Tag: Round nursery pots

Posted on

Magnesium is a key regulator of metabolic processes such as glycolysis and the Krebs cycle

Studies in yeasts and animal cells suggest that RPA-coating of single stranded DNA act as a signal to activate ATR dependent downstream phosphorylation, primarily through an associated protein called ATRIP . SUV2 is required for repair of UV induced damage, as is its namesake, and suv2 mutants are also sensitive to HU, MMC and CDDP. In sum, ATR, ALT2, SOG1 and SUV2 are all required for DNA damage response as a result of replication fork stalls. This requirement of ATR, ALT2, SOG1 and SUV2 for a plant’s survival in the response to specific genotoxins as seen with HU, MMC and CDDP heightens the curiosity as to what the true nature of Al’s impact on DNA is. As the sensitivity of atr,alt2, sog1 and suv2 mutant roots to defined genotoxins demonstrates, loss of these factors should lead to sensitivity to Al rather than tolerance if Al directly causes DNA damage. It seems counter intuitive that a plant gains Al tolerance by reducing the function of factors necessary for DNA damage response. If atr, alt2, sog1 and suv2 mutant roots can maintain root growth even in the presence of Al, what actual damage is detected by ATR, ALT2, SOG1 and SUV2 in the presence of Al? Identification of four factors that have clear roles in DNA damage responses suggests that a primary effect of Al toxicity is directly related to compromised genomic integrity, with Al possibly serving as a genotoxic agent, whether real or perceived. It is curious that loss of any one cell cycle checkpoint results in increased tolerance to Al rather than sensitivity as is observed with known genotoxic agents like HU, MMC and CDDP. This may suggest that these checkpoints are either so sensitive that even the limited amount of genomic stress that might directly or indirectly occur with Al could activate these factors yet in reality be relatively inconsequential to growth, or that Al is inappropriately perceived as a genotoxic agent by ATR, ALT2, SOG1 and SUV2. Based on the current findings on SOG1 and SUV2 in conjunction with the previous reports on ATR and ALT2,plastic nursery plant pot a DNA damage response is the primary cause of Al dependent root growth inhibition in Arabidopsis.

What kind of Al dependent DNA damage these factors are detecting is still unknown; however, in concurrence with research from the fields of Al toxicity and DNA damage responses, there could be a multitude of sources of this damage, including but not limited to: Reactive Oxygen Species, phosphate deficiency leading to dNTP depletion, competition with Mg2+ causing ATP depletion and enzymatic dysfunction, and topological strain affecting replication fork stalls or reduced transcriptional capabilities.Chronic high levels of Al exposure have been shown to result in peroxidation of lipids within the membranes of cells . Lipid peroxidation is likely a downstream result of Al damage, perhaps caused by Reactive Oxygen Species known to be generated by Al . ROS can cause DNA damage, where damage to individual bases may also be implicated, and could be tested for in a comet assay. In peripheral blood lymphocytes treated with Al, a high incidence of oxidized bases, particularly purines and apurinic/apyramidinic sites, were attributed to Al-generated ROS . In the human genome such base lesions are indeed repaired by ATR-mediated nucleotide excision repair . In Arabidopsis, over expression of a variety of factors in the antioxidant pathway have resulted in increased Al tolerance in Arabidopsis such as glutathione S-transferase and peroxidase . However, a loss-of-function mutant of At4g10500, an uncharacterized member of the 2-oxoglutarate and Fe-dependent oxygenase super family, tested for possible scavenging of ROS in response to Al exposure did not show any phenotypic changes in response to Al . Additionally, if Al-generated ROS were indeed primarily responsible for root growth inhibition, theloss-of-function mutants for DNA damage response factors would result in heightened sensitivity rather than tolerance to Al. Unrepaired damage caused by ROS leads to oxidative damage of lipids, amino acids, and DNA which can lead to cell death. Although ROS is likely a detrimental symptom of Al exposure, as Al toxicity is a complex and widely destructive biological assault, it is unlikely that ATR, ALT2, SOG1, and SUV2 are detecting damage caused by ROS.Al toxicity and phosphate deficiency typically coexist due to acid soil conditions that promote Al bio-availability while simultaneously reduce Pi uptake by the roots , and ALS3 has been identified as a required factor in a Pi starvation response in a sucrose-dependent manner . While Pi deficiency has many varied symptoms as phosphorous is required for photophosphorylation, genetic metabolism, transportation of nutrients, and phospholipid composition of cell membranes , it has dire effects on DNA replication as massive quantities of Pi are needed in the form of dNTP’s that are polymerized in order to form DNA as well as in the ATP consumed in the polymerization reaction. dNTP depletion caused by Pi deficiency would cause replication fork stalls, and lead to replication catastrophe, much like HU treatment.

A Pi starvation growth assay where seedlings of Col-0 wild type, als3-1, and atr- 4;als3-1 could be tested for Pi starvation responses. If atr-4 is capable of suppressing the als3-1 phosphate sensitivity, this could resolve whether or not Pi deficiency plays a predominant role in the Al-dependent damage response. However, damage caused by Pi deficiency would logically result in heightened Al sensitivity rather than tolerance as is observed for the loss-of-function mutants for ATR, ALT2, SOG1 and SUV2. Alternatively, with sucrose being a required factor for Pi sensitivity in als3-1, an Al growth assay where seedlings of Col-0 wild type and als3-1 are grown in the absence of sucrose with increasing amounts of AlCl3 could assess the ALS3-dependent PI starvation response. If Al toxicity is actually the result of Pi starvation, als3-1 should grow similarly to Col-0 wild type in the absence of sucrose, rather than a hypersensitive response to Al. It seems highly improbable given that Al exposure forces terminal differentiation through means of endore duplication, requiring rounds of DNA replication, as possible mechanism to inhibit root growth caused by Pi deficiency. Al3+ ions complete with magnesium ions for binding sites on the plasma membrane and decrease the uptake of magnesium into the root. Increasing concentrations of available magnesium in soil or nutrient media or over expressing magnesium transport genes enhance Al resistance as increased magnesium released into the rhizosphere competes with Al. Magnesium is the predominant ionic regulator of metabolism, largely through its role as a cofactor for all phosphoryl transfers in the cell.It also acts as a second messenger for growth factors in regulation of protein synthesis and is required to maintain genomic stability. Besides its stabilizing effect on DNA and chromatin structure, magnesium is an essential cofactor in almost all enzymatic systems involved in DNA processing. Most obvious in DNA replication, its function is not only charge-related, but very specific with respect to the high fidelity of DNA synthesis . Furthermore, as an essential cofactor in nucleotide excision repair, base excision repair and mismatch repair, magnesium is required for the removal of DNA damage generated by environmental mutagens, endogenous processes, and DNA replication . More studies are warranted to study how Al interferes with the function of magnesium in plants under Al toxic conditions. Al growth responses in the presence of excess magnesium could be tested for atr-4;als3-1, alt2;als3-1, sog1-7;als3-1, and suv2;als3-1 in comparison with Col-0 wild type and als3-1 to determine if magnesium supplementation can alleviate the effects of Al toxicity.

This test would likely be indicative of Al resistance, showing magnesium outcompeting Al for entry into the cells of the root. If Al interferes with DNA replication machinery,seedling starter pot in vitro investigations such as PCR assays could be tested for amplicon lengths in the presence of Al to measure processivity, as such assays has been demonstrated in yeast . Such assays could be performed with commercially available DNA ploymerases, as Arabidopsis polymerase enzymes are not readily available.The effects of Al at the nuclear level are poorly understood. Al rapidly accumulates at high levels in root meristem nuclei and is especially concentrated around interphase chromatin as well as mitotic figures . Al does not appear to have a base composition preference and it is likely that this lack of base discrimination is due Al binding to the phosphate backbone of DNA . This could result from an electrostatic attraction of Al3+ to the negative charges of the phosphodiester bonds. Such an association could increase the rigidity of euchromatin and relax supercoiled heterochromatin destabilizing genome topology through an ever-fluxing torsional tug-of-war. DNA gyrases and topoisomerases regulate topological strains such as supercoiled and relaxed DNA, especially caused by replisome progression, and are required to prevent replication fork stalls caused by supercoiled DNA in front of unwinding . Loss-of-function mutants of topI or topII could be tested for growth responses to Al. Perhaps in reality, overexpression mutants of a topoisomerase would be capable of counteracting the strain Al exerts on DNA and should be generated and tested. However, despite the effectiveness of topoisomerases to alleviate replication induced topological strains, their functions would not rid the nucleus of Al3+ and would likely be unable to ameliorate the affect of Al on the whole genome. Such a topological strain caused by binding of Al to DNA or chromatin could condense DNA molecules and inhibit cell division by reducing its capacity to provide proper DNA separation as is necessary for DNA replication and mitotically relevant transcriptional events . Others have shown that Al causes DNA compaction, as well as compaction of chromatin, potentially through inhibition of unwinding of genomic DNA since Al3+ will raise the Tm of the double helix . Al has been shown to precipitate DNA out of solution in vitro, especially templates normally found in transcriptionally active euchromatin, extending the implication that Al compaction could lead to transcriptional repression. Importantly, many of these gene-silencing effects can be explained due to the extraordinary charge density of the Al ion, and perhaps this alone is reason enough to disrupt DNA processes that activate checkpoint responses. While gene silencing may indeed result from internalized Al, this is likely not a significant consequence of the effect of Al on DNA, as Al-inducible gene expression is a confirmed response to the internalization of Al . However, it is not unreasonable to predict that such conformational changes to the DNA could be perceived by ATR as being deleterious to replication fork progression. Such a topological strain on DNA caused by Al may cause a conformational change reminiscent of covalent crosslinkers, such as MMC and CDDP. Since Al3+ is expected to have high affinity for the negatively charged phosphodiester backbone of DNA and would presumably interact with this backbone differently than divalent cations and could cause an electrostatic interaction where Al acts as a non-covalent pseudo-crosslink. If Al acted as a pseudo-crosslinker that disrupts or restricts unwinding of DNA during processes such as DNA replication and RNA transcription, this may trigger ATR-dependent checkpoint activation, similar to a replication fork stall caused by a true DNA crosslink. As discussed, ATR, ALT2, SOG1 and SUV2 are all required to respond to DNA cross linking agents and all are linked to Al-dependent stoppage of root growth .Many uncertainties persist in regards to the nature of the unknown damage detected by ATR, ALT2, SOG1 and SUV2. It seems counter intuitive that a plant gains tolerance to an agent that causes DNA damage by reducing the function of a factor necessary for DNA-damage detection. Peculiarly, the genomic stability of Arabidopsis roots is secure enough to endore duplicate following Al exposure, indicating that unlike true DNA cross links, the effect of Al on the DNA is not severe enough to inhibit multiple rounds of DNA synthesis phases that would be required for the endore duplication cycle. Yet, visualization of micro-nuclei formation in cells of the root tip indicates that real breakage of chromosomes occurs following long term exposure to Al. Once more, it is perplexing that ATM is largely uninvolved with the Al response despite the consequent DNA breakage in the formation of micro-nuclei. Perhaps these factors force root growth inhibition in the presence of Al in order to prevent passage of minor damage to subsequent generations.

Posted on

Exposures to pesticides in the third trimester did not increase risk for preterm birth

Each PUR record includes the name of the pesticide’s active ingredient, the poundage applied, the crop type, and the location and the date of application. The California Department of Water Resources performs countywide, large-scale surveys of land use and crop cover every 7–10 years. Land use maps increase spatial resolution because they provide more detailed land use geography that allows us to refine the pesticide applications . We then combined PUR records, land use maps, and geocoded birth addresses to produce estimates of pesticide exposure during pregnancy. Monthly exposure estimates were calculated by adding the poundage of pesticide applied in a 2-kilometer buffer surrounding each address and weighting the total poundage by the proportion of acreage treated within the buffer. Previous pesticide studies relied on different buffer sizes from 500m , half a mile , 1000m , 1250m , 1600m , 5000m , to up to 8000m distances , depending on the pesticide of interest, landscape, and weather conditions. In light of previous research, the buffer of 2-km we chose, will provide a reasonable distance for assessing pesticide applications around residential addresses. For each calendar month, our integrated GIS-system returned continuous measures for each specific chemical applied within 2-km of individuals’ residences. We defined the first, second, and third trimesters as 0-12 weeks, 13-25 weeks, and ≥26 weeks of pregnancy, respectively. For preterm birth, the length of gestation and hence exposure period are shorter than term birth by design; to account for that, we assessed the third trimester exposures using 27- 32 weeks of gestation only since more than 88% of all preterm births had a gestational length longer than 32 weeks. For each pesticide, daily poundage for each gestational day of pregnancy was calculated based on monthly values,seedling starter pot and then averaged across all days in each trimester. We then categorized prenatal exposure as ever/never exposed to a specific chemical in each trimester.

We selected 17 individual chemicals previously observed to have reproductive toxicity . Additionally, we also considered all pesticides from three widely used chemical classes that have been linked to reproductive toxicity based on the Pesticide Action Network pesticide database , i.e. 24 n-methyl carbamate/dithiocarbamates, 50 organophosphates, and 29 pyrethroid pesticides to which one or more study subjects were exposed according to our 2km buffer criterion . For each class, we used the sum of the total number of individual chemicals that each subject was ever exposed to in each time period of interest. We divided subjects into high , low , and no exposure to the respective pesticide, and compared high and low with the no exposure group as the reference. Since information about the specific location of non-agricultural pesticide applications are not provided by the PUR and because some individuals in urban areas are highly exposed to traffic-related air pollution or hazardous air toxics that are known risk factors for adverse birth outcomes , we restricted our analyses to individuals born in agricultural regions, defining those as residences within 2km buffer of any type of agricultural pesticide application during pregnancy . We conducted unconditional logistic regression analyses adjusting for matching factors and the source of control subjects and estimated odds ratios and 95% confidence intervals . To account for the unbalanced gender ratio and birth year distribution in this combined sample, we included the inverse of the sampling fraction as a stabilized weighting factor to reflect the sex and birth year distribution of all California births. Statistical analyses were performed using SAS software, Version 9.4 . We additionally adjusted for covariates as potential confounders and effect measure modifiers based on the literature : including maternal age at delivery , maternal race/ethnicity , maternal birthplace , maternal education , parity , payment source for prenatal care as a proxy for family income , prenatal care in the first trimester , and a previously developed neighborhood-level SES metric . Furthermore, we conducted stratified analyses by maternal race/ethnicity since exposures may be higher among Hispanics, especially recent immigrants, who may live close to agricultural fields and have poor housing conditions ; by infant sex because males are more likely to be born preterm ; as well as by season of conception , estimated from the last menstrual period and length of gestation, because of seasonal variations in pesticide applications .

In sensitivity analyses, we compared effect estimates with and without adjusting for two risk factors for adverse birth outcomes, maternal cigarette smoking during pregnancy and prepregnancy Body Mass Index , calculated as maternal pre-pregnancy weight divided by maternal height  for births in 2007-2010 only, since these variables are only available on the birth certificate from 2007 onward. We also investigated the potential confounding effects from outdoor air pollution that can impact fetal growth during critical periods among the autism controls only due to data availability. We estimated trimester-specific exposures to local, traffic-derived NOx, PM2.5, and CO, including roadways within 1.5 km of subjects’ birth addresses, i.e. inter-quartile range -scaled measure of NOx as a local traffic marker derived from the CAlifornia LINE source dispersion model model . Additionally, we adjusted for co-exposure to at least one of other individual chemicals as a single variable when assessing each individual chemical, and estimated mutually adjusted ORs for the three chemical class exposures during the same exposure window. When evaluating later trimester exposures we adjusted for exposure during prior pregnancy periods, because these effect estimates may be altered by earlier exposures . Since a low geocode quality is likely to introduce spatial exposure misclassification, we excluded those with a geocode quality at the USPS Zip Code Area centroid level or coarser. Lastly, we examined spontaneous vaginal deliveries only, excluding medically indicated preterm deliveries more likely to be due to severe maternal pregnancy complications including pre-eclampsia and gestational diabetes that might or might not be in the causal pathway for pesticide exposures and the outcome. Infants born preterm or born term with low birthweight were more likely to have mothers of younger age, less education, lower neighborhood SES, starting prenatal care after the first trimester, and using Medi-Cal or other government programs instead of private insurance. In addition, infants born preterm were more likely to be a third or later born child, and have mothers with Hispanic or Black race/ethnic background; infants born term but with low birthweight were more likely to be female and a first born child, and born to Black and Asian mothers . First- and second trimester exposures to some pesticides we have selected were associated with a small increase in risk for preterm birth. Specifically, in multivariate adjusted models, first trimester exposures to glyphosate compounds, paraquat dichloride, chlorpyrifos, imidacloprid, permethrin, dimethoate, and methyl bromide, and second trimester exposures to chlorothalonil, glyphosate compounds, paraquat dichloride, simazine, and imidacloprid, yielded adjusted ORs between 1.03 and 1.07 with 95% CIs excluding the null value .

Maternal education changed the OR estimates the most among all covariates.Effect estimates were generally slightly stronger in female infants, except for simazine, which shows stronger effect in males with an OR of 1.06~1.07 . Stratified analysis by season of conception suggested that effect estimates were generally stronger when the peak season of pesticide application concurred with the first or second trimester of pregnancy . When examining chemical classes,round nursery pots first trimester exposures to carbamates , or pyrethroids increased ORs for preterm birth in the high exposure group, compared with the no exposure group, while second trimester exposures to carbamates, organophosphates, or pyrethroids were all associated with small increases in ORs for preterm birth . We generally did not observe elevated ORs for preterm birth among male infants, but observed a stronger 7–11% increase with exposure during the first or second trimester among female infants . Exposure prevalence and effect estimates were generally stronger in infants born to the foreign born or US-born Hispanic mothers than White mothers . Associations between the selected individual pesticides or chemical classes and term low birthweight for each trimester in pregnancy were mostly null. In multivariate adjusted models, we only saw increased ORs for second or third-trimester exposures to myclobutanil ; similarly, exposures to the three chemical classes were not associated with term low birthweight in general, except for marginally elevated odds in infants exposed to 2 or more pyrethroids . Results were similar in our sensitivity analyses, with additional adjustment for maternal prepregnancy BMI and maternal smoking in the years 2007-2010, for NOx as traffic-related air pollution, or restricting to those with a high geocode quality only. For each individual pesticide, adjusting for co-exposure to other pesticides resulted in attenuation of odds by 2-3%; ORs mutually adjusted of three chemical classes or adjusted for prior exposures were mostly similar to or slightly decreased; the mutually adjusted OR for pyrethroids was most stable, suggesting a more robust association with pyrethroids, which were used more in recent years . ORs were generally stronger when we restricted to spontaneous preterm births only for both individual chemicals and chemical classes. In this large California study of women living within 2km distance from agricultural fields on which pesticides were applied, we found that early and mid-pregnancy exposure to selected pesticides known or suspected to be reproductive toxicants and chemicals in the classes of pyrethroids and possibly also carbamates or organophosphates, are associated with a small to moderate size increase in risk of preterm birth between 1998 and 2010.

We found little evidence for pesticides being related to term low birthweight, except for exposures to pyrethroids as a class further corroborating their adverse influence on pregnancy observed for preterm birth and possibly one single pesticide myclobutanil – however, this might have been a chance observation given that we tested 17 individual chemicals. Yet, term low birth weight is a much rarer event than preterm birth and we had less statistical power to estimate small effects accurately. Our positive findings for preterm birth are consistent with biomarker-based studies with measured organophosphates, or pyrethroids and their metabolic breakdown products in maternal blood or urine or umbilical cord blood , though most of the literature assessing environmental exposures to pesticide found inadequate evidence for associations with preterm birth . Less than a handful of studies conducted in the US examined associations for environmental exposures to pesticides from agricultural applications and preterm birth and/or low birthweight and provided month- or trimester-specific estimates . These studies were almost exclusively conducted using California’s unique PUR system, nevertheless they differed in terms of how they assessed exposures and pregnancy outcomes. Our study was in line with an earlier study in the San Joaquin Valley that assessed pesticides labeled with EPA signal word toxicity by summing up their active ingredients applied in the 2.6 km2 section surrounding maternal residences and reported high exposure to pesticides increased risks of preterm birth and low birthweight by 5-9% overall . In contrast, one study reported mostly negative associations between spontaneous preterm deliveries and exposure to 69 chemical groups or 543 specific chemicals in 1998-2011 in the San Joaquin Valley , perhaps because this study focused on late pregnancy instead of early or mid-pregnancy, which is believed to be the critical period for exposures causing preterm birth , and in addition a ‘live-birth selection bias’ could in part explain the negative effect. The other study in northern California reported methyl bromide use within 5 km of mother’s home was also positively associated with gestational age in the first trimester; yet their results were sensitive to buffer size and could potentially be confounded by chloropicrin or diazinon, often used conjunctively with methyl bromide . Maternal, placental, and fetal factors are thought to determine risk of preterm birth and may be affected by prenatal exposure to environmental chemicals . For example, it is known that chlorpyrifos can cross the placenta and enter the fetus, possibly altering the growth and development of the fetus . Mechanisms by which pesticides may affect risks of preterm birth include interference with immune pathways and inflammation , or with metabolic and endocrine regulatory pathways as well as oxidative stress . For example, in-vitro study results suggested that phosmet and chlorpyrifos alter cell viability and induce an inflammatory cytokine profile, indicating that organophosphates may adversely affect trophoblast cells .