Pesticides and environmental injustice in the USA: root causes, current regulatory reinforcement and a path forward.

Many environmental pollutants are known to have disproportionate effects on Black, Indigenous and People of Color (BIPOC) as well as communities of low-income and wealth. The reasons for these disproportionate effects are complex and involve hundreds of years of systematic oppression kept in place through structural racism and classism in the USA. Here we analyze the available literature and existing datasets to determine the extent to which disparities in exposure and harm exist for one of the most widespread pollutants in the world - pesticides. Our objective was to identify and discuss not only the historical injustices that have led to these disparities, but also the current laws, policies and regulatory practices that perpetuate them to this day with the ultimate goal of proposing achievable solutions. Disparities in exposures and harms from pesticides are widespread, impacting BIPOC and low-income communities in both rural and urban settings and occurring throughout the entire lifecycle of the pesticide from production to end-use. These disparities are being perpetuated by current laws and regulations through 1) a pesticide safety double standard, 2) inadequate worker protections, and 3) export of dangerous pesticides to developing countries. Racial, ethnic and income disparities are also maintained through policies and regulatory practices that 4) fail to implement environmental justice Executive Orders, 5) fail to account for unintended pesticide use or provide adequate training and support, 6) fail to effectively monitor and follow-up with vulnerable communities post-approval, and 7) fail to implement essential protections for children. Here we've identified federal laws, regulations, policies, and practices that allow for disparities in pesticide exposure and harm to remain entrenched in everyday life for environmental justice communities. This is not simply a pesticides issue, but a broader public health and civil rights issue. The true fix is to shift the USA to a more just system based on the Precautionary Principle to prevent harmful pollution exposure to everyone, regardless of skin tone or income. However, there are actions that can be taken within our existing framework in the short term to make our unjust regulatory system work better for everyone.

The USA lags behind other agricultural nations in banning harmful pesticides.

BACKGROUND: The United States of America (USA), European Union (EU), Brazil and China are four of the largest agricultural producers and users of agricultural pesticides in the world. Comparing the inclination and ability of different regulatory agencies to ban or eliminate pesticides that have the most potential for harm to humans and the environment can provide a glimpse into the effectiveness of each nation's pesticide regulatory laws and oversight. METHODS: The approval status of more than 500 agricultural pesticides was identified in the USA, EU, Brazil and China and compared between nations. The amount of pesticides that were used in the USA and banned in these other nations was compiled and linear regression was used to identify trends in use. RESULTS: There are 72, 17, and 11 pesticides approved for outdoor agricultural applications in the USA that are banned or in the process of complete phase out in the EU, Brazil, and China, respectively. Of the pesticides used in USA agriculture in 2016, 322 million pounds were of pesticides banned in the EU, 26 million pounds were of pesticides banned in Brazil and 40 million pounds were of pesticides banned in China. Pesticides banned in the EU account for more than a quarter of all agricultural pesticide use in the USA. The majority of pesticides banned in at least two of these three nations have not appreciably decreased in the USA over the last 25 years and almost all have stayed constant or increased over the last 10 years. CONCLUSIONS: Many pesticides still widely used in the USA, at the level of tens to hundreds of millions of pounds annually, have been banned or are being phased out in the EU, China and Brazil. Of the pesticides banned in at least two of these nations, many have been implicated in acute pesticide poisonings in the USA and some are further restricted by individual states. The United States Environmental Protection Agency (US EPA) has all but abandoned its use of non-voluntary cancellations in recent years, making pesticide cancellation in the USA largely an exercise that requires consent by the regulated industry.

ASAR15, A cis-acting locus that controls chromosome-wide replication timing and stability of human chromosome 15.

DNA replication initiates at multiple sites along each mammalian chromosome at different times during each S phase, following a temporal replication program. We have used a Cre/loxP-based strategy to identify cis-acting elements that control this replication-timing program on individual human chromosomes. In this report, we show that rearrangements at a complex locus at chromosome 15q24.3 result in delayed replication and structural instability of human chromosome 15. Characterization of this locus identified long, RNA transcripts that are retained in the nucleus and form a "cloud" on one homolog of chromosome 15. We also found that this locus displays asynchronous replication that is coordinated with other random monoallelic genes on chromosome 15. We have named this locus ASynchronous replication and Autosomal RNA on chromosome 15, or ASAR15. Previously, we found that disruption of the ASAR6 lincRNA gene results in delayed replication, delayed mitotic condensation and structural instability of human chromosome 6. Previous studies in the mouse found that deletion of the Xist gene, from the X chromosome in adult somatic cells, results in a delayed replication and instability phenotype that is indistinguishable from the phenotype caused by disruption of either ASAR6 or ASAR15. In addition, delayed replication and chromosome instability were detected following structural rearrangement of many different human or mouse chromosomes. These observations suggest that all mammalian chromosomes contain similar cis-acting loci. Thus, under this scenario, all mammalian chromosomes contain four distinct types of essential cis-acting elements: origins, telomeres, centromeres and "inactivation/stability centers", all functioning to promote proper replication, segregation and structural stability of each chromosome.

The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo.

Bone morphogenetic proteins 4 and 7 (BMP4 and BMP7) are morphogens that signal as either homodimers or heterodimers to regulate embryonic development and adult homeostasis. BMP4/7 heterodimers exhibit markedly higher signaling activity than either homodimer, but the mechanism underlying the enhanced activity is unknown. BMPs are synthesized as inactive precursors that dimerize and are then cleaved to generate both the bioactive ligand and prodomain fragments, which lack signaling activity. Our study reveals a previously unknown requirement for the BMP4 prodomain in promoting heterodimer activity. We show that BMP4 and BMP7 precursor proteins preferentially or exclusively form heterodimers when coexpressed in vivo. In addition, we show that the BMP4 prodomain is both necessary and sufficient for generation of stable heterodimeric ligands with enhanced activity and can enable homodimers to signal in a context in which they normally lack activity. Our results suggest that intrinsic properties of the BMP4 prodomain contribute to the relative bioactivities of homodimers versus heterodimers in vivo. These findings have clinical implications for the use of BMPs as regenerative agents for the treatment of bone injury and disease.

Simultaneous rather than ordered cleavage of two sites within the BMP4 prodomain leads to loss of ligand in mice.

ProBMP4 is generated as a latent precursor that is sequentially cleaved at two sites within the prodomain to generate an active ligand. An initial cleavage occurs adjacent to the ligand domain, which generates a non-covalently associated prodomain/ligand complex that is subsequently dissociated by cleavage at an upstream site. An outstanding question is whether the two sites need to be cleaved sequentially and in the correct order to achieve proper control of BMP4 signaling during development. In the current studies, we demonstrate that mice carrying a knock-in point mutation that causes simultaneous rather than sequential cleavage of both prodomain sites show loss of BMP4 function and die during mid-embryogenesis. Levels of mature BMP4 are severely reduced in mutants, although levels of precursor and cleaved prodomain are unchanged compared with wild type. Our biochemical analysis supports a model in which the transient prodomain/ligand complex that forms during sequential cleavage plays an essential role in prodomain-mediated stabilization of the mature ligand until it can acquire protection from degradation by other means. By contrast, simultaneous cleavage causes premature release of the ligand from the prodomain, leading to destabilization of the ligand and loss of signaling in vivo.

Asynchronous replication, mono-allelic expression, and long range Cis-effects of ASAR6.

Mammalian chromosomes initiate DNA replication at multiple sites along their length during each S phase following a temporal replication program. The majority of genes on homologous chromosomes replicate synchronously. However, mono-allelically expressed genes such as imprinted genes, allelically excluded genes, and genes on female X chromosomes replicate asynchronously. We have identified a cis-acting locus on human chromosome 6 that controls this replication-timing program. This locus encodes a large intergenic non-coding RNA gene named Asynchronous replication and Autosomal RNA on chromosome 6, or ASAR6. Disruption of ASAR6 results in delayed replication, delayed mitotic chromosome condensation, and activation of the previously silent alleles of mono-allelic genes on chromosome 6. The ASAR6 gene resides within an ∼1.2 megabase domain of asynchronously replicating DNA that is coordinated with other random asynchronously replicating loci along chromosome 6. In contrast to other nearby mono-allelic genes, ASAR6 RNA is expressed from the later-replicating allele. ASAR6 RNA is synthesized by RNA Polymerase II, is not polyadenlyated, is restricted to the nucleus, and is subject to random mono-allelic expression. Disruption of ASAR6 leads to the formation of bridged chromosomes, micronuclei, and structural instability of chromosome 6. Finally, ectopic integration of cloned genomic DNA containing ASAR6 causes delayed replication of entire mouse chromosomes.

DNA replication timing, genome stability and cancer: late and/or delayed DNA replication timing is associated with increased genomic instability.

Normal cellular division requires that the genome be faithfully replicated to ensure that unaltered genomic information is passed from one generation to the next. DNA replication initiates from thousands of origins scattered throughout the genome every cell cycle; however, not all origins initiate replication at the same time. A vast amount of work over the years indicates that different origins along each eukaryotic chromosome are activated in early, middle or late S phase. This temporal control of DNA replication is referred to as the replication-timing program. The replication-timing program represents a very stable epigenetic feature of chromosomes. Recent evidence has indicated that the replication-timing program can influence the spatial distribution of mutagenic events such that certain regions of the genome experience increased spontaneous mutagenesis compared to surrounding regions. This influence has helped shape the genomes of humans and other multicellular organisms and can affect the distribution of mutations in somatic cells. It is also becoming clear that the replication-timing program is deregulated in many disease states, including cancer. Aberrant DNA replication timing is associated with changes in gene expression, changes in epigenetic modifications and an increased frequency of structural rearrangements. Furthermore, certain replication timing changes can directly lead to overt genomic instability and may explain unique mutational signatures that are present in cells that have undergone the recently described processes of "chromothripsis" and "kataegis". In this review, we will discuss how the normal replication timing program, as well as how alterations to this program, can contribute to the evolution of the genomic landscape in normal and cancerous cells.

Forever Pesticides: A Growing Source of PFAS Contamination in the Environment.

BACKGROUND: Environmental contamination by fluorinated chemicals, in particular chemicals from the per- and polyfluoroalkyl substances (PFAS) class, has raised concerns around the globe because of documented adverse impacts on human health, wildlife, and ecosystem quality. Recent studies have indicated that pesticide products may contain a variety of chemicals that meet the PFAS definition, including the active pesticide ingredients themselves. Given that pesticides are some of the most widely distributed pollutants across the world, the legacy impacts of PFAS addition into pesticide products could be widespread and have wide-ranging implications on agriculture and food and water contamination, as well as the presence of PFAS in rural environments. OBJECTIVES: The purpose of this commentary is to explore different ways that PFAS can be introduced into pesticide products, the extent of PFAS contamination of pesticide products, and the implications this could have for human and environmental health. METHODS: We submitted multiple public records requests to state and federal agencies in the United States and Canada and extracted relevant data from those records. We also compiled data from publicly accessible databases for our analyses. DISCUSSION: We found that the biggest contributor to PFAS in pesticide products was active ingredients and their degradates. Nearly a quarter of all US conventional pesticide active ingredients were organofluorines and 14% were PFAS, and for active ingredients approved in the last 10 y, this had increased to 61% organofluorines and 30% PFAS. Another major contributing source was through PFAS leaching from fluorinated containers into pesticide products. Fluorination of adjuvant products and "inert" ingredients appeared to be limited, although this represents a major knowledge gap. We explored aspects of immunotoxicity, persistence, water contamination, and total fluorine load in the environment and conclude that the recent trend of using fluorinated active ingredients in pesticides may be having effects on chemical toxicity and persistence that are not given adequate oversight in the United States. We recommend a more stringent risk assessment approach for fluorinated pesticides, transparent disclosure of "inert" ingredients on pesticide labels, a complete phase-out of post-mold fluorination of plastic containers, and greater monitoring in the United States. https://doi.org/10.1289/EHP13954.

An autosomal locus that controls chromosome-wide replication timing and mono-allelic expression.

Mammalian DNA replication initiates at multiple sites along chromosomes at different times, following a temporal replication program. Homologous alleles typically replicate synchronously; however, mono-allelically expressed genes such as imprinted genes, allelically excluded genes and genes on the female X chromosome replicate asynchronously. We have used a chromosome engineering strategy to identify a human autosomal locus that controls this replication timing program in cis. We show that Cre/loxP-mediated rearrangements at a discrete locus at 6q16.1 result in delayed replication of the entire chromosome. This locus displays asynchronous replication timing that is coordinated with other mono-allelically expressed genes on chromosome 6. Characterization of this locus revealed mono-allelic expression of a large intergenic non-coding RNA, which we have named asynchronous replication and autosomal RNA on chromosome 6, ASAR6. Finally, disruption of this locus results in the activation of the previously silent alleles of linked mono-allelically expressed genes. We previously found that chromosome rearrangements involving eight different autosomes display delayed replication timing, and that cells containing chromosomes with delayed replication timing have a 30-80-fold increase in the rate at which new gross chromosomal rearrangements occurred. Taken together, these observations indicate that human autosomes contain discrete cis-acting loci that control chromosome-wide replication timing, mono-allelic expression and the stability of entire chromosomes.

Catalysts of DNA Strand Cleavage at Apurinic/Apyrimidinic Sites.

Apurinic/apyrimidinic (AP) sites are constantly formed in cellular DNA due to instability of the glycosidic bond, particularly at purines and various oxidized, alkylated, or otherwise damaged nucleobases. AP sites are also generated by DNA glycosylases that initiate DNA base excision repair. These lesions represent a significant block to DNA replication and are extremely mutagenic. Some DNA glycosylases possess AP lyase activities that nick the DNA strand at the deoxyribose moiety via a ß- or ß,δ-elimination reaction. Various amines can incise AP sites via a similar mechanism, but this non-enzymatic cleavage typically requires high reagent concentrations. Herein, we describe a new class of small molecules that function at low micromolar concentrations as both ß- and ß,δ-elimination catalysts at AP sites. Structure-activity relationships have established several characteristics that appear to be necessary for the formation of an iminium ion intermediate that self-catalyzes the elimination at the deoxyribose ring.

Small Molecule Inhibitors of 8-Oxoguanine DNA Glycosylase-1 (OGG1).

The DNA base excision repair (BER) pathway, which utilizes DNA glycosylases to initiate repair of specific DNA lesions, is the major pathway for the repair of DNA damage induced by oxidation, alkylation, and deamination. Early results from clinical trials suggest that inhibiting certain enzymes in the BER pathway can be a useful anticancer strategy when combined with certain DNA-damaging agents or tumor-specific genetic deficiencies. Despite this general validation of BER enzymes as drug targets, there are many enzymes that function in the BER pathway that have few, if any, specific inhibitors. There is a growing body of evidence that suggests inhibition of 8-oxoguanine DNA glycosylase-1 (OGG1) could be useful as a monotherapy or in combination therapy to treat certain types of cancer. To identify inhibitors of OGG1, a fluorescence-based screen was developed to analyze OGG1 activity in a high-throughput manner. From a primary screen of ∼50,000 molecules, 13 inhibitors were identified, 12 of which were hydrazides or acyl hydrazones. Five inhibitors with an IC50 value of less than 1 µM were chosen for further experimentation and verified using two additional biochemical assays. None of the five OGG1 inhibitors reduced DNA binding of OGG1 to a 7,8-dihydro-8-oxoguanine (8-oxo-Gua)-containing substrate, but all five inhibited Schiff base formation during OGG1-mediated catalysis. All of these inhibitors displayed a >100-fold selectivity for OGG1 relative to several other DNA glycosylases involved in repair of oxidatively damaged bases. These inhibitors represent the most potent and selective OGG1 inhibitors identified to date.

Genetic interaction between Bmp2 and Bmp4 reveals shared functions during multiple aspects of mouse organogenesis.

Vertebrate Bmp2 and Bmp4 diverged from a common ancestral gene and encode closely related proteins. Mice homozygous for null mutations in either gene show early embryonic lethality, thereby precluding analysis of shared functions. In the current studies, we present phenotypic analysis of compound mutant mice heterozygous for a null allele of Bmp2 in combination with null or hypomorphic alleles of Bmp4. Whereas mice lacking a single copy of Bmp2 or Bmp4 are viable and have subtle developmental defects, compound mutants show embryonic and postnatal lethality due to defects in multiple organ systems including the allantois, placental vasculature, ventral body wall, skeleton, eye and heart. Within the heart, BMP2 and BMP4 function coordinately to direct normal lengthening of the outflow tract, proper positioning of the outflow vessels, and septation of the atria, ventricle and atrioventricular canal. Our results identify numerous BMP4-dependent developmental processes that are also very sensitive to BMP2 dosage, thus revealing novel functions of Bmp2.

GATA-2 functions downstream of BMPs and CaM KIV in ectodermal cells during primitive hematopoiesis.

In Xenopus, primitive blood originates from the mesoderm, but extrinsic signals from the ectoderm are required during gastrulation to enable these cells to differentiate as erythrocytes. The nature of these signals, and how they are transcriptionally regulated, is not well understood. We have previously shown that bone morphogenetic proteins (BMPs) are required to signal to ectodermal cells to generate secondary non-cell-autonomous signal(s) necessary for primitive erythropoiesis, and that calmodulin-dependent protein kinase IV (CaM KIV) antagonizes BMP signaling. The current studies demonstrate that Gata-2 functions downstream of BMP receptor activation in these same cells, and is a direct target for antagonism by CaM KIV. We show, using loss of function analysis in whole embryos and in explants, that ectodermal Gata-2 is required for primitive erythropoiesis, and that BMP signals cannot rescue blood defects caused by ectoderm removal or loss of ectodermal GATA-2. Furthermore, we provide evidence that acetylation of GATA-2 is required for its function in primitive blood formation in vivo. Our data support a model in which Gata-2 is a transcriptional target downstream of BMPs within ectodermal cells, while activation of the CaM KIV signaling pathway alters GATA-2 function posttranslationally, by inhibiting its acetylation.