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.

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.

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".

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.

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.

Role of pregnane X receptor and aryl hydrocarbon receptor in transcriptional regulation of pxr, CYP2, and CYP3 genes in developing zebrafish.

Ligand-activated receptors regulate numerous genes, and mediate effects of a broad set of endogenous and exogenous chemicals in vertebrates. Understanding the roles of these transcription factors in zebrafish (Danio rerio) is important to the use of this non-mammalian model in toxicological, pharmacological, and carcinogenesis research. Response to a potential agonist for the pregnane X receptor (Pxr) [pregnenolone (PN)] was examined in developing zebrafish, to assess involvement of Pxr in regulation of selected genes, including genes in cytochrome P450 subfamilies CYP2 and CYP3. We also examined interaction of Pxr and the aryl hydrocarbon receptor (Ahr) signaling pathways. Pregnenolone caused a dose-dependent increase in mRNA levels of pxr, ahr2, CYP1A, CYP2AA1, CYP2AA12, CYP3A65, and CYP3C1, most of which peaked at 3 µM PN. The well-known Ahr agonist 3,3',4,4',5-pentachlorobiphenyl (PCB126) also upregulated expression of pxr, ahr2, CYP1A, CYP2AA12, CYP3A65, and CYP3C1 in a dose-dependent manner. Inhibition of pxr translation by morpholino antisense oligonucleotides (MO) suppressed PN-induced expression of pxr, ahr2, CYP3A65, and CYP3C1 genes. Levels of CYP2AA1 and CYP2AA12 mRNA were increased in the control-MO group exposed to PN; this was prevented by knocking down Pxr. Similarly, Ahr2-MO treatment blocked PCB126-induced mRNA expression of pxr, CYP1A, CYP2AA12, CYP3A65, and CYP3C1. The present study shows self-regulation of pxr by PN in developing zebrafish. Selected zebrafish CYP1, CYP2 (including several CYP2AAs) and CYP3 genes appear to be under the regulation of both Pxr and Ahr2.

Glutathione redox dynamics and expression of glutathione-related genes in the developing embryo.

Embryonic development involves dramatic changes in cell proliferation and differentiation that must be highly coordinated and tightly regulated. Cellular redox balance is critical for cell fate decisions, but it is susceptible to disruption by endogenous and exogenous sources of oxidative stress. The most abundant endogenous nonprotein antioxidant defense molecule is the tripeptide glutathione (γ-glutamylcysteinylglycine, GSH), but the ontogeny of GSH concentration and redox state during early life stages is poorly understood. Here, we describe the GSH redox dynamics during embryonic and early larval development (0-5 days postfertilization) in the zebrafish (Danio rerio), a model vertebrate embryo. We measured reduced and oxidized glutathione using HPLC and calculated the whole embryo total glutathione (GSHT) concentrations and redox potentials (Eh) over 0-120 h of zebrafish development (including mature oocytes, fertilization, midblastula transition, gastrulation, somitogenesis, pharyngula, prehatch embryos, and hatched eleutheroembryos). GSHT concentration doubled between 12h postfertilization (hpf) and hatching. The GSH Eh increased, becoming more oxidizing during the first 12h, and then oscillated around -190 mV through organogenesis, followed by a rapid change, associated with hatching, to a more negative (more reducing) Eh (-220 mV). After hatching, Eh stabilized and remained steady through 120 hpf. The dynamic changes in GSH redox status and concentration defined discrete windows of development: primary organogenesis, organ differentiation, and larval growth. We identified the set of zebrafish genes involved in the synthesis, utilization, and recycling of GSH, including several novel paralogs, and measured how expression of these genes changes during development. Ontogenic changes in the expression of GSH-related genes support the hypothesis that GSH redox state is tightly regulated early in development. This study provides a foundation for understanding the redox regulation of developmental signaling and investigating the effects of oxidative stress during embryogenesis.

Ahr2-dependence of PCB126 effects on the swim bladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish.

The teleost swim bladder is assumed a homolog of the tetrapod lung. Both swim bladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR(2)) agonists; in zebrafish (Danio rerio) the swim bladder fails to inflate with exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P450 1 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swim bladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on ß-catenin dependent transcription, histological effects, and Ahr2 dependence of the effect of PCB126 on swim bladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24h and then held in clean water until day 4, a normal time for swim bladder inflation. The effects of PCB126 were concentration-dependent with EC(50) values of 1.4 to 2.0 nM for induction of the CYP1s, 3.7 and 5.1 nM (or higher) for cox-2a and cox-2b induction, and 2.5 nM for inhibition of swim bladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5 nM) on swim bladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2nM PCB126 approximately 30% of eleutheroembryos(3) failed to inflate the swim bladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swim bladder. Our results indicate that PCB126 blocks swim bladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swim bladder cells.

Cytochrome p450 1 genes in birds: evolutionary relationships and transcription profiles in chicken and Japanese quail embryos.

BACKGROUND: Cytochrome P450 1 (CYP1) genes are biomarkers for aryl hydrocarbon receptor (AHR) agonists and may be involved in some of their toxic effects. CYP1s other than the CYP1As are poorly studied in birds. Here we characterize avian CYP1B and CYP1C genes and the expression of the identified CYP1 genes and AHR1, comparing basal and induced levels in chicken and quail embryos. METHODOLOGY/PRINCIPAL FINDINGS: We cloned cDNAs of chicken CYP1C1 and quail CYP1B1 and AHR1. CYP1Cs occur in several bird genomes, but we found no CYP1C gene in quail. The CYP1C genomic region is highly conserved among vertebrates. This region also shares some synteny with the CYP1B region, consistent with CYP1B and CYP1C genes deriving from duplication of a common ancestor gene. Real-time RT-PCR analyses revealed similar tissue distribution patterns for CYP1A4, CYP1A5, CYP1B1, and AHR1 mRNA in chicken and quail embryos, with the highest basal expression of the CYP1As in liver, and of CYP1B1 in eye, brain, and heart. Chicken CYP1C1 mRNA levels were appreciable in eye and heart but relatively low in other organs. Basal transcript levels of the CYP1As were higher in quail than in chicken, while CYP1B1 levels were similar in the two species. 3,3',4,5,5'-Pentachlorobiphenyl induced all CYP1s in chicken; in quail a 1000-fold higher dose induced the CYP1As, but not CYP1B1. CONCLUSIONS/SIGNIFICANCE: The apparent absence of CYP1C1 in quail, and weak expression and induction of CYP1C1 in chicken suggest that CYP1Cs have diminishing roles in tetrapods; similar tissue expression suggests that such roles may be met by CYP1B1. Tissue distribution of CYP1B and CYP1C transcripts in birds resembles that previously found in zebrafish, suggesting that these genes serve similar functions in diverse vertebrates. Determining CYP1 catalytic functions in different species should indicate the evolving roles of these duplicated genes in physiological and toxicological processes.

Pacific Ocean-wide profile of CYP1A1 expression, stable carbon and nitrogen isotope ratios, and organic contaminant burden in sperm whale skin biopsies.

BACKGROUND: Ocean pollution affects marine organisms and ecosystems as well as humans. The International Oceanographic Commission recommends ocean health monitoring programs to investigate the presence of marine contaminants and the health of threatened species and the use of multiple and early-warning biomarker approaches. OBJECTIVE: We explored the hypothesis that biomarker and contaminant analyses in skin biopsies of the threatened sperm whale (Physeter macrocephalus) could reveal geographical trends in exposure on an oceanwide scale. METHODS: We analyzed cytochrome P450 1A1 (CYP1A1) expression (by immunohistochemistry), stable nitrogen and carbon isotope ratios (as general indicators of trophic position and latitude, respectively), and contaminant burdens in skin biopsies to explore regional trends in the Pacific Ocean. RESULTS: Biomarker analyses revealed significant regional differences within the Pacific Ocean. CYP1A1 expression was highest in whales from the Galapagos, a United Nations Educational, Scientific, and Cultural Organization World Heritage marine reserve, and was lowest in the sampling sites farthest away from continents. We examined the possible influence of the whales' sex, diet, or range and other parameters on regional variation in CYP1A1 expression, but data were inconclusive. In general, CYP1A1 expression was not significantly correlated with contaminant burdens in blubber. However, small sample sizes precluded detailed chemical analyses, and power to detect significant associations was limited. CONCLUSIONS: Our large-scale monitoring study was successful at identifying regional differences in CYP1A1 expression, providing a baseline for this known biomarker of exposure to aryl hydrocarbon receptor agonists. However, we could not identify factors that explained this variation. Future oceanwide CYP1A1 expression profiles in cetacean skin biopsies are warranted and could reveal whether globally distributed chemicals occur at biochemically relevant concentrations on a global basis, which may provide a measure of ocean integrity.

Cytochrome P4501A biomarker indication of the timeline of chronic exposure of Barrow's goldeneyes to residual Exxon Valdez oil.

We examined hepatic EROD activity, as an indicator of CYP1A induction, in Barrow's goldeneyes captured in areas oiled during the 1989 Exxon Valdez spill and those from nearby unoiled areas. We found that average EROD activity differed between areas during 2005, although the magnitude of the difference was reduced relative to a previous study from 1996/1997, and we found that areas did not differ by 2009. Similarly, we found that the proportion of individuals captured from oiled areas with elevated EROD activity (≥ 2 times unoiled average) declined from 41% in winter 1996/1997 to 10% in 2005 and 15% in 2009. This work adds to a body of literature describing the timelines over which vertebrates were exposed to residual Exxon Valdez oil and indicates that, for Barrow's goldeneyes in Prince William Sound, exposure persisted for many years with evidence of substantially reduced exposure by 2 decades after the spill.

Cytochrome P4501A biomarker indication of oil exposure in harlequin ducks up to 20 years after the Exxon Valdez oil spill.

Hydrocarbon-inducible cytochrome P4501A (CYP1A) expression was measured, as ethoxyresorufin-O-deethylase (EROD) activity, in livers of wintering harlequin ducks (Histrionicus histrionicus) captured in areas of Prince William Sound, Alaska, USA, oiled by the 1989 Exxon Valdez spill and in birds from nearby unoiled areas, during 2005 to 2009 (up to 20 years following the spill). The present work repeated studies conducted in 1998 that demonstrated that in harlequin ducks using areas that received Exxon Valdez oil, EROD activity was elevated nearly a decade after the spill. The present findings strongly supported the conclusion that average levels of hepatic EROD activity were higher in ducks from oiled areas than those from unoiled areas during 2005 to 2009. This result was consistent across four sampling periods; furthermore, results generated from two independent laboratories using paired liver samples from one of the sampling periods were similar. The EROD activity did not vary in relation to age, sex, or body mass of individuals, nor did it vary strongly by season in birds collected early and late in the winter of 2006 to 2007, indicating that these factors did not confound inferences about observed differences between oiled and unoiled areas. We interpret these results to indicate that harlequin ducks continued to be exposed to residual Exxon Valdez oil up to 20 years after the original spill. This adds to a growing body of literature suggesting that oil spills have the potential to affect wildlife for much longer time frames than previously assumed.

Induction of cytochrome P450 1 genes and stress response genes in developing zebrafish exposed to ultraviolet radiation.

Ultraviolet (UV) radiation damages cell molecules, and has been suggested to up-regulate mammalian cytochrome P4501 (CYP1) genes through an aryl hydrocarbon receptor (AHR) mediated mechanism. In this study, embryos and larvae of zebrafish (Danio rerio) were exposed to UV to determine the effects on expression of CYP1 and stress response genes in vivo in these fish. Zebrafish embryos were exposed for varying times to UV on two consecutive days, with exposure beginning at 24 and 48h post-fertilization (hpf). Embryos exposed for 2, 4 or 6h twice over 2 days to UVB (0.62 W/m(2); 8.9-26.7 kJ/m(2)) plus UVA (2.05 W/m(2); 29.5-144.6 kJ/m(2)) had moderately (2.4+/-0.8-fold) but significantly up-regulated levels of CYP1A. UVA alone had no effect on CYP1A expression. Proliferating cellular nuclear antigen (PCNA) and Cu-Zn superoxide dismutase (SOD1) transcript levels were induced (2.1+/-0.2 and 2.3+/-0.5-fold, respectively) in embryos exposed to two 6-h pulses of 0.62 W/m(2) UVB (26.8 kJ/m(2)). CYP1A was induced also in embryos exposed to higher intensity UVB (0.93 W/m(2)) for two 3-h or two 4-h pulses (20.1 or 26.8 kJ/m(2)). CYP1B1, SOD1 and PCNA expression was induced by the two 3-h pulses of the higher intensity UVB, but not after two 4-h pulses of the higher intensity UVB, possibly due to impaired condition of surviving embryos, reflected in a mortality of 34% at that UVB dose. A single 8-h long exposure of zebrafish larvae (8dpf) to UVB at 0.93 W/m(2) (26.8 kJ/m(2)) significantly induced CYP1A and CYP1B1 expression, but other CYP1 genes (CYP1C1, CYP1C2 and CYP1D1) showed no significant increase. The results show that UVB can induce expression of CYP1 genes as well stress response genes in developing zebrafish, and that UVB intensity and duration influence the responses.

New cytochrome P450 1B1, 1C2 and 1D1 genes in the killifish Fundulus heteroclitus: Basal expression and response of five killifish CYP1s to the AHR agonist PCB126.

Knowledge of the complement of cytochrome P450 (CYP) genes is essential to understanding detoxification and bioactivation mechanisms for organic contaminants. We cloned three new CYP1 genes, CYP1B1, CYP1C2 and CYP1D1, from the killifish Fundulus heteroclitus, an important model in environmental toxicology. Expression of the new CYP1s along with previously known CYP1A and CYP1C1 was measured by qPCR in eight different organs. Organ distribution was similar for the two CYP1Cs, but otherwise patterns and extent of expression differed among the genes. The AHR agonist 3,3',4,4',5-pentachlorobiphenyl (PCB126) (31 pmol/g fish) induced expression of CYP1A and CYP1B1 in all organs examined, while CYP1C1 was induced in all organs except testis. The largest changes in response to PCB126 were induction of CYP1A in testis (approximately 700-fold) and induction of CYP1C1 in liver (approximately 500-fold). CYP1B1 in liver and gut, CYP1A in brain and CYP1C1 in gill also were induced strongly by PCB126 (> 100-fold). CYP1C1 expression levels were higher than CYP1C2 in almost all tissues and CYP1C2 was much less responsive to PCB126. In contrast to the other genes, CYP1D1 was not induced by PCB126 in any of the organs. The organ-specific response of CYP1s to PCB126 implies differential involvement in effects of halogenated aromatic hydrocarbons in different organs. The suite of inducible CYP1s could enhance the use of F. heteroclitus in assessing aquatic contamination by AHR agonists. Determining basal and induced levels of protein and the substrate specificity for all five CYP1s will be necessary to better understand their roles in chemical effects and physiology.

Cytochrome P4501A1 expression, polychlorinated biphenyls and hydroxylated metabolites, and adipocyte size of bottlenose dolphins from the Southeast United States.

Persistent organic pollutants (POPs) bioaccumulate in blubber of marine mammals. Therefore, it is important to understand the structure and dynamics of blubber layers and how they affect the accumulation of POPs and subsequent biochemical responses. We used established histological and immunohistochemical methods to document the structure of bottlenose dolphin (Tursiops truncatus) blubber and to assess the expression of cytochrome P4501A1 (CYP1A1) in skin-blubber biopsies of dolphins sampled in the waters off Charleston, SC (CHS) (N=38), and Indian River Lagoon, FL (IRL) (N=36). CYP1A1 expression was strongest and most frequent in capillary endothelial cells and was stratified in blubber; the greatest CYP1A1 staining was in the deepest layer. CYP1A1 expression in deep blubber and 2,3,7,8-TCDD Toxic Equivalents measured in the entire blubber were significantly higher in dolphins from CHS as compared to those from IRL. Adipocyte size was associated with the extent of CYP1A1 expression. Male dolphins with smaller adipocytes from CHS and IRL had higher levels of CYP1A1 expression in deep blubber. In CHS females, CYP1A1 expression in vascular endothelial cells varied with reproductive status. CYP1A1 expression in the deep layer was highest in simultaneously pregnant-lactating dolphins, and these dolphins had the smallest adipocytes in deep blubber. In all dolphins, CYP1A1 expression in the deep blubber layer was positively related to concentrations of hydroxylated PCBs (OH-PCBs) in plasma. In summary, redistribution of AHR agonists from blubber into the circulatory system may enhance PCB metabolism and production of OH-PCBs by induction of CYP1A1 in hepatocytes and, possibly, by induction of CYP1A1 in endothelial cells of the deep blubber. The OH-PCBs thus formed have the potential to interfere with thyroid hormone homeostasis.

Role of AHR2 in the expression of novel cytochrome P450 1 family genes, cell cycle genes, and morphological defects in developing zebra fish exposed to 3,3',4,4',5-pentachlorobiphenyl or 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Halogenated agonists for the aryl hydrocarbon receptor (AHR), such as 3,3',4,4',5-pentachlorobiphenyl (PCB126) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), cause developmental toxicity in fish. AHR dependence of these effects is known for TCDD but only presumed for PCB126, and the AHR-regulated genes involved are known only in part. We defined the role of AHR in regulation of four cytochrome P450 1 (CYP1) genes and the effect of PCB126 on cell cycle genes (i.e., PCNA and cyclin E) in zebra fish (Danio rerio) embryos. Basal and PCB126-induced expression of CYP1A, CYP1B1, CYP1C1, and CYP1C2 was examined over time as well as in relation to cell cycle gene expression and morphological effects of PCB126 in developing zebra fish. The four CYP1 genes differed in the time for maximal basal and induced expression, i.e., CYP1B1 peaked within 2 days postfertilization (dpf), the CYP1Cs around hatching (3 dpf), and CYP1A after hatching (14-21 dpf). These results indicate developmental periods when the CYP1s may play physiological roles. PCB126 (0.3-100nM) caused concentration-dependent CYP1 gene induction (EC50: 1.4-2.7nM, Lowest observed effect concentration [LOEC]: 0.3-1nM) and pericardial edema (EC50: 4.4nM, LOEC: 3nM) in 3-dpf embryos. Blockage of AHR2 translation significantly inhibited these effects of PCB126 and TCDD. PCNA gene expression was reduced by PCB126 in a concentration-dependent manner, suggesting that PCB126 could suppress cell proliferation. Our results indicate that the four CYP1 genes examined are regulated by AHR2 and that the effect of PCB126 on morphology in zebra fish embryos is AHR2 dependent. Moreover, the developmental patterns of expression and induction suggest that CYP1 enzymes could function in normal development and in developmental toxicity of PCB126 in fish embryos.

Basal and 3,3',4,4',5-pentachlorobiphenyl-induced expression of cytochrome P450 1A, 1B and 1C genes in zebrafish.

The cytochrome P4501C (CYP1C) gene subfamily was recently discovered in fish, and zebrafish (Danio rerio) CYP1C1 transcript has been cloned. Here we cloned the paralogous CYP1C2, showing that the amino acid sequence is 78% identical to CYP1C1, and examined gene structure and expression of CYP1A, CYP1B1, CYP1C1, and CYP1C2. Xenobiotic response elements were observed upstream of the coding regions in all four genes. Zebrafish adults and embryos were exposed (24 h) to 100 nM 3,3',4,4',5-polychlorinated biphenyl (PCB126) or 20 ppm acetone and subsequently held in clean water for 24 h (adults) or 48 h (embryos). All adult organs examined (eye, gill, heart, liver, kidney, brain, gut, and gonads) and embryos showed basal expression of the four genes. CYP1A was most strongly expressed in liver, whereas CYP1B1, CYP1C1, and CYP1C2 were most strongly expressed in heart and eye. CYP1B1 and the CYP1C genes showed an expression pattern similar to one another and to mammalian CYP1B1. In embryos CYP1C1 and CYP1C2 tended to have a higher basal expression than CYP1A and CYP1B1. PCB126 induced CYP1A in all organs, and CYP1B1 and CYP1C1 in all organs except gonads, or gonads and brain, respectively. CYP1C2 induction was significant only in the liver. However, in embryos all four genes were induced strongly by PCB126. The results are consistent with CYP1C1 and CYP1C2, as well as CYP1A and CYP1B1, being regulated by the aryl hydrocarbon receptor. While CYP1A may have a protective role against AHR agonists in liver and gut, CYP1B1, CYP1C1, and CYP1C2 may also play endogenous roles in eye and heart and possibly other organs, as well as during development.

Correlates of cytochrome P450 1A1 expression in bottlenose dolphin (Tursiops truncatus) integument biopsies.

Integument biopsy is a nondestructive method for sampling free-ranging cetaceans, which allows for the determination of both contaminant concentrations and biomarker responses. Cytochrome P450 1A1 (CYP1A1) expression is induced by polycyclic aromatic hydrocarbons and planar halogenated aromatic hydrocarbons such as the non-ortho and mono-ortho polychlorinated biphenyls (PCBs). CYP1A induction has been used extensively as a biomarker of exposure to such compounds in vertebrates. We measured PCB concentrations and CYP1A1 expression in integument biopsies from bottlenose dolphins (Tursiops truncatus) resident in Sarasota Bay, FL. This population of dolphins has been the subject of long-term population and health assessment, affording the opportunity to evaluate the influence of age, sex, and reproductive status on CYP1A1 expression. CYP1A1 expression was seen in endothelial cells, vascular smooth muscle, and nerve cells in the dermis, similar to what has been observed in other cetacean species. Endothelial CYP1A1 expression varied along the length of the biopsy, which could be related to differences in the structure and functionality of the blubber in different parts of the integument. Neither age nor sex was related to CYP1A1 expression in these biopsies, and reproductive status did not relate to levels of CYP1A1 in females. Total PCB and toxic equivalent quotient concentrations in blubber were positively correlated with dermal endothelial CYP1A1 expression, although Sigmamono-ortho PCBs concentrations did not show this relationship. Contaminant concentrations appear to be stronger determinants of CYP1A1 expression in integument of these dolphins, than are age, sex, or reproductive status.

Molecular mechanisms of 2,3,7,8-tetrachlorodibenzo-p-dioxin cardiovascular embryotoxicity.

2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons are widespread environmental contaminants and potent developmental toxicants. Hallmarks of embryonic exposure include edema, hemorrhage, and mortality. Recent studies in zebrafish and chicken have revealed direct impairment of cardiac muscle growth that may underlie these overt symptoms. TCDD toxicity is mediated by the aryl hydrocarbon receptor, but downstream targets remain unclear. Oxidative stress and growth factor modulation have been implicated in TCDD cardiovascular toxicity. Gene expression profiling is elucidating additional pathways by which TCDD might act. We review our understanding of the mechanism of TCDD embryotoxicity at morphological and molecular levels.