Isolation and Establishment of a Highly Proliferative, Cancer Stem Cell-Like, and Naturally Immortalized Triple-Negative Breast Cancer Cell Line, KAIMRC2.

In vitro studies of a disease are key to any in vivo investigation in understanding the disease and developing new therapy regimens. Immortalized cancer cell lines are the best and easiest model for studying cancer in vitro. Here, we report the establishment of a naturally immortalized highly tumorigenic and triple-negative breast cancer cell line, KAIMRC2. This cell line is derived from a Saudi Arabian female breast cancer patient with invasive ductal carcinoma. Immunocytochemistry showed a significant ratio of the KAIMRC2 cells' expressing key breast epithelial and cancer stem cells (CSCs) markers, including CD47, CD133, CD49f, CD44, and ALDH-1A1. Gene and protein expression analysis showed overexpression of ABC transporter and AKT-PI3Kinase as well as JAK/STAT signaling pathways. In contrast, the absence of the tumor suppressor genes p53 and p73 may explain their high proliferative index. The mice model also confirmed the tumorigenic potential of the KAIMRC2 cell line, and drug tolerance studies revealed few very potent candidates. Our results confirmed an aggressive phenotype with metastatic potential and cancer stem cell-like characteristics of the KAIMR2 cell line. Furthermore, we have also presented potent small molecule inhibitors, especially Ryuvidine, that can be further developed, alone or in synergy with other potent inhibitors, to target multiple cancer-related pathways.

A Drug Repositioning Approach Identifies a Combination of Compounds as a Potential Regimen for Chronic Lymphocytic Leukemia Treatment.

Drug repositioning is a promising and powerful innovative strategy in the field of drug discovery. In this study, we screened a compound-library containing 800 Food and Drug Administration approved drugs for their anti-leukemic effect. All screening activities made use of human peripheral blood mononuclear cells (PBMCs), isolated from healthy or leukemic donors. Compounds with confirmed cytotoxicity were selected and classified in three groups: i) anti-neoplastic compounds which are drugs used in leukemia treatment, ii) compounds known to have an anti-cancer effect and iii) compounds demonstrating an anti-leukemic potential for the first time. The latter group was the most interesting from a drug repositioning perspective and yielded a single compound, namely Isoprenaline which is a non-selective ß-adrenergic agonist. Analysis of the cytotoxic effect of this drug indicated that it induces sustainable intracellular ATP depletion leading, over time, to necrotic cell death. We exploited the Isoprenaline-induced intracellular ATP depletion to sensitize primary leukemic cells to fludarabine (purine analogue) and Ibrutinib (Bruton's tyrosine kinase inhibitor) treatment. In-vitro treatment of primary leukemic cells with a combination of Isoprenaline/fludarabine or Isoprenaline/Ibrutinib showed a very high synergistic effect. These combinations could constitute a new efficient regimen for CLL treatment following successful evaluation in animal models and clinical trials.

Therapeutic Efficacy of Nyctanthes arbor-tristis Flowers to Inhibit Proliferation of Acute and Chronic Primary Human Leukemia Cells, with Adipocyte Differentiation and in Silico Analysis of Interactions between Survivin Protein and Selected Secondary Metabolites.

Although the antidiabetic efficacy of Nyctanthes arbor-tristis flowers has been reported, antiproliferative and anti-obesity activities are yet to be explored. We examined the anti-obesity and antiproliferative potentials of different fractions (hexane, chloroform, ethyl acetate, methanol) of N. abor-tristis flower extract for the first time using 3T3-L1 cells, primary peripheral blood mononuclear cells (PBMC) isolated from healthy and adult acute myeloid (AML) and chronic lymphocytic leukemia (CLL) patients, recombinant Jurkat T cells, and MCF7 cell lines. The in vitro hypoglycemic activity was evaluated using the inhibition of -amylase enzyme and glucose uptake by yeast cells. The percentage glucose uptake and -amylase inhibitory activity increased in a dose-dependent manner in the crude and the tested fractions (hexane and ethyl acetate). Inhibition of the 3T3-L1 cells' differentiation was observed in the ethyl acetate and chloroform fractions, followed by the hexane fraction. Antiproliferative analyses revealed that Nyctanthes exerted a high specific activity against anti-AML and anti-CLL PBMC cells, especially by the hexane and ethyl acetate fractions. The gas chromatography/mass spectrometry analysis indicated the presence of 1-heptacosanol (hexane fraction), 1-octadecene (hexane and chloroform fractions), and other organic compounds. Molecular docking demonstrated that phenol,2,5-bis(1,1-dimethylethyl) and 4-hydroxypyridine 1-oxide compounds showed specificity toward survivin protein, indicating the feasibility of N. abor-tristis in developing new drug leads against leukemia.

Nuclear Receptors Are Differentially Expressed and Activated in KAIMRC1 Compared to MCF7 and MDA-MB231 Breast Cancer Cells.

We recently established a KAIMRC1 cell line that has unique features compared to the known breast cancer cell lines, MCF7 and MDA-MB231. To characterize it further, we investigated the expression profile of nuclear receptors and their respective co-factors in these cell lines. We confirm that in contrast to the triple negative cell line MDA-MB231, the MCF7 and KAIMRC1 are estrogen receptor alpha (ERa) and progesterone receptor alpha (PRa) positive, with significant lower expression of these receptors in KAIMRC1. KAIMRC1 cell is a vitamin D receptor (VDR) negative and V-ErbA-Related Protein 2 (EAR2) positive in contrast to MCF7 and MDA-MB231. Remarkably, the histone deacetylases (HDACs) are highly expressed in KAIRMC1 with HDAC6 and HDAC 7 are exclusively expressed in KAIMRC1 while thyroid hormone receptor-associated protein 80 (TRAP80), telomeric DNA binding protein 1 (TBP1) and TGF-beta receptor interacting protein (TRIP1) are absent in KAIMRC1 but present in MCF7 and MDA-MB231. In a luciferase reporter assay, the ERa coexpression is needed for estrogen receptor element (ERE)-luciferase activation by estradiol in KAIMRC1 but not in MCF7. The co-expression of exogenous Liver X receptor alpha (LXRa)/retinoid X receptor alpha (RXRa) are necessary for LXR responsive element (LXRE) activation by the GW3696 in the three cell lines. However, the activity of peroxisome proliferator-activated receptor response element (PPARE)-tk-luciferase reporter increased when peroxisome proliferator-activated receptors alpha (PPARa)/RXRa were coexpressed but the addition of PPARa agonist (GW7647) did not stimulate further the reporter. The signal of the PPARE reporter increased in a dose-dependent manner with rosiglitazone (PPARg agonist) in KAIMRC1, MCF7, and MDA-MB231 when the proliferator-activated receptors gamma (PPARg)/RXRa receptors were cotransfected. Retinoic acid-induced activation of retinoic acid receptor response element (RARE)-tk-luciferase is dependent on exogenous expression of retinoic acid receptor alpha (RARa)/RXRa heterodimer in MDA-MB 231 but not in MCF7 and KAIMRC1 cell lines. In the three cell lines, Bexarotene-induced retinoid X receptor response element (RXRE)-luciferase reporter activation was induced only if the RXRa/LXRa heterodimer were co-expressed. The vitamin D receptor response element (VDRE)-luciferase reporter activity showed another distinct feature of KAIMRC1, where only co-expression of exogenous vitamin D receptor (VDR)/RXRa heterodimer was sufficient to reach the maximum rate of activation of VDRE reporter. In the proliferation assay, nuclear receptors ligands showed a distinct effect on KAIMRC1 compared to MCF7 and MDA-MB231. Growth inhibition effects of used ligands suggest that KAIMRC1 correlate more closely to MDA-MB231 than MCF7. Vitamin D3, rosiglitazone, novel RXR compound (RXRc) and PPARa compound (GW6471) have the most profound effects. In conclusion, we showed that nuclear receptors are differentially expressed, activated and also their ligand produced distinct effects in KAIMRC1 compared to MCF7 and MDA-MB231. This finding gives us confidence that KAIMRC1 has a unique biological phenotype.

P19 Cells as a Model for Studying the Circadian Clock in Stem Cells before and after Cell Differentiation.

In mammals, circadian rhythmicity is sustained via a transcriptional/translational feedback loop referred to as the canonical molecular circadian clock. Circadian rhythm is absent in undifferentiated embryonic stem cells; it begins only after differentiation. We used pluripotent P19 embryonal carcinoma stem cells to check the biological clock before and after differentiation into neurons using retinoic acid. We show that the central clock genes ARNTL (Bmal), Per2 and Per3, and the peripheral clock genes Rev-erb-α and ROR-α, oscillate before and after differentiation, as does the expression of the neuronal differentiation markers Hes5, ß-3-tubulin (Tubb3) and Stra13, but not Neurod1. Furthermore, the known clock-modulating compounds ERK, EGFR, Pi3K, p38, DNA methylation and Sirtiun inhibitors, in addition to Rev-erb-α ligands, modulate the expression of central and peripheral clock genes. Interestingly Sirtinol, Sirt1 and Sirt2 inhibitors had the greatest significant effect on the expression of clock genes, and increased Hes5 and Tubb3 expression during neuronal differentiation. Our findings reveal a new frontier of circadian clock research in stem cells: contrary to what has been published previously, we have shown the clock to be functional and to oscillate, even in undifferentiated stem cells. Modulating the expression of clock genes using small molecules could affect stem cell differentiation.

Association analysis between four vitamin D receptor gene polymorphisms and developmental dysplasia of the hip.

Developmental dysplasia of the hip (DDH) is a congenital condition characterized by abnormality in acetabulum size and/or shape. The incidence rate of DDH differs between different populations with risk factors including positive family history, breech presentation, sex, firstborn status, side of the hip, mode of delivery and oligohydramnios. It is recognized that DDH has a genetic component that exhibit autosomal dominant patterns. Many candidate genes have been studied and found to be associated with the disease; most of them are normally involved in cartilage development and joint metabolism. In this study, the association of four single-nucleotide polymorphisms (SNPs) (rs731236, rs1544410, rs7975232 and rs2228570) in the vitamin D receptor (VDR) gene was studied by a case-control analysis. The study sample involves 50 cases with confirmed DDH presentation and 50 nonDDH controls. SNPs were genotyped using conventional polymerase chain reaction (PCR) and restriction fragment-length polymorphism (RFLP) techniques. Genotype and allele frequencies were analysed using SPSS software. No significant associations were found between the VDR polymorphisms analysed and DDH. Further work need to be performed using genomewide analysis to elucidate the genetic basis of DDH.

Interaction between cadmium (Cd), selenium (Se) and oxidative stress biomarkers in healthy mothers and its impact on birth anthropometric measures.

To our knowledge, this study may be the first to examine the antagonistic role of selenium (Se) on oxidative stress induced by cadmium (Cd) and its impact on birth measures. Cd and Se levels were measured in umbilical-cord blood and the placentas of a subsample of 250 healthy mothers who participated between 2005 and 2006 in the project "Prenatal Exposure to Pollutants". The median Cd levels in cord and maternal blood and placental tissue were 0.78µg/l, 0.976µg/l and 0.037µg/g dry wt., respectively. The median levels of Se in cord serum and placental tissue were 65.68µg/l and 1.052µg/g dry wt., respectively. Se was more than 100-fold in molar excess over Cd in both cord serum and placental tissue. The median molar Cd/Se ratios in cord serum and placental tissue were 0.008 and 0.024, respectively, which were much lower than unity. This study suggests that both Cd and Se play a role in the mechanism of oxidative stress, but, the process underlying this mechanism remains unclear. Nevertheless, three biomarkers of oxidative stress had inconsistent relationships with Cd and/or Se in various matrices, perhaps due to potential untested confounders. Our results generally support an association between low in utero exposure to Cd and the anthropometric development of the fetus. Adjusted regression models indicated a negative association of cord blood Cd levels ≥0.78µg/l with Apgar 5-min scores and birth height. Maternal Cd levels ≥0.976µg/l were associated with a 5.94-fold increased risk of small-for-gestational-age births, which increased to 7.48-fold after excluding preterm births. Placenta weight decreased with increasing placental Cd levels ≥0.037µg/g dry wt. (p=0.045), an association that became stronger after excluding preterm births or adjusting for birth weight. Cord Se levels ≥65.68µg/l were positively associated with placenta weight (p=0.041) and thickness (p=0.031), an association that remained unchanged after excluding preterm births. Cord Se levels, however, were negatively associated with cephalization index, but only after excluding preterm births (p=0.017). Each birth measure was again modeled as a function of the Cd/Se ratios in cord blood and placenta tissue. Interestingly cord ratios ≥0.008 were negatively associated with Apgar-5min score (p=0.047), birth weight (p=0.034) and placenta thickness (p=0.022). After excluding preterm births, only the association with placenta thickness remained significant (p=0.021), while birth weight (p=0.053) was marginally significant. In contrast, cephalization index increased with Cd/Se ratios ≥0.008 (p=0.033), an association that became marginally significant after excluding preterm births (p=0.058). For placental Cd/Se ratios ≥0.024, only placenta weight was reduced with (p=0.037) and without (p=0.009) the inclusion of preterm births. These findings do not support an antagonistic mechanism between Cd and Se. The role of oxidative mechanisms either induced by Cd exposure or alleviated by Se on these birth anthropometric measures was examined by principal component analysis. Se did not have a clear protective role against Cd-induced adverse effects despite its substantial excess over Cd, and its role in alleviating oxidative stress by reducing malondialdehyde levels. The results may suggest that the extent of the Se beneficial effects is not governed only by its concentration but also by the chemical forms of Se that interact with various proteins. Consequently, the speciation of Se in such studies is essential for understanding and predicting Se availability for absorption.

Mercury (Hg) and oxidative stress status in healthy mothers and its effect on birth anthropometric measures.

This study was conducted to: (a) investigate the antagonistic interaction between selenium (Se) and mercury (Hg) in mothers and their newborns, (b) delineate the role of oxidative mechanisms induced by Hg exposure and (c) examine the protective effect of Se on Hg-induced oxidative stress and birth outcomes. Levels of Hg and Se were measured in umbilical cord blood and the placentas of 250 healthy mothers who participated in a study between 2006 and 2006 assessing prenatal exposure various pollutants. Levels of malondialdehyde (MDA) in cord and maternal blood and of 8-hydroxy-2-deoxyguanosine in urine were measured for assessing oxidative stress. Tail moment (TM) in the comet assay, as a biomarker of DNA damage was measured in samples of cord and maternal blood. The mean Se levels in umbilical cord blood (67.618±12.897µg/l) were lower than those reported in many countries, but none of the newborns had Se levels <20µg/l (the threshold limit of Keshan disease). More than 80% of the newborns, though, had Se levels below the 80µg/l needed for maximum glutathione peroxidase activity. Even though 18.6% of the newborns had levels of Hg ≥5.8µg/l (the reference dose of the Environmental Protection Agency), no relationship was observed with the biomarkers of oxidative stress. The mean placental Hg levels (0.056±0.075µg/g dry wt.) were higher than those reported for newborns with abnormal fetal development. Our study also documented significant placental transfer of Hg and Se to the fetus. The Hg/Se molar ratio in both cord blood and placental tissue was well below 1. The average amount of Se in both matrices was approximately 50-fold in molar excess over Hg. The molar excess of Se in the umbilical cord (0.843µmol/l), however, was lower than in placental tissues (13.098µmol/kg dry wt.). In further support of the relationships of Hg and Se on oxidative stress, we observed significantly lower levels of maternal MDA associated with Se levels in both cord blood and placental tissues and significantly higher TM levels associated with placental Hg in both newborns and their mothers. In contrast, Se/Hg molar ratios in placental tissues were positively associated with MDA and negatively with TM. The disproportion between Hg and Se might be influenced by the length of Hg exposure that in turn might affect Se bioavailability. Each birth anthropometric outcome was modeled as a function of Hg, Se and their interactions. After an adjustment for confounding variables, Hg in cord blood had a significantly positive rather than the expected negative association with crown-heel length. Placental Hg was associated with reduced birth height. Both associations were independent of prematurity. The status of Se in newborns was positively associated with crown-heel length and placental weight, with and without preterm births, and with birth weight, but only without preterm births. In contrast, a lower cephalization index was correlated with Se levels in cord blood, which may be an indicator of a detrimental effect on health. Our study, however, revealed associations between significantly lower levels of placental Se and several birth anthropometric measures (head circumference, birth weight and birth height) but the significance disappeared after excluding preterm births. Regression analyses generally indicated either significant or marginally significant Hg-Se antagonistic interactions that may have moderated the toxic effect of Hg on head circumference and birth weight. This finding may be due to chance or residual confounding and so may not be clinically relevant, but it may also suggest that Hg, Se and Hg-Se interactions are important factors for understanding Hg-induced adverse birth outcomes. Additional research will be necessary to evaluate the biological impact of combined metals in the assessment of fetal growth and development.

Birth outcome measures and maternal exposure to heavy metals (lead, cadmium and mercury) in Saudi Arabian population.

This cross-sectional study was conducted to assess the association between exposure to heavy metals (lead, cadmium and mercury) during pregnancy and birth outcomes in 1578 women aged 16-50 years who delivered in Al-Kharj hospital, Saudi Arabia, in 2005 and 2006. The levels of lead, cadmium and mercury were measured in umbilical cord blood, maternal blood and the placenta. Outcome variables were anthropometric measures taken at birth, along with the risk of being small-for-gestational age (SGA). We selected the 10th percentile as the cutoff for dichotomizing measures of birth outcome. Cadmium, despite its partial passage through the placenta had the most prominent effect on several measures of birth outcome. After adjustment for potential confounders, logistic regression models revealed that crown-heel length (p=0.034), the Apgar 5-minute score (p=0.004), birth weight (p=0.015) and SGA (p=0.049) were influenced by cadmium in the umbilical cord blood. Significant decreases in crown-heel length (p=0.007) and placental thickness (p=0.022) were seen with higher levels of cadmium in maternal blood. As placental cadmium increased, cord length increased (p=0.012) and placental thickness decreased (p=0.032). Only lead levels in maternal blood influenced placental thickness (p=0.011). Mercury in both umbilical cord and maternal blood was marginally associated with placental thickness and placental weight, respectively. Conversely, placental mercury levels significantly influenced head circumference (p=0.017), the Apgar 5-minute score (p=0.01) and cord length (p=0.026). The predictions of these models were further assessed with the area under the curve (AUC) of the receiver operating curves (ROCs), which were modest (larger than 0.5 and smaller than 0.7). The independence of gestational age or preterm births on the observed effect of metals on some measures of birth outcome, suggested detrimental effects of exposure on fetal development. The magnitude of the estimated effects might not necessarily be of clinical significance for infants but may have a considerable public-health relevance given the high prevalence of exposure to heavy metals. Further research should be conducted to confirm these findings and to evaluate their long-term risks, if any.

Polycyclic aromatic hydrocarbons (PAHs) as determinants of various anthropometric measures of birth outcome.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that are known to induce oxidative stress. There have been several reports about the link between PAH exposure and complications in pregnancy. This cross-sectional study was conducted to: (1) measure the levels of benzo(a)anthracene (BaA), chrysene (Ch), benzo(b)fluoranthene (BbF), benzo(a)pyrene (BaP), and dibenzo(a,h)anthracene (DBahA) in placentas and maternal and -umbilical cord blood obtained at delivery from 1578 women between June 2005 and 2006 in the area of Al-Kharj, Saudi Arabia; (2) assess their influence on various anthropometric measures of birth outcome taking into consideration the carcinogenic properties of these PAHs; and (3) determine the degree of PAH-related oxidative DNA damage and birth outcome. Among the five tested PAHs, only BaP was carcinogenic; therefore, the levels of the other four probable or possible carcinogenic PAHs (BaA, Ch, BaF, and DBahA) were summed as ∑4-PAHs. Levels of 1-hydroxypyrene (1-HP) were determined in maternal urine samples as a biomarker of PAH internal dose. Urinary cotinine (COT) was measured as an index of smoking. The following markers of oxidative stress were selected: malondialdehyde (MDA) in cord (C-MDA) and maternal (M-MDA) serum and 8-hydroxy-2-deoxyguanosine (8-OHdG) in maternal urine. None of the tested PAHs was found in maternal or cord blood. However, all five PAH compounds were detected in placentas; Ch was the highest (6.582 µg/kg dry wt.), and BaA was the lowest (0.236 µg/kg dry wt.). The mean concentration of urinary 1-HP found in this study was 0.216 ± 0.856 µg/g Cr. After adjusting for gestational age and other confounding variables, regression models revealed an inverse relationship between placental weight, cord length and placental BaP. A similar trend was observed between cord length and ∑4-PAHs in placental tissues. Urinary 1-HP, though, cannot be used as an unequivocal biomarker of PAH exposure, but it can be an appropriate indicator of exposure to environmental tobacco smoke (ETS). The data demonstrate that ETS exposure (as measured by urinary COT) may adversely affect birth outcome as shown by reduced head circumference, birth weight, and birth length, as well as increased cephalization index. The positive relationship between 8-OHdG levels and 1-HP in urine provides evidence of an oxidative stress mechanism. Although this study provides no direct evidence of an association between PAH exposure and DNA damage, increased oxidative stress in the form of lipid peroxidation significantly affected various birth measures. Therefore, there is a need for studies regarding PAH exposure and its associated biological effects to determine the extent of potential fetal damage as well as possible long-term effects, such as cancer.

Heavy metals (lead, cadmium and mercury) in maternal, cord blood and placenta of healthy women.

Lead, cadmium and mercury were measured in placental tissue, umbilical cord and maternal blood samples of 1578 women who delivered at the Al-Kharj King Khalid Hospital between 2005 and 2006. The aim of this study was to evaluate the status of heavy metal exposure in mothers and their newborns and to identify predictors of maternal exposure. Lead was detected in all cord and maternal blood and in 96% of placental tissues. Only in 0.89% and 0.83% of cord and maternal blood samples were the levels of lead above the CDC threshold limit of 10 µg/dl. Maternal blood lead was also higher (2.3%) than the German Reference value in women of 7 µg/dl. Approximately 9.3% of women had a placental lead above the 95th percentile in the range of 0.83-78 µg/g dry wt., a level of possible developmental toxicity. Cadmium was detected in 94.8% and 97.9% of cord and maternal blood samples respectively, though only five newborns had a cadmium level above the OSHA threshold limit of 5 µg/l. Comparing our results to the newly revised German Reference value for nonsmokers, 48.6% of mothers had blood cadmium levels >1.0 µg/l. We found as well that 25% of women had placental cadmium in the >75th percentile, in the range of 0.048-4.36 µg/g dry wt., which is likely to affect fetal growth and development. Of the maternal and cord blood samples, 11.2% and 13%, respectively, had mercury levels >5.8 µg/l, which is the EPA reference dose. Nearly 49% of women had mercury levels >2.0 µg/l, the German Reference value for those who consume fish ≤3 times a month. Around 50% of the mothers had placental mercury in the range of 0.031-13.0 µg/g dry wt. Regression analyses indicated that the levels of metals in the blood and placenta were influenced by several factors. This study provides informative baseline biomonitoring data and reveals a substantial exposure to heavy metals in non-occupationally exposed Saudi mothers and their newborns that might jeopardize the health of both. Additional research is also urgently needed to explore factors such as environment, diet, lifestyle and/or cultural habits contributing to maternal and fetal exposures. Preventive measures to eliminate or minimize the unnecessary risk of fetus exposure to heavy metals or other pollutants during pregnancy should be initiated once these factors are identified.

Outcome of in-vitro fertilization treatment and DDT levels in serum and follicular fluid.

BACKGROUND: Although p,p'-dichlorodiphenyltrichloroethane (DDT) is banned for agricultural purpose in Saudi Arabia, it is occasionally used to control vector-borne diseases in certain regions of the country. MATERIAL/METHODS: A case-control study was designed to investigate the possible effects of DDT and its metabolites on pregnancy and fertilization rate outcome. The study population was composed of 619 Saudi women (age 19-50 years) who sought in-vitro fertilization (IVF) treatment between 2002 and 2003. RESULTS: p,p'-DDE, the main metabolite of DDT, was the most frequently detected residue in serum or follicular fluid, with mean values of 1.646 microg/L and 0.407 microg/L, respectively. After controlling for many potential confounding variables, multiple logistic regression analysis revealed no association between pregnancy outcome or fertilization rate and p,p'-DDE levels in serum or follicular fluid. CONCLUSIONS: The inability to identify an effect may be related to the comparatively low concentrations of DDE in our population. But because p,p'-DDE was detected in the serum of 77.7% our participants, it should be considered as a matter of public heath concern. Currently there is no active source of DDT in our region; therefore, further studies are needed to identify sources in order to develop preventive measures because we can not exclude its potential reproductive toxicity.

Prenatal and postnatal lead exposure and early cognitive development: longitudinal study in Saudi Arabia.

The goal of this study was to assess the effect of prenatal and postnatal lead exposure on early cognitive development of infants using the Bayley Scale of Infant Development (BSID-I) at the age of 6, 12, 18, and 24 months in a longitudinal study. Based on the results of 653 cord blood lead levels, infants were classified into three groups for neuropsychological assessments: low lead risk (<10th percentile) and high lead risk (>10th percentile) of the distribution of cord blood lead level were designated as low (< or = 1.045 microg/dL) and high (> or = 3.466 microg/dL) lead risk groups. Blood lead levels in between the <10th and >90th percentile were designated as mid lead risk group. A total of 66 infants were supposed to be selected from each group for a follow-up study. Of these, only 106 participated 6 months after the study. During the follow-up study, the dropout was very high with attrition rates of 74.5%, 52.8%, and 39.6% during the 12, 18, and 24 months. Mean blood lead levels increased from 3.36 to 4.45 microg/dL between the ages of 6 and 24 months, but the standardized Mental Development Index (MDI) and Psychomotor Development Index (PDI) decreased from 99.26 and 98.13 (6 months old) to 93.29 and 82.52, respectively (24 months old). Due to the high rate of attrition, most of the infants in the low group were lost. Therefore, we used the 75th percentile of blood lead levels as a cutoff in the statistical analyses. After adjustment for a large number of confounding variables, prenatal lead exposure was found to be significantly associated with the standardized MDI and PDI scores at the age of 6 months old with a P value of 0.02 for both. A borderline significant effect of prenatal lead exposure was also seen on standardized PDI scores at the age of 24 months (P = 0.09). On the other hand, no relationship was seen between postnatal blood lead levels and concurrent cognitive development scores. Such an observation is not conclusive because of low statistical power due to small sample size. Our results provide additional evidence for low prenatal lead exposure effects on cognitive development in Saudi infants living in a rural area.

Role of Nigella sativa and a number of its antioxidant constituents towards azoxymethane-induced genotoxic effects and colon cancer in rats.

This study examined the chemopreventive effect of Nigella sativa and some of its antioxidant constituents on a number of colon cancer biomarkers in rats induced with azoxymethane (AOM). Sixty male Sprague-Dawley rats were randomly assigned into ten subgroups: vehicle (1-5) and experimental (6-10). The rats in each group were fed one of the following diets: basal diet, (200 mg/kg) Nigella sativa, (0.2 mg/kg) selenium, (1.2 mg/kg) all-trans-retinol plus (100 mg/kg) DL-alpha-tocopherol and (10 mg/kg) thymoquinone, respectively. Only rats in subgroups 6-10 were injected with AOM (15 mg/kg) once per week for 2 weeks. Both groups were fed their respective diets for 5 weeks. Then they were killed and examined for colonic aberrant crypt foci (ACF). Our result showed that only vitamin supplementation was effective on ACF. Nigella sativa revealed inhibitory effects only on DNA damage (day 34) in the AOM-treated rat group. Alternatively, selenium, thymoquinone and vitamins inhibited the MDA content in the liver. Although the exact mechanisms involved in the protective role of Nigella sativa against the initiation of colon carcinogenesis are not clearly understood, the results suggest that its inhibitory effects might depend on the combined competitive inhibition of various antioxidant constituents of this plant.

Longitudinal study of prenatal and postnatal lead exposure and early cognitive development in Al-Kharj, Saudi Arabia: a preliminary results of cord blood lead levels.

Extensive data shows a direct link between low-level lead exposure during early development and deficits in neurobehavioral-cognitive performance evident late in childhood through adolescence. Our previous studies confirmed the transfer of lead from the mother to the fetus as well as the effect of low lead exposure on neuropsychological behavior in school children. Such results led us to design a longitudinal survey to evaluate the effect of prenatal and/or postnatal lead exposure on early cognitive development among selected group of children from birth to 2 years of age. During the first stage of this study (between March and July 2004), we measured lead levels in 653 umbilical cord blood samples taken from healthy Saudi mothers delivering at King Khalid Hospital, Al-Kharj. This gave a good opportunity to look at the prevalence of increased blood lead levels in umbilical cord blood and to identify risk factors for prenatal lead exposure. The mean cord lead levels were 2.21 +/- 1.691 microg/dl in the range of 0.284-17.276 microg/dl. Only 1.23% of the newborns had blood lead levels >10 microg/dl, the Center for Disease Control level of concern. To investigate risk factors affecting cord blood lead levels, only subjects with lead levels above the 75th percentile (2.475 microg/dl) were selected in the multiple regression models. We observed that cord blood lead levels were significantly influenced by maternal age, the location of residence and intake of prenatal supplements. Controlling for newborn's head circumferences confounders, it was found that cord blood lead levels were significantly and negatively associated with newborn's head circumference (beta = -0.158, p = 0.036). The negative association was seen between intake of prenatal supplements and cord blood lead levels emphasizing the role of prenatal supplementations during pregnancy. The significant reductions in newborns, head circumferences due to lead exposure may have serious implications for their future performance and achievement. This study reveals that even at low prenatal lead exposure, all possible measures to inspect lead sources in our environment and reduce lead exposure should be taken.

Exposure to heavy metals (lead, cadmium and mercury) and its effect on the outcome of in-vitro fertilization treatment.

We investigated the effect of lead, cadmium and mercury exposure on pregnancy and fertilization rate outcome among 619 Saudi women (age 19-50 years) who sought in-vitro fertilization (IVF) treatment between 2002 and 2003. The concentrations of lead, cadmium and mercury were measured in both blood and follicular fluids. At levels well below the current US occupational exposure limit guidelines (40microg/dL) and even less than the current Centers for Disease Control and Prevention level of concern for preventing lead poisoning in children (10microg/dL), blood lead level was negatively associated with fertilization outcome in both adjusted and unadjusted logistic regression models. We found that among various demographic, socioeconomic and environmental factors, fish consumption was positively associated with blood lead levels. These results support the hypothesis that a raised blood lead level affects infertility and intervention to reduce the lead exposure might be needed for women of reproductive age. The present results also revealed unexpected finding - the positive relationship between follicular cadmium levels and fertilization outcome, which points to the necessity for further investigation. Though adverse effect of mercury on pregnancy outcome or fertilization rate was not evident in this study, mercury5.8microg/L (EPA safety limit) was found in the blood and follicular fluid of 18.7% and 8.3% of the women, respectively. Concerns about its possible adverse effects on the physiology of reproduction or fetal development cannot be ruled out. It should be noted that skin-lightening creams and dental amalgam were important contributors to mercury exposure. Such finding is alarming and priority for further studies are, urgently, needed.

Cadmium and mercury levels in Saudi women and its possible relationship with hypertension.

The association between elevated blood pressure and blood cadmium and mercury levels was examined (2001-2002) in 185 Saudi women previously selected for a case-control study of lead and hypertension risk. Blood pressure was measured twice according to the World Health Organization recommendations. Cadmium and mercury were determined with graphite furnace and hydride system-atomic absorption spectrometry, respectively. Mean blood cadmium concentrations were 0.874 +/- 0.995 microg/L in hypertensive and 0.785 +/- 0.665 microg/L in controls. While blood mercury concentrations for hypertensives and controls were 3.506 +/- 3.617 microg/L and 3.687 +/- 3.186 microg/L, respectively. Participants were classified according to the median of blood cadmium and mercury levels. After adjustment for potentially confounding variables, the final logistic regression analyses revealed that women with blood cadmium > or = 0.627 microg/L were 3.934 times were more likely to be hypertensive than those with blood cadmium levels < 0.627 microg/L, although this was marginally significant (p = 0.098). This was likely the result of the small number of subjects, resulting in the weak power to detect a strong significant difference between hypertensives and control cases. On the other hand, the final regression model showed no association between hypertension and mercury. However, this finding should not be conclusive because of the inappropriate choice of the biomarker indicator. Nevertheless, our study supports the hypothesis that exposure to cadmium might increase the risk of hypertension.

Heavy metal concentrations in the breast milk of Saudi women.

Lead, cadmium, and mercury concentrations were determined in breast milk of Saudi lactating mothers from Riyadh and Al-Ehssa regions in Saudi Arabia who were not occupationally exposed. The mean levels for cadmium, lead, and mercury were 1.732 microg/L, 31.671 microg/L, and 3.100 microg/L, respectively. In contrast to mercury, mothers living in the Al-Ehssa region had significantly higher cadmium and lead concentrations in their breast milk than those in the Riyadh region. The estimated weekly intakes of cadmium, lead, and mercury of breast-fed infants in this study were in some cases higher than the Provisional Tolerance Weekly Intake (PTWI) recommended by FAO/WHO, which pose a threat to their health. This necessitates the urgent need to undertake a comprehensive study to determine the sources of exposure to these heavy metals. Breast-feeding is of great beneficial value for the infant's development; therefore, efforts should be made to prevent its contamination with environmental pollutants.