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MMC is an interstrand cross-linking agent , and CDDP is an intrastrand cross-linking agent

At the subcellular level, SOG1 is present in the nucleus, consistent with previous findings; however, unlike any previous data in response to other genotoxins, SOG1 does show a change following Al exposure that is ATR-dependent and may be the result of relocalization, morphological changes to the cell identity, as well as possible diminished visualization due to protein degradation. Post-translational modification of SOG1 was determined to be crucial to the regulation of its function. There is no significant transcriptional change to SOG1 expression in the presence of Al, despite protein accumulation subsiding following Al exposure. This suggests that there may be an undetermined mechanism for protein turnover following SOG1 activation. As the p53 functional homologue in Arabidopsis, SOG1 turnover could prove to be a conserved regulatory mechanism as p53 is ubiquitinated by the E3 ligase, MDM2, in mammals . Perhaps this is even a speculative role for ALT2, as WD40 proteins have been shown to associate with E3 ubiquitin ligases. It is unlikely that ALT2 would be responsible for SOG1 turnover as part of an ubiquitin-proteasome pathway as loss of ALT2 would result in inappropriate persistence of SOG1 and lead to hypersensitivity rather than tolerance as is observed. As of yet, evidence only supports that the activation mechanism in response to Al is dependent on ATR, likely through phosphorylation of SOG1 by ATR. Future studies are needed to determine how SOG1 is degraded following ATR-dependent activation in response to Al as well as to other stresses. As shown in Chapter 5, the fourth Al tolerance gene isolated from the als3-1 suppressor screen was SUV2, nursery pots which encodes the Arabidopsis homologue of the mammalian ATR-interacting protein, ATRIP. The suv2-3 mutation represents a premature stop codon in the eighth exon of SUV2 at amino acid 359 of 646.

After establishing the als3-1 suppressor as an Al tolerant suv2 mutation, it was characterized as being part of the same ATR-mediated pathway, further supporting SUV2 as an interacting partner of ATR in Arabidopsis. Additionally, both cell cycle arrest and differentiation of the QC were established to be dependent on SUV2 following Al exposure, and that these responses ultimately lead to endore duplication as a means to terminal differentiation of the root meristem. Thissue- and cell-specific localization of SUV2 was shown to be present within the cytoplasm and nucleus of actively dividing cells of the root tip. There is accumulation of SUV2 throughout the meristematic regions of the root tip in the absence of Al. When grown in an Al toxic environment, SUV2 persists in the meristematic region of the root tip, but this zone has significantly been reduced in size and therefore there is a concomitant reduction of SUV2 in the root organ. At the sub-cellular level, SUV2 is present in the cytoplasm and the nucleus of cells at the root tip in both the absence and presence of Al. It is likely that as Al causes differentiation of the root meristem up to the point of complete stem cell consumption, the zone of cell division becomes reduced while the zone of elongation encroaches closer to the apex i.e. a morphological reduction in zones of root development. This reduction in the meristematic zone may account for the insignificant but observable progressive reduction in SUV2 transcript levels following treatment with increasing amounts of Al.As with SOG1, SUV2 is phosphorylated by ATR in vitro. While in vivo studies are needed in both cases to confirm this post-translational modification, this would be considered a conserved relationship between ATR and SUV2, as this phosphorylation is known to occur in the homologous proteins in yeast and humans . This begs the question: what are the bona fide substrates of ATR in the Al response pathway? While SOG1 has been demonstrated to have 5 ATM/ATR phosphorylation sites, defined as TQ or SQ motifs, and SUV2 is speculated to contain two sites, in a recent phosphoproteomics study, SOG1 was not even identified as a substrate of ATR or ATM in response to ionizing radiation, let alone SUV2 . Some DNA repair factors such as LIG4, UVH3 and MRE11 as well other DNA maintenance and metabolism factors like CHR4, HTA7/HTA10, PCNA1, MCM4 and H2AXA were proven to be ATR and ATM targets in this kinase target study .

While the authors acknowledge that their experimental technique using adverse tryptic cleavage likely was responsible for not identifying SOG1 in their large-scale identification of kinase targets, this large-scale proteomic endeavor only tested IR stress and did not distinguish between ATR versus ATM phosphorylated targets . Despite the substantial contribution these findings offer to the field of plant DNA maintenance and repair, more in depth studies are needed not just for Al responses, but also for the myriad types of damage to plant DNA that must be repaired. Al treatment leads to root growth inhibition due to terminal differentiation by means of endore duplication as visualized by increases in cell and nucleus size of cells of the root tip. Al treatment results in substantial increases in both cell and nuclear size for als3-1 roots, which is consistent with terminal differentiation in conjunction with endore duplication. This increase in size was shown to be dependent on the Al tolerance factors: ATR, ALT2, SOG1 and SUV2. This shows that all four genetic factors control terminal differentiation and endore duplication in response to Al. Previous studies of a sog1 loss-of-function mutant defined a set of SOG1- mediated genes that were inducible by γ-radiation . In response to Al toxicity, SOG1 and ATR have now been established to be acting within the same response pathway, so it was of great interest to determine if SOG1, ATR, ALT2, and SUV2 were responsible for the induction of this established gene set after treatment with Al. The proper conditions to determine whether Al causes changes in known SOG1-mediates genes was determined by a time course study of the persistence of SOG1 accumulation without completed QC differentiation in response to high Al concentrations. After 4 days of growth on highly inhibitory concentrations of Al, the pool of stem cells in the root meristem differentiate into cells no longer capable of dividing; while SOG1 is completely absent from the root tip after 5 to 6 days of growth on high concentrations of Al. Taken together, this established that 3 days of growth in Al was the optimal point at which expression changes would be analyzed, satisfying the need for SOG1 actively inducing transcriptional changes that would lead to root growth inhibition in response to Al. Genes that were selected for expression analysis were found in a previous study as being highly induced following γ-radiation in a SOG1-dependent manner .

From the γ-radiation study, a number of DNA repair genes like BRCA1, the ortholog for the human breast cancer susceptibility gene; PARP2, a key component of microhomology-mediated DNA repair; GMI1, involved in homologous recombination and chromosome maintenance; and members of the RAD family of genes, RAD17 and RAD51. CYCLINB1;1accumulates in the G2 phase of cell cycle progression and is regulated by transcriptional activation. During normal cell cycle progression, in a population of cells like the root rip, a few would be in the G2 phase at a given time expressing CYCB1;1, but an increase in expression would suggest more cells within the cell population were halted in the G2 phase, indicative of a G2 cell cycle arrest. Other transcription factors were induced in this γ-radiation study,plastic planters like TRFL3 and TRFL10, MYB family transcription factors known to have roles in developmental processes, defense responses and DNA maintenance . In total, 16 genes were assayed . Treatment with Al resulted in a measurable increase in expression of the subset of assayed genes in Col-0 wild type compared with no Al, and for als3-1 there was an even larger increase of gene expression. In contrast, an increase in expression of these genes was not observed for any of the Al tolerant single mutants or double mutants in comparison to the respective controls. This indicates that Al triggers an ATR-, ALT2, SUV2- and SOG1- dependent transcriptional program that is similar to that observed following treatment with γ-radiation, providing an important link between the cell cycle checkpoints involved in DNA damage detection and transcription in response to Al. Clearly, cell cycle checkpoints are emerging as key regulators of Al response, indicating that Al-dependent activation of these factors is central to terminal differentiation following chronic exposure to Al. Unlike γ-radiation, this stoppage of root growth is ATM-independent, as demonstrated by real-time PCR analysis of an atm loss-of-function mutant, indicating that at least in respect to Al stress, SOG1 functions downstream of ATR rather than ATM. This is of particular importance since it is indicative of the type of damage that ATR, ALT2, SOG1 and SUV2 are detecting in the context of Al. There are clear transcriptional differences between Al treatment and γ-radiation. Examples of these differences would be the lack of induction of AtRAD21 in the absence or presence of Al, as well as the ATM independent manner of SOG1-dependent transcript induction in Al treated seedlings while they are ATM-dependent upon exposure to γ-radiation. The difference in the role of ATM in response to these two stresses is interesting, especially considering that ATM is largely uninvolved in the Al response despite the requirement of functional ATR, ALT2, SOG1 and SUV2. This indicates that ATR, ALT2, SOG1 and SUV2 comprise an Al-response pathway that does not primarily require ATM. This suggests that each DNA stress results in a unique transcriptional profile that may be revealing in relation to their respective impacts on genomic integrity. With the overwhelming evidence that the DNA damage response factors ATR, ALT2, SOG1 and SUV2 all play a role in detecting Al dependent damage, it was of interest to examine their roles in response to known genotoxins. HU is an inhibitor of ribonucleotide reductase by scavenging free radicals that are used for the reduction of ribonucleotides . This stalls the replication fork due to depletion of deoxyribonucelotides. ATR functions to detect replication fork blocks and single stranded DNA breaks and atr mutants are sensitive to HU .

MMC is an aziridine containing antibiotic isolated from Streptomyces caepitosus . MMC itself does not react with DNA, but once it becomes reduced by quinone, the aziridine opens and allows MMC to attack the DNA . This reaction forms crosslinks across the complementary strands of the DNA double helix, or interstrand cross links . DNA cross linking can also occur between adjacent bases, called intrastrand DNA cross linking. The chemotherapy drug CDDP is a platinum-containing drug that is a neutral molecule until it is activated through a series of spontaneous aquation reactions, which involve the sequential replacement of the cis-chloro ligands of CDDP with water molecules . When the aquation event occurs, this allows the platinum atom to bind to DNA, preferentially guanine bases, which forms DNA–DNA intrastrand crosslinks . DNA cross links block replication because when they go unrepaired, collapse of the replication fork occurs blocking replication and leading to cell death . ALT2 is the Arabidopsis homologue of the human protein, CSA, which monitors conformational changes in DNA as assessed by blockage ofDNA replication and RNA transcription, a known effect caused by DNA cross links. alt2-1 is hypersensitive to MMC and CDDP . SOG1, the plant functional homologue of the human transcription factor p53, is a transcription factor in Arabidopsis responsible for the initiation of DNA damage responses including DNA repair as well as the initiation of endore duplication in plants. sog1 loss-of-function mutants are sensitive to both the replication fork poison, HU, and the DNA cross linking agents, MMC and CDDP. SUV2 is the Arabidopsis functional homologue to the human protein, ATRIP, which is required for recruiting ATR to sites of DNA damage, presumably to regions coated in Replication Protein A . RPA is a heterotrimeritc protein complex, comprised of sub-units RPA1, RPA2, and RPA3, which binds to single stranded DNA to protect it from nuceolytic degradation and hairpin formation, similar to the prokaryotic Single Stranded Binding protein .

We geocoded maternal residential addresses listed on the birth certificates using an automated approach

Since most pesticide applications are spatially explicit and the usage may vary over years, these studies face challenges of lacking high spatial and temporal resolution for exposure assessment, on top of the known ecological fallacy. On the other hand, individual level GIS-based studies that assessed exposure to agricultural pesticides in proximity to residences, have shown inconsistent findings. A recent Spanish study explored the possible association between childhood renal tumors and residential proximity to environmental pollution sources by calculating the percentage of total crop surface within a 1-km buffer around each individual’s last known residence and discovered that children living in the proximity of agricultural crops have higher risk of developing renal cancer . However, US studies of individual measures of agricultural pesticides in proximity to residences, mostly suggested no association with childhood cancers, or at best modest associations for certain types of cancers and chemicals or chemical groups. A Texas study measured crop field density within a 1-km buffer of residence at birth for both cases and controls born in 1990-1998 to study the risk for childhood cancers by sub-types and found no evidence of elevated risk associated with residential proximity to cropland for most childhood cancers, except for modestly positive associations with non-Hodgkin lymphoma , Burkitt lymphoma , and other gliomas. A California population-based case-control study of early childhood cancer used mothers’ residential addresses at the time of birth to evaluate risks associated with residential proximity to agricultural applications of pesticides during pregnancy and also found no associations with most specific chemicals and chemicals groups,plastic planters except for modestly elevated ORs for leukemia associated with probable and possible carcinogen use and with nearby agricultural applications of organochlorines and organophosphates.

They suggested that the few elevated risk associations for specific chemicals including metam sodium and dicofol in this study are likely due to chance from multiple comparisons . Another Northern California study assessed residential proximity within a half-mile of pesticide applications by linking address histories with reports of agricultural pesticide use and examined the association of the first year of life or early childhood pesticide exposures and childhood acute lymphoblastic leukemia and suggested elevated ALL risk was associated with lifetime moderate exposure to certain physicochemical groups of pesticides, including organophosphates ,cholorinated phenols , and triazines , and with pesticides classified as insecticides or fumigants.These results vary by chemicals or chemical groups examined, and methods of exposure assessment, and therefore produced inconsistent results. In addition, many of the above studies found no or week associations between all cancer types grouped together and proximity to crop fields, agricultural activities, specific agents, or groups of chemicals. It may problematic because the etiologies of most childhood cancer sub-types remain largely unknown and may not share the same causal pathway mediated by pesticides. As mentioned before, these studies of ambient pesticide exposures in pregnancy or early childhood, often assign exposures based upon the child’s or mother’s residence. While large scale record-linkage studies can avoid selection and recall biases that often impact smaller studies with active subject recruitment, previous record-based studies often relied solely on maternal residential address at birth, which is readily available on many birth certificates and/or residential address at diagnosis, as was done in some childhood cancer studies . Previous evidence suggested that exposure to pesticides before or during pregnancy may harm the developing fetus. Nevertheless, increased risks were seen for postnatal period as well. For instance, a study of childhood leukemia tried to distinguish between pre-pregnancy, pregnancy and postnatal exposures as critical windows for household pesticide exposure, and insecticide exposures early in life appear to be significant, though the effect is not as strong as prenatal exposures .

After birth, children may also be more susceptible to the harmful effects of pesticides than adults, as they have more actively dividing cells , providing the rationale to studies of childhood cancers not only focus on prenatal but also early life exposures. The reliance on one address for assessment of exposures in the first year of life or early childhood implicitly makes the assumption that a child’s residence remained the same throughout the entire period of interest, or if they moved, that the exposure levels remained the same. Even if some studies assessed exposures as the children’s residential proximity to agricultural fields or pesticide applications at the time of birth, these exposure indicators at birth are assumed to reflect exposures in prenatal and/or postnatal period, which are believed to be critical windows of exposures for childhood cancers. Consequently, the “one-address” approach may lead to exposure misclassification for those who moved in early childhood especially for exposures with high spatial heterogeneity. In a 2003-2007 California statewide representative survey, only 14% of all women moved in the 2-7 months post-partum , but with increasing age of the child, the frequency of residential moves also increased. For more than 50% of childhood cancer cases under age 5 diagnosed in California between 1988 and 2005, address at birth differed from the address at cancer diagnosis , which raises concerns about using residence at birth to assess exposures in early childhood. Exposure misclassification due to moving is a ubiquitous problem encountered by nearly all record-based studies that lack a complete residential history for each child. Previous studies suggested that residential mobility may be associated with certain risk factors for childhood cancers such as maternal age, marital status, parity, family income, and other socioeconomic status metrics , resulting in non-differential or even differential misclassification of exposures. Currently, in the US, the privilege of having an accurate complete residential history still only belongs to interview-based environmental epidemiological studies, which are often quite small with hundreds of subjects because of high time and monetary costs of such interviews and likely under powered.

Small case-control studies typically asked for individuals’ lifetime residential histories , including the beginning and end dates for each address. Large cohort studies follow participants over for a long time and often update their addresses periodically from follow-up questionnaires, so they may not know the exact moving dates. Sometimes these studies additionally collect information from the US Postal Service change-of-address forms or major credit reporting agencies, but the date associated with each address does not necessarily capture an accurate “move-in” date, but rather reflects the first known date . While it is not feasible to acquire complete residential histories from interviews for all subjects in large record-based studies as a gold standard to compare against the recorded birth or diagnosis address, databases containing public records of individuals collected by commercial companies have become available in recent years, allowing us to trace individuals without a self reported residential history. For example, LexisNexis Public Records, Inc. , a commercial credit reporting company,plastic nursery plant pot provides all known addresses for a set of individuals upon request. If the commercial residential history data has relatively high accuracy, their low cost and broad coverage would provide valuable information to all studies requiring residential history data . The basic service provided by LexisNexis returns the latest three known addresses while the enhanced service, with a higher cost, returns all known addresses from at least 1995. This database was maintained primarily for the purpose of contacting study participants but not for scientific research use, and therefore may not be as accurate as residential history obtained from interviews or self-administrated questionnaires. Several earlier studies have attempted to compare reconstructed residential history based on LexisNexis records with interview-based residential history for enrolled subjects and validate its use for research purposes. A Michigan case-control study of bladder cancer first compared lifetime residential histories collected through written surveys and 3 residential addresses available in LexisNexis and reported 71.5% match rate . Their bladder cancer cases were less than 80 years of age upon diagnosis, and controls were selected from similar age groups. Both cases and controls had lived in 1 of the 11 counties in Michigan for more than 5 years before recruitment in 2008-2009. Another US-wide study selected a random sample of 1000 subjects originally enrolled in the National Institutes of Health-American Association of Retired Persons Diet and Health Study, with AARP members aged 50–69 years and living in one of six US states or two metropolitan areas at the time of enrollment. Authors found 72% and 87% detailed address match rates with the basic and enhanced services provided by LexisNexis, respectively . The most recent LexisNexis validation study looked into participants in California Teachers Study, a prospective cohort study initiated in 1995-1996 and originally designed to study breast cancer. These women aged between 22 to 104 years at enrollment , and lived throughout California. The study pointed out that though the overall match rate between the two sources of addresses was good , it was diminished among black women and younger women . In summary, such residential information from LexisNexis, if of high quality, could potentially augment existing address information and help us reconstruct residential histories for subjects in large record-linkage studies and provide more accurate exposure estimates.

However, researchers should be aware that residential mobility of young children or their mothers may be different from that of mid-aged or older population, who were the majority in all three validation studies discussed above and believed to have a more stable residence. In addition, differences in distributions of race/ethnicity, socioeconomic status, and geographic regions may influence residential mobility in various study populations as well.During the 1st decade of the 21st century, the estimated rates of preterm birthand low birth weightpeaked at 11%-13% and 7%-8% in the US, respectively . Though survival of infants born preterm and/or low birthweight has improved in the last decades due to advancements in prenatal and neonatal care, they are more susceptible to adverse health outcomes such as neurodevelopmental impairment, respiratory and gastrointestinal complications , obesity, diabetes mellitus, hypertension, and kidney disease ; they also result in substantially higher infant and childhood mortality rates . California is the largest agricultural state in the United States, with more than 150 million pesticide active ingredients applied every year . Previous experimental studies show that various pesticides, including organophosphates and pyrethroids, can influence prenatal development related to adverse birth outcomes .Proposed mechanisms include disturbance of placental functions , endocrine disruption , immune regulation and inflammatory mechanisms . Pesticides have been found in indoor residential dust in residences near agricultural fields, and may persist for years . However, epidemiologic studies yielded inconsistent results, specifically while ecological and cross-sectional studies reported positive associations for PTB and LBW and pesticide use in agriculture , results from studies assessing self-reported or occupational use of pesticides were inconsistent . A systematic review of 25 studies examining agriculture-related exposures from residential proximity to pesticide applications suggested weak or no effects on preterm birth and low birth weight, possibly due to the methodological difficulties of exposure assessment . More recent residential proximity studies using simple or aggregate-level exposure assessments provided some evidence for pesticide influencing birth outcomes . Two recent Geographic Information System -based studies restricted to the San Joaquin Valley of California reported conflicting results – one found pesticide exposures to increase preterm birth and low birthweight by 5-9% in those highly exposed to chemicals with acute toxicity as based on the US EPA Signal Word , while the other assessed 543 individual chemicals and 69 physicochemical groupings found negative associations for spontaneous preterm birth . Nevertheless, various small pesticide biomarker-based studies with measured organochlorines, organophosphates, or pyrethroids and their metabolic breakdown products in maternal blood or urine or umbilical cord blood suggested positive associations with preterm birth or with lower birthweight but results varied by chemicals and outcomes assessed . Here, we assessed GIS-derived exposures during pregnancy to selected agricultural pesticides applied near maternal residences and risks of preterm birth and term low birthweight, considering trimester-specific exposure windows in a large sample of births in agricultural regions of California.Birth addresses with a low geocode quality due to missing or non-geocodeable fields on the birth certificates accounted for ~12% of all addresses geocoded. We then calculated measures of residential ambient pesticide exposures using a GIS-based Residential Ambient Pesticide Estimation System, as previously described . In brief, since 1974 agricultural pesticide applications for commercial use are recorded in Pesticide Use Reports mandated by the CA Department of Pesticide Regulation .