammals have shown a requirement for PXR in ligand-dependent activation of CYP3A expression or reporter gene activity. Morpholino knockdown of Pxr in zebrafish indicated a similar requirement. Here, we report on the generation of 2 zebrafish lines each carrying a heritable deletion in the pxr coding region, predicted to result in loss of a functional gene product. To our surprise, larvae homozygous for either of the pxr mutant alleles retain their ability to induce cyp3a65 mRNA expression following exposure to the established zebrafish Pxr ligand, pregnenolone. Thus, zebrafish carrying pxr alleles with deletions in either the DNA binding or the ligand-binding domains did not yield a loss-of-function phenotype, suggesting that a compensatory mechanism is responsible for cyp3a65 induction. Alternative possibilities are that Pxr is not required for the induction of selected genes, or that truncated yet functional mutant Pxr is sufficient for the downstream transcriptional effects. It is crucial that we develop a better understanding for the role of Pxr in this important biomedical test species. This study highlights the potential for compensatory mechanisms to avoid deleterious effects arising from gene mutations.

On the occurrence of cytochrome P450 in viruses.

Genes encoding cytochrome P450 (CYP; P450) enzymes occur widely in the Archaea, Bacteria, and Eukarya, where they play important roles in metabolism of endogenous regulatory molecules and exogenous chemicals. We now report that genes for multiple and unique P450s occur commonly in giant viruses in the Mimiviridae, Pandoraviridae, and other families in the proposed order Megavirales. P450 genes were also identified in a herpesvirus (Ranid herpesvirus 3) and a phage (Mycobacterium phage Adler). The Adler phage P450 was classified as CYP102L1, and the crystal structure of the open form was solved at 2.5 Å. Genes encoding known redox partners for P450s (cytochrome P450 reductase, ferredoxin and ferredoxin reductase, and flavodoxin and flavodoxin reductase) were not found in any viral genome so far described, implying that host redox partners may drive viral P450 activities. Giant virus P450 proteins share no more than 25% identity with the P450 gene products we identified in Acanthamoeba castellanii, an amoeba host for many giant viruses. Thus, the origin of the unique P450 genes in giant viruses remains unknown. If giant virus P450 genes were acquired from a host, we suggest it could have been from an as yet unknown and possibly ancient host. These studies expand the horizon in the evolution and diversity of the enormously important P450 superfamily. Determining the origin and function of P450s in giant viruses may help to discern the origin of the giant viruses themselves.

Sequence Variations in pxr (nr1i2) From Zebrafish (Danio rerio) Strains Affect Nuclear Receptor Function.

Regulators of biotransformation are of particular interest in pharmacology and toxicology, determining in part the metabolism, disposition, and toxicity of chemicals. The nuclear receptor NR1I2 (pregnane X receptor, PXR) is a prominent xenosensor that regulates the expression of biotransformation enzymes governing elimination of many exogenous as well as endogenous compounds. Zebrafish (Danio rerio) has only one gene locus for pxr, but different genetic variants have been identified in zebrafish. However, the prevalence and significance of these variants are unknown. We hypothesize that sequence variation occurring in the Pxr gene of zebrafish may affect the action and fate of many chemicals in this species, a key model organism in various fields of research, including environmental toxicology. Here, we examine variation in Pxr sequences from four different strains of zebrafish and assess the responses of each Pxr to clotrimazole and butyl-4-aminobenzoate. The Pxr variants differed in both their ability to bind these structurally different ligands and to regulate reporter gene expression in vitro. We infer that the observed sequence variations in zebrafish Pxrs likely affect the response to putative Pxr agonists in vivo and potentially cause strain-specific biotransformation of xenobiotics in zebrafish. Thus, the choice of zebrafish strain could affect the outcome of downstream toxicological studies.

Molecular adaptation to high pressure in cytochrome P450 1A and aryl hydrocarbon receptor systems of the deep-sea fish Coryphaenoides armatus.

Limited knowledge of the molecular evolution of deep-sea fish proteomes so far suggests that a few widespread residue substitutions in cytosolic proteins binding hydrophilic ligands contribute to resistance to the effects of high hydrostatic pressure (HP). Structure-function studies with additional protein systems, including membrane bound proteins, are essential to provide a more general picture of adaptation in these extremophiles. We explored molecular features of HP adaptation in proteins binding hydrophobic ligands, either in lipid bilayers (cytochrome P450 1A - CYP1A) or in the cytosol (the aryl hydrocarbon receptor - AHR), and their partners P450 oxidoreductase (POR) and AHR nuclear translocator (ARNT), respectively. Cloning studies identified the full-length coding sequence of AHR, CYP1A and POR, and a partial sequence of ARNT from Coryphaenoides armatus, an abyssal gadiform fish thriving down to 5000m depth. Inferred protein sequences were aligned with many non-deep-sea homologs to identify unique amino acid substitutions of possible relevance in HP adaptation. Positionally unique substitutions of various physicochemical properties were found in all four proteins, usually at sites of strong-to-absolute residue conservation. Some were in domains deemed important for protein-protein interaction or ligand binding. In addition, some involved removal or addition of beta-branched residues; local modifications of beta-branched residue patterns could be important to HP adaptation. In silico predictions further suggested that some unique substitutions might substantially modulate the flexibility of the polypeptide segment in which they are found. Repetitive motifs unique to the abyssal fish AHR were predicted to be rich in glycosylation sites, suggesting that post-translational changes could be involved in adaptation as well. Recombinant CYP1A and AHR showed functional properties (spectral characteristics, catalytic activity and ligand binding) that demonstrate proper folding at 1atm, indicating that they could be used as deep-sea fish protein models to further evaluate protein function under pressure. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone".

Ryanodine receptor and FK506 binding protein 1 in the Atlantic killifish (Fundulus heteroclitus): A phylogenetic and population-based comparison.

Non-dioxin-like polychlorinated biphenyls (NDL PCBs) activate ryanodine receptors (RyR), microsomal Ca2+ channels of broad significance. Teleost fish may be important models for NDL PCB neurotoxicity, and we used sequencing databases to characterize teleost RyR and FK506 binding protein 12 or 12.6kDa (genes FKBP1A; FKBP1B), which promote NDL PCB-triggered Ca2+ dysregulation. Particular focus was placed on describing genes in the Atlantic killifish (Fundulus heteroclitus) genome and searching available RNA-sequencing datasets for single nucleotide variants (SNV) between PCB tolerant killifish from New Bedford Harbor (NBH) versus sensitive killifish from Scorton Creek (SC), MA. Consistent with the teleost whole genome duplication (tWGD), killifish have six RyR genes, corresponding to a and b paralogs of mammalian RyR1, 2 and 3. The presence of six RyR genes was consistent in all teleosts investigated including zebrafish. Killifish have four FKBP1; one FKBP1b and three FKBP1a named FKBP1aa, FKBP1ab, likely from the tWGD and a single gene duplicate FKBP1a3 suggested to have arisen in Atherinomorphae. The RyR and FKBP1 genes displayed tissue and developmental stage-specific mRNA expression, and the previously uncharacterized RyR3, herein named RyR3b, and all FKBP1 genes were prominent in brain. We identified a SNV in RyR3b encoding missense mutation E1458D. In NBH killifish, 57% were heterozygous and 28% were homozygous for this SNV, whereas almost all SC killifish (94%) lacked the variant (n≥39 per population). The outlined sequence differences between mammalian and teleost RyR and FKBP1 together with outlined population differences in SNV frequency may contribute to our understanding of NDL PCB neurotoxicity.

The role of Nrf1 and Nrf2 in the regulation of glutathione and redox dynamics in the developing zebrafish embryo.

Redox signaling is important for embryogenesis, guiding pathways that govern processes crucial for embryo patterning, including cell polarization, proliferation, and apoptosis. Exposure to pro-oxidants during this period can be deleterious, resulting in altered physiology, teratogenesis, later-life diseases, or lethality. We previously reported that the glutathione antioxidant defense system becomes increasingly robust, including a doubling of total glutathione and dynamic shifts in the glutathione redox potential at specific stages during embryonic development in the zebrafish, Danio rerio. However, the mechanisms underlying these changes are unclear, as is the effectiveness of the glutathione system in ameliorating oxidative insults to the embryo at different stages. Here, we examine how the glutathione system responds to the model pro-oxidants tert-butylhydroperoxide and tert-butylhydroquinone at different developmental stages, and the role of Nuclear factor erythroid 2-related factor (Nrf) proteins in regulating developmental glutathione redox status. Embryos became increasingly sensitive to pro-oxidants after 72h post-fertilization (hpf), after which the duration of the recovery period for the glutathione redox potential was increased. To determine whether the doubling of glutathione or the dynamic changes in glutathione redox potential are mediated by zebrafish paralogs of Nrf transcription factors, morpholino oligonucleotides were used to knock down translation of Nrf1 and Nrf2 (nrf1a, nrf1b, nrf2a, nrf2b). Knockdown of Nrf1a or Nrf1b perturbed glutathione redox state until 72 hpf. Knockdown of Nrf2 paralogs also perturbed glutathione redox state but did not significantly affect the response of glutathione to pro-oxidants. Nrf1b morphants had decreased gene expression of glutathione synthesis enzymes, while hsp70 increased in Nrf2b morphants. This work demonstrates that despite having a more robust glutathione system, embryos become more sensitive to oxidative stress later in development, and that neither Nrf1 nor Nrf2 alone appear to be essential for the response and recovery of glutathione to oxidative insults.

The liver transcriptome of suckermouth armoured catfish (Pterygoplichthys anisitsi, Loricariidae): Identification of expansions in defensome gene families.

Pterygoplichthys is a genus of related suckermouth armoured catfishes native to South America, which have invaded tropical and subtropical regions worldwide. Physiological features, including an augmented resistance to organic xenobiotics, may have aided their settlement in foreign habitats. The liver transcriptome of Pterygoplichthys anisitsi was sequenced and used to characterize the diversity of mRNAs potentially involved in the responses to natural and anthropogenic chemicals. In total, 66,642 transcripts were assembled. Among the identified defensome genes, cytochromes P450 (CYP) were the most abundant, followed by sulfotransferases (SULT), nuclear receptors (NR) and ATP binding cassette transporters (ABC). A novel expansion in the CYP2Y subfamily was identified, as well as an independent expansion of the CYP2AAs. Two expansions were also observed among SULT1. Thirty-two transcripts were classified into twelve subfamilies of NR, while 21 encoded ABC transporters. The diversity of defensome transcripts sequenced herein could contribute to this species' resistance to organic xenobiotics.

Plant lignan secoisolariciresinol suppresses pericardial edema caused by dioxin-like compounds in developing zebrafish: Implications for suppression of morphological abnormalities.

Dioxins and dioxin-like compounds (DLCs) enter the body mainly through diet and cause various toxicological effects through activation of the aryl hydrocarbon receptor (AhR), a ligand activated transcription factor. Some plant extracts and phytochemicals are reported to suppress this transformation. However, most of these reports have been from in vitro experiments and few reports have been from in vivo experiments. In addition, there has been no report of foodstuffs that effectively prevent AhR-associated morphological abnormalities such as deformities caused by dioxins and DLCs in vivo. In this study, we show that secoisolariciresinol (SECO), a natural lignan-type polyphenolic phytochemical found mainly in flaxseed, has a rescuing effect, actually suppressing morphological abnormalities (pericardial edema) in zebrafish embryos exposed to 3,3',4,4',5-pentachlorobiphenyl (PCB126), a dioxin-like PCB congener. Importantly, the rescuing effect of SECO was still evident when it was applied 16 h after the beginning of exposure to PCB126. This study suggests that SECO may be useful as a natural suppressive agent for morphological abnormalities caused by dioxins and DLCs.

Biological effects of 6-formylindolo[3,2-b]carbazole (FICZ) in vivo are enhanced by loss of CYP1A function in an Ahr2-dependent manner.

6-Formylindolo[3,2-b]carbazole (FICZ) is a potent aryl hydrocarbon receptor (AHR) agonist that is efficiently metabolized by AHR-regulated cytochrome P4501 enzymes. FICZ is a proposed physiological AHR ligand that induces its own degradation as part of a regulatory negative feedback loop. In vitro studies in cells show that CYP1 inhibition in the presence of FICZ results in enhanced AHR activation, suggesting that FICZ accumulates in the cell when its metabolism is blocked. We used zebrafish (Danio rerio) embryos to investigate the in vivo effects of FICZ when CYP1A is knocked down or inhibited. Embryos were injected with morpholino antisense oligonucleotides targeting CYP1A (CYP1A-MO), Ahr2, or a combination of both. FICZ exposure of non-injected embryos or embryos injected with control morpholino had little effect. In CYP1A-MO-injected embryos, however, FICZ dramatically increased mortality, incidence and severity of pericardial edema and circulation failure, reduced hatching frequency, blocked swim bladder inflation, and strongly potentiated expression of Ahr2-regulated genes. These effects were substantially reduced in embryos with a combined knockdown of Ahr2 and CYP1A, indicating that the toxicity was mediated at least partly by Ahr2. Co-exposure to the CYP1 inhibitor alpha-naphthoflavone (αNF) and FICZ had similar effects as the combination of CYP1A-MO and FICZ. HPLC analysis of FICZ-exposed embryos showed increased levels of FICZ after concomitant CYP1A-MO injection or αNF co-exposure. Together, these results show that a functioning CYP1/AHR feedback loop is crucial for regulation of AHR signaling by a potential physiological ligand in vivo and further highlights the role of CYP1 enzymes in regulating biological effects of FICZ.

Cytochrome P450 20A1 in zebrafish: Cloning, regulation and potential involvement in hyperactivity disorders.

Cytochrome P450 (CYP) enzymes for which there is no functional information are considered "orphan" CYPs. Previous studies showed that CYP20A1, an orphan, is expressed in human hippocampus and substantia nigra, and in zebrafish (Danio rerio) CYP20A1 maternal transcript occurs in eggs, suggesting involvement in brain and in early development. Moreover, hyperactivity is reported in humans with chromosome 2 microdeletions including CYP20A1. We examined CYP20A1 in zebrafish, including impacts of chemical exposure on expression. Zebrafish CYP20A1 cDNA was cloned, sequenced, and aligned with cloned human CYP20A1 and predicted vertebrate orthologs. CYP20A1s share a highly conserved N-terminal region and unusual sequences in the I-helix and the heme-binding CYP signature motifs. CYP20A1 mRNA expression was observed in adult zebrafish organs including the liver, heart, gonads, spleen and brain, as well as the eye and optic nerve. Putative binding sites in proximal promoter regions of CYP20A1s, and response of zebrafish CYP20A1 to selected nuclear and xenobiotic receptor agonists, point to up-regulation by agents involved in steroid hormone response, cholesterol and lipid metabolism. There also was a dose-dependent reduction of CYP20A1 expression in embryos exposed to environmentally relevant levels of methylmercury. Morpholino knockdown of CYP20A1 in developing zebrafish resulted in behavioral effects, including hyperactivity and a slowing of the optomotor response in larvae. The results suggest that altered expression of CYP20A1 might be part of a mechanism linking methylmercury exposure to neurobehavioral deficits. The expanded information on CYP20A1 brings us closer to "deorphanization", that is, identifying CYP20A1 functions and its roles in health and disease.