It Takes Time to Unravel the Ecology of War in Gaza, Palestine: Long-Term Changes in Maternal, Newborn and Toddlers' Heavy Metal Loads, and Infant and Toddler Developmental Milestones in the Aftermath of the 2014 Military Attacks.

Toxicant, teratogen and carcinogen metal war remnants negatively affect human health. The current study analyzes, first, the persistence of heavy metal contamination in newborn hair in four cohorts across time in Gaza Palestine; second, the change in mothers' and infants' heavy metal contamination from birth to toddlerhood; and third, the impact of heavy metal contamination on infants' and toddlers' growth and development. The hair of newborns was analyzed for twelve heavy metals by Inductively Coupled Plasma Mass Spectrometry (ICP/MS) in cohorts recruited at delivery in 2011, 2015, 2016, and 2018-2019. In the 2015 cohort, mothers' hair samples were taken at delivery, and toddlers and mothers hair were also analyzed 18 months later. Growth levels of infants at six months and toddlers at 18 months were assessed according to World Health Organization (WHO) standards according to a mother report and pediatric check-up, respectively. 1. The level of metal contamination in utero was persistently high across 8 years, 2011, 2015, 2016, 2019, following three major military attacks (2009, 2012, 2014). 2. The 2015 cohort babies exposed in utero to attacks in 2014 at six months showed association of high load at birth in mother of arsenic and in newborn of barium with underweight, of barium and molybdenum in newborn with stunting. 3. Eighteen months after birth, toddlers had a higher level of metals in hairs than when they were born, while, in their mothers, such levels were similar to those at delivery, confirming persistence in the environment of war remnants. Underweight and stunting, both in infants and toddlers, were higher than reported for previous years, as well as being progressive within the cohort. Severe environmental factors, metal contamination and food insecurity put Gaza's infant health at risk.

Long Term Risks to Neonatal Health from Exposure to War-9 Years Long Survey of Reproductive Health and Contamination by Weapon-Delivered Heavy Metals in Gaza, Palestine.

Introduction: High levels of environmental contaminants with long term effects and teratogenic and carcinogenic potential, such as heavy metals, were introduced by weaponry in war areas in the last decades. Poorer reproductive health and increases in non-communicable diseases were reported after wars and are the suspected long term effects of contamination by stable war remnants. Although potentially affecting millions of people, this is still an understudied issue of public health. Background: Gaza, Palestine since 2006 has been an object of repeated severe military attacks that left heavy metals remnants in the environment, in wound tissues and that were assumed by the population. Retrospective studies showed a progressive increase in birth defects since the 2006 attacks. In 2011 we started surveillance at birth alongside analysis of the heavy metals load carried by pregnant women and their babies. Methods: We used protocols for birth registration which also document the extent of exposures to attacks, war remnants and to other environmental risks that allow comparison of 3 data sets-2011, 2016 and 2018-2019 (4000-6000 women in each set). By ICP/MS analysis we determined the content of 23 metals in mothers' hair. Appropriate statistical analysis was performed. Results: Comparison of data in birth registers showed a major increase in the prevalence in birth defects and preterm babies between 2011 and 2016, respectively from 1.1 to 1.8% and from 1.1 to 7.9%, values remaining stable in 2019. Negative outcomes at birth in 2016 up to 2019 were associated with exposure of the mothers to the attacks in 2014 and/or to hot spots of heavy metals contamination. Metal loads since the attacks in 2014 were consistently high until 2018-2019 for barium, arsenic, cobalt, cadmium, chrome, vanadium and uranium, pointing to these metals as potential inducers for the increased prevalence of negative health outcomes at birth since 2016. Conclusions: Bodily accumulation of metals following exposure whilst residing in attacked buildings predispose women to negative birth outcomes. We do not know if the metals act in synergy. Trial for mitigation of the documented negative effects of high metal load on reproductive health, and ensuing perinatal deaths, could now be done in Gaza, based on this documentary record. High load of heavy metals may explain recent increases in non-communicable diseases and cancers at all ages in Gaza. Modern war's legacy of diseases and deaths extends in time to populations and demands monitoring.

Hospital centered surveillance of births in Gaza, Palestine, 2011-2017 and heavy metal contamination of the mothers reveals long-term impact of wars.

Prevalence of preterm, low birth weight and birth defects increased significantly since 2011 in Gaza, Palestine. No change in known co-factors of reproductive health justified this rise. Two military attacks in 2012 and 2014 introduced novel risk factors for outcomes at birth: contamination by teratogenic and carcinogenic heavy metals weapon-remnants, ongoing impoverishment, and impaired rehabilitation of waste management. It was previously shown that mothers exposed to military attacks had higher metal load than those unexposed and mother's heavy metals trans-pass placenta. We investigated association in time of heavy metal contamination and reproductive health using hospital-based surveillance (2011-2016-2017) of births, accompanied by assessment in 2016 of metal load in mother and newborn hair. Mother's housing proximal to unmanaged waste predicted preterm birth and birth defects, and these women had highest load of heavy metals. Poor diet predicted low birth weight. Circumstances prevent investigation of heavy metals molecular impact(s) during fetal development.

Toxic Environment of war: Maternal prenatal heavy metal load predicts infant emotional development.

BACKGROUND: People in war zones are exposed to heavy metal contamination deriving from new-generation weapons, in addition to exposure to psychologically traumatizing war events. Pregnant women and their children-to-be are particularly vulnerable to both biological and psychological war effects. OBJECTIVE: The aim of the current study was to analyse the impact of maternal prenatal heavy metal contamination on infant emotional development and to examine the potential moderating role of maternal symptoms of post-traumatic stress disorder (PTSD) in the association between heavy metal load and infant emotional development. METHODS: The participants were 502 Palestinian mothers, pregnant in their first trimester during the 2014 War on Gaza. The mothers were recruited at their delivery (T1) and followed at the infants' age of 6-7 months (T2; N = 392). The load of five weapon-related heavy metals (chromium, mercury, vanadium, strontium, and uranium) was analysed by Inductively Coupled Plasma Mass Spectrometry (ICP/MS) from mothers' hair samples at childbirth (T1). Assessment of maternal PTSD symptoms was based on the Harvard Trauma Questionnaire (HTQ) and infant emotional development on the Infant Behavior Questionnaire (IBQ), both reported by mothers (T2). RESULTS: Two of the analysed metals, chromium and uranium, adversely predicted children's early emotional development, indicated by decreased positive affectivity, increased negative emotionality, and problems in early orientation and regulation. Mother's PTSD did not moderate the impact of heavy metal contamination on children's emotional development. CONCLUSIONS: Adverse impact of war is not limited to those who experience it directly, but is passed on to future generations through multiple mechanisms. International organizations are obliged to protect parents and infants from the modern weaponry in wars.

A cross sectional study of the relationship between the exposure of pregnant women to military attacks in 2014 in Gaza and the load of heavy metal contaminants in the hair of mothers and newborns.

OBJECTIVE: Metal contamination of humans in war areas has rarely been investigated. Weaponry's heavy metals become environmentally stable war remnants and accumulate in living things. They also pose health risks in terms of prenatal intake, with potential long term risks for reproductive and children's health. We studied the contribution of military attacks to the load of 23 metals in the hair of Palestinian women in the Gaza Strip, who were pregnant at the time of the military attacks in 2014, and their newborns. We compared the metal load in the mothers with values for adult hair from outside the war area (RHS) as the reference. We investigated heavy metals trans-passing in utero, and assessed if the heavy metal intake could derive from sources unrelated to the war. DESIGN: Cross sectional study. PARTICIPANTS AND SETTING: Cross sectional convenience sample of 502 mothers delivering in the Gaza Strip and their newborns. MAIN OUTCOME MEASURED: Measure of the load of heavy metals in mother and newborn hair by inductively coupled plasma-mass spectrometry (ICP-MS). Comparison of metal loads with the reference RHS, between groups with different exposures to attacks and house/agriculture chemicals, and between mothers and newborns. Data for birth registry and for exposures to war and other known risk factors were obtained at interview with the mothers. Photographic documentation of damage from military attacks was obtained. RESULTS: The whole cross sectional convenience sample had a significantly higher load of heavy metals than the reference RHS. Women exposed to military attacks had a significantly higher load of heavy metals than those not exposed; the load in newborns correlated positively with the mothers' load. No significant difference was found between users/non-users of house/agriculture chemicals. No other known confounder was identified. CONCLUSIONS: High heavy metal loads in mothers, reflected in those of their newborns, were associated with exposure to military attacks, posing a risk of immediate and long term negative outcomes for pregnancy and child health. Surveillance, biomonitoring and further research are recommended. Implications for general and public health are discussed.

Specific association of teratogen and toxicant metals in hair of newborns with congenital birth defects or developmentally premature birth in a cohort of couples with documented parental exposure to military attacks: observational study at Al Shifa Hospital, Gaza, Palestine.

This study was undertaken in Gaza, Palestine, in a cohort of babies born in 2011. Hair samples of newborns were analyzed for metal load by DRC-ICP-MS. We report specific level of contamination by teratogen/toxicants metals of newborn babies, environmentally unexposed, according to their phenotypes at birth: normal full term babies, birth defects or developmentally premature. The occurrence of birth defects was previously shown to be correlated in this cohort to documented exposure of parents to weapons containing metal contaminants, during attacks in 2009. We detect, in significantly higher amounts than in normal babies, different specific teratogen or toxicant elements, known weapons' components, characteristic for each of birth defect or premature babies. This is the first attempt to our knowledge to directly link a phenotype at birth with the in utero presence of specific teratogen and/or toxicant metals in a cohort with known episodes of acute exposure of parents to environmental contamination by these same metals, in this case delivered by weaponry The babies were conceived 20-25 months after the major known parental exposure; the specific link of newborn phenotypes to war-remnant metal contaminants, suggests that mothers' contamination persists in time, and that the exposure may have a long term effect.

Birth defects in Gaza: prevalence, types, familiarity and correlation with environmental factors.

This is the first report of registration at birth, and of incidence of major structural birth defects (BD) obtained in Gaza at Al Shifa Hospital, where 28% of total births in Gaza Strip occur. Doctors registered 4,027 deliveries, with a protocol comprehensive of clinical, demographic, kin and environmental questions. Prevalence of BD is 14/1,000, without association with intermarriage or gender of the child. Prevalence of late miscarriages and still births are respectively 23.3/1,000 and 7.4/1,000, and of premature births 19.6/1,000. Couples with a BD child have about 10 times higher frequency of recurrence of a BD in their progeny than those with normal children, but none of their 694 siblings and only 10/1,000 of their 1,423 progeny had BD, similar to the frequency in general population. These data suggest occurrence of novel genetic and epigenetic events in determination of BD. Children with BD were born with higher frequency (p < 0 001) in families where one or both parents were under "white phosphorus" attack, that in the general population. Bombing of the family home and removal of the rubble were also frequently reported by couples with BD occurrence. These data suggests a causative/favoring role of acute exposure of parents to the weapons-associated contaminants, and/or of their chronic exposure from their persistence in the environment on the embryonic development of their children.

Four polygamous families with congenital birth defects from Fallujah, Iraq.

Since 2003, congenital malformations have increased to account for 15% of all births in Fallujah, Iraq. Congenital heart defects have the highest incidence, followed by neural tube defects. Similar birth defects were reported in other populations exposed to war contaminants. While the causes of increased prevalence of birth defects are under investigation, we opted to release this communication to contribute to exploration of these issues. By using a questionnaire, containing residential history and activities that may have led to exposure to war contaminants, retrospective reproductive history of four polygamous Fallujah families were documented. Our findings point to sporadic, untargeted events, with different phenotypes in each family and increased recurrence. The prevalence of familial birth defects after 2003 highlights the relevance of epigenetic mechanisms and offers insights to focus research, with the aim of reducing further damage to people's health.

Hind limb unloading of mice modulates gene expression at the protein and mRNA level in mesenchymal bone cells.

BACKGROUND: We investigated the extent, modalities and reversibility of changes at cellular level in the expression of genes and proteins occurring upon Hind limb unloading (HU) in the tibiae of young C57BL/6J male mice. We focused on the effects of HU in chondrogenic, osteogenic, and marrow mesenchymal cells. METHODS: We analyzed for expression of genes and proteins at two time points after HU (7 and 14 days), and at 14 days after recovery from HU. Levels of mRNAs were tested by in situ hybridization. Protein levels were tested by immunohistochemistry. We studied genes involved in osteogenesis (alkaline phosphatase (AP), osteocalcin (OC), bonesialoprotein (BSP), membrane type1 matrix metalloproteinase (MT1-MMP)), in extracellular matrix (ECM) formation (procollagenases (BMP1), procollagenase enhancer proteins (PCOLCE)) and remodeling (metalloproteinase-9 (MMP9), RECK), and in bone homeostasis (Stro-1, CXCL12, CXCR4, CD146). RESULTS: We report the following patterns and timing of changes in gene expression induced by HU: 1) transient or stable down modulations of differentiation-associated genes (AP, OC), genes of matrix formation, maturation and remodelling, (BMP1, PCOLCEs MMP9) in osteogenic, chondrogenic and bone marrow cells; 2) up modulation of MT1-MMP in these same cells, and uncoupling of its expression from that of AP; 3) transient down modulation of the osteoblast specific expression of BSP; 4) for genes involved in bone homeostasis, up modulation in bone marrow cells at distal epiphysis for CXCR4, down modulation of CXCL12, and transient increases in osteoblasts and marrow cells for Stro1. 14 days after limb reloading expression returned to control levels for most genes and proteins in most cell types, except AP in all cells, and CXCL12, only in bone marrow. CONCLUSIONS: HU induces the coordinated modulation of gene expression in different mesenchymal cell types and microenvironments of tibia. HU also induces specific patterns of expression for homeostasis related genes and modulation of mRNAs and proteins for ECM deposition, maturation and remodeling which may be key factors for bone maintenance.

Metals detected by ICP/MS in wound tissue of war injuries without fragments in Gaza.

BACKGROUND: The amount and identity of metals incorporated into "weapons without fragments" remain undisclosed to health personnel. This poses a long-term risk of assumption and contributes to additional hazards for victims because of increased difficulties with clinical management. We assessed if there was evidence that metals are embedded in "wounds without fragments" of victims of the Israeli military operations in Gaza in 2006 and 2009. METHODS: Biopsies of "wounds without fragments" from clinically classified injuries, amputation (A), charred (C), burns (B), multiple piercing wounds by White Phosphorus (WP) (M), were analyzed by ICP/MS for content in 32 metals. RESULTS: Toxic and carcinogenic metals were detected in folds over control tissues in wound tissues from all injuries: in A and C wounds (Al, Ti, Cu, Sr, Ba, Co, Hg, V, Cs and Sn), in M wounds (Al, Ti, Cu, Sr, Ba, Co and Hg) and in B wounds (Co, Hg, Cs, and Sn); Pb and U in wounds of all classes; B, As, Mn, Rb, Cd, Cr, Zn in wounds of all classes, but M; Ni was in wounds of class A. Kind and amounts of metals correlate with clinical classification of injuries, exposing a specific metal signature, similar for 2006 and 2009 samples. CONCLUSIONS: The presence of toxic and carcinogenic metals in wound tissue is indicative of the presence in weapon inducing the injury. Metal contamination of wounds carries unknown long term risks for survivors, and can imply effects on populations from environmental contamination. We discuss remediation strategies, and believe that these data suggest the need for epidemiological and environmental surveys.

The carboxyl terminal trimer of procollagen I induces pro-metastatic changes and vascularization in breast cancer cells xenografts.

BACKGROUND: The COOH terminal peptide of Pro-collagen type I (PICP, also called C3) is chemotactic for endothelial melanoma and breast cancer cells. PICP induces the expression of Metalloproteinases-2 and -9, of Vascular endothelial growth factor and of the chemokine CXCL-12 receptor CXCR4 in MDA MB231 breast carcinoma cells in vitro. METHODS: We used a model of xenografts in BalbC/nude mice obtaining tumors by implanting in contro-lateral subcutaneous positions MDA MB231 cells added or not with purified PICP and studied the earlier phases of tumor development, up to 48 days from implant, by histology, immunostain and in situ hybridization. RESULTS: Addition of PICP promotes rapid vascularization of the tumors while does not affect mitotic and apoptotic indexes and overall tumor growth. PICP-treated, relative to control tumors, show up-modulation of Vascular endothelial factor, Metalloproteinase-9 and CXCR4, all tumor prognostic genes; they also show down-modulation of the endogenous Metalloproteinase inhibitor, reversion-inducing-cysteine-rich protein with kazal motifs, and a different pattern of modulation of Tissue Inhibitor of Metalloproteinase-2. These changes occur in absence of detectable expression of CXCL-12, up to 38 days, in control and treated tumors. CONCLUSION: PICP has an early promoting effect in the acquisition by the tumors of prometastatic phenotype. PICP may be play a relevant role in the productive interactions between stroma and tumor cells by predisposing the tumor cells to respond to the proliferation stimuli ensuing the activation of signaling by engagement of CXCR4 by cytokines and by fostering their extravasion, due to the induction of increased vascular development.

Role of MT1-MMP in the osteogenic differentiation.

Metalloproteinase MT1-MMP is induced and Pro-MMP-2 up modulated early in rat preosteoblasts (ROB) set to differentiate. We here show that the induction of MMPs, accompanied by activation of Pro-MMP-2, occurs by 6 h of adhesion on endogenous extracellular matrix (ECM), Fibronectin (FN) and Collagen type I (CI). These events do not occur after adhesion on Collagen III (CIII), Vitronectin (VN) or BSA. Within the first hour on inducing substrata or plastic, FAK is unchanged and ERK(1,2), is activated, but this activation is not sufficient for MT1-MMP induction. The function of p38 MAPK and PTKs is not required for the induction by substrata of MMPs. Six hours after plating preosteoblasts on MMP-inducing substrata, complexes of beta1 integrin with MT1-MMP are formed, that contain integrin dimers specifically engaged by the substratum, alpha4 and alpha5 chains for cells plated on FN, and alpha2 chain for cells plated on CI and ECM. Induction of MT1-MMP and its expression during osteogenesis pleiotropically regulate alkaline phosphatase (AP) expression. During differentiation, variant clones derived from preosteoblasts and MMPs-over-expressing osteoblasts show high MT1-MMP level associated with high AP level both persisting in time, while inhibition of MMPs is accompanied by inhibition of AP. Up or down modulation of AP, transcriptionally or by inhibition of the enzyme activity, has no effect on level or timing of expression of MT1-MMP and Pro-MMP-2. The persistence in expression of MT1-MMP during differentiation, and the associated persistence in expression of AP, as well as their inhibition, both impair the formation of nodules and mineral deposition. A transient pattern of expression of MT1-MMP is required for the establishment of nodules, and MT1-MMP decrease is permissive for nodule mineralization. The expression of AP is required for nodule formation and its level modulates the mineralization. MT1-MMP has multiple functions and is implicated in multiple steps of the differentiation process, acting to regulate homeostasis of the osteogenic differentiation.

Potentiating role of IGFBP-2 on IGF-II-stimulated alkaline phosphatase activity in differentiating osteoblasts.

The insulin-like growth factor (IGF) system plays an important role in the autocrine and paracrine regulation of bone formation and remodeling. The aim of this study was to evaluate the role of the autocrine IGF system during osteogenic differentiation in rat tibial osteoblasts (ROB) in culture. In this in vitro model, the stages of osteogenesis studied were S1, corresponding to the onset of alkaline phosphatase (AP) expression (days 0-3); S2, coincident with the peak of AP expression in differentiation culture conditions (days 4-6), and S3, corresponding to the onset of mineral deposition in the extracellular matrix (days 7-9). The results showed that conditioned medium of ROB contains greater amounts of IGF-II than IGF-I at all differentiation stages. Both peptides showed the highest concentrations on day 3 of differentiation (end of S1). All IGF-binding proteins (IGFBPs), except IGFBP-1 and -6, were detected, and IGFBP-2 was the most abundant IGFBP present in the conditioned media, and its degradation increased from S1 to S3. By semiquantitative RT-PCR, IGF-I and IGF-II were highly expressed on days 3 and 6, whereas IGFBP-2 was constantly expressed. We focused our study on the role of IGF-II and IGFBP-2 on the synthesis of AP, an early marker of osteoblast maturation. The results showed that a significant increase in AP expression was induced by IGF-II added to the differentiating osteoblasts continuously or in S1 but not in S2 or S3. IGFBP-2 was able to potentiate endogenous and exogenous IGF-II-dependent stimulation of AP activity, and its proteolytic degradation in late stages of osteogenesis (S2 and S3) was highly correlated with the increase of active matrix metalloproteinase-2 in the CM and with the decreased efficacy of IGF-II action. These data suggest that IGFBP-2, at nearly equimolar concentration with IGF-II, plays a potentiating role in IGF-II action on ROB differentiation in vitro.

Pro-collagen I COOH-terminal trimer induces directional migration and metalloproteinases in breast cancer cells.

Breast and prostatic carcinomas, melanoma, and endothelial cell lines are chemoattracted by medium conditioned by mature osteoblasts. The chemoattractant for endothelial cells was identified with C3, carboxyl-terminal trimer of pro-collagen type I. We report that C3 induces directional migration and proliferation, the expression of tissue inhibitor of metalloproteinases-2, pro-metalloproteinase-2 and -9, and their activation in MDA MB231 cells, without changing the expression of tissue inhibitor of metalloproteinases-1 and of metalloproteinase-14. Antiserum against metalloproteinase-2 or -9 or -14, tissue inhibitor of metalloproteinases-1, or GM6001 inhibits the C3-induced migration. Urokinase and its receptor are detected and unchanged upon exposure to C3. The antibody against urokinase or addition of plasminogen activator inhibitor inhibits migration. Blocking antibodies to integrins alpha(2), alpha(6), beta(1), and beta(3) inhibit chemotaxis and do not change urokinase and urokinase receptor expression. Blockage of alpha(2), beta(1), and beta(3) integrins affect differently the induction by C3 of pro-metalloproteinase-2 and -9 and of tissue inhibitor of metalloproteinases-2. Chemotaxis to C3 is also inhibited by genistein, by pertussis toxin, which also inhibits C3-induced pro-metalloproteinase -2 and -9, but not urokinase expression. Wortmannin partially inhibits C3-induced cell migration. Other, but not all, breast carcinoma lines tested responded to C3 with migration and pro-metalloproteinase-2 induction. Presently C3 is the only agent known to induce migration specifically of both endothelial and breast carcinoma cells. The mitogenic and motogenic role of C3 in vitro might prefigure a role in in vivo carcinogenesis and in the establishment of metastasis.