Julio Landero-Figueroa

Sex- and Tissue-Specific Methylome Changes in Brains of Mice Perinatally Exposed to Lead

Changes in DNA methylation and subsequent changes in gene expression regulation are the hallmarks of age- and tissue-dependent epigenetic drift and plasticity resulting from the combinatorial integration of genetic determinants and environmental cues. To determine whether perinatal lead exposure caused persistent DNA methylation changes in target tissues, we exposed mouse dams to 0, 3 or 30 ppm of lead acetate in drinking water for a period extending from 2 months prior to mating, through gestation, until weaning of pups at postnatal day-21, and analyzed whole-genome DNA methylation in brain cortex and hippocampus of 2-month old exposed and unexposed progeny. Lead exposure resulted in hypermethylation of three differentially methylated regions in the hippocampus of females, but not males. These regions mapped to Rn4.5s, Sfi1, and Rn45s loci in mouse chromosomes 2, 11 and 17, respectively. At a conservative fdr<0.001, 1623 additional CpG sites were differentially methylated in female hippocampus, corresponding to 117 unique genes. Sixty of these genes were tested for mRNA expression and showed a trend toward negative correlation between mRNA expression and methylation in exposed females but not males. No statistically significant methylome changes were detected in male hippocampus or in cortex of either sex. We conclude that exposure to lead during embryonic life, a time when the organism is most sensitive to environmental cues, appears to have a sex- and tissue-specific effect on DNA methylation that may produce pathological or physiological deviations from the epigenetic plasticity operative in unexposed mice.

Developing ICP-MS/MS for the detection and determination of synthetic DNA-protein crosslink models via phosphorus and sulfur detection

Various endogenous and exogenous agents drive the un-directed formation of covalent bonds between proteins and DNA. These complex molecules are of great biological relevance, as can derive in mutations, but are difficult to study because of their heterogeneous chemical properties. New analytical approaches with sufficient detection capabilities to detect and quantify these compounds can help to standardize study models based on synthesized standards. The use of atomic spectrometry can provide quantitative information on the DNA-protein cross-link reaction yield along with basic stoichiometry of the products, based on internal elemental tags, sulfur from Cys and Met amino acids, and phosphorus from the DNA. A new instrumental approach to remove isobaric and polyatomic interferences from 31P+ and 32S+ was developed recently, with state-of-the-art for interference removal that captures 31P+ in Q1; it reacts with O2 in an octopole collision-reaction cell generating 47PO+, therefore allowing detection in Q3 without 31NOH+/48Ca/47Ti interferences. Similarly, 32S+ is reacted to 48SO+, eliminating the polyatomic interferences at m/z = 32. In conjunction with the high resolving power of high-performance liquid chromatography (HPLC), this newer technology was applied by to the product purification of a DNA-protein cross link model and some preliminary structural studies.

Design of a hydrogen peroxide-activatable agent that specifically targets cancer cells.

Some cancers, like acute myeloid leukemia (AML), use reactive oxygen species to endogenously activate cell proliferation and angiogenic signaling cascades. Thus many cancers display increases in reactive oxygen like hydrogen peroxide concentrations. To translate this finding into a therapeutic strategy we designed new hydrogen peroxide-activated agents with two key molecular pharmacophores. The first pharmacophore is a peroxide-acceptor and the second is a pendant amine. The acceptor is an N-(2,5-dihydroxyphenyl)acetamide susceptible to hydrogen peroxide oxidation. We hypothesized that selectivity between AML and normal cells could be achieved by tuning the pendant amine. Synthesis and testing of fourteen compounds that differed at the pendent amine led to the identification of an agent (14) with 2μM activity against AML cancer cells and an eleven fold-lower activity in healthy CD34+ blood stem cells. Interestingly, analysis shows that upon oxidation the pendant amine cyclizes, ejecting water, with the acceptor to give a bicyclic compound capable of reacting with nucleophiles. Preliminary mechanistic investigations show that AML cells made from addition of two oncogenes (NrasG12D and MLL-AF9) increase the ROS-status, is initially an anti-oxidant as hydrogen peroxide is consumed to activate the pro-drug, and cells respond by upregulating electrophilic defense as visualized by Western blotting of KEAP1. Thus, using this chemical approach we have obtained a simple, potent, and selective ROS-activated anti-AML agent.

Identification of selenium-containing proteins in HEK 293 kidney cells using multiple chromatographies, LC–ICPMS and nano-LC–ESIMS

Our previous studies using HeLa and HEK 293 cells demonstrated that selenomethionine, SeMet, exerts more of an antagonistic effect on arsenic than other selenium species. These studies attributed the antagonistic effect of SeMet to decreased levels of reactive oxygen species, ROS, changes in protein phosphorylation and possible incorporation of SeMet into proteins. The present study employs a metallomics approach to identify the selenium-containing proteins in HEK 293 cells raised with SeMet. The proteins were screened and separated using two dimensional high performance liquid chromatography (HPLC)-inductively coupled plasma mass spectrometry (ICPMS), size exclusion chromatography (SEC) and reversed-phase chromatography (RPC). The Se-containing proteins were identified by peptide mapping using nano-HPLC-Chip-electrospray ionization mass spectrometry (ESIMS).

The metal and metalloprotein profile of human plasma as biomarkers for stroke diagnosis

Stroke, a major cause of disability and mortality, affects someone in the United States every 40s. Stroke biomarkers, including those that could be used as a blood test for diagnosis of stroke, have been particularly elusive. We performed a double blind study to identify human plasma biomarkers for the diagnosis of stroke, including acute ischemic stroke (AIS) and intracerebral hemorrhage (ICH). We utilized a three-track approach based on the total metal profile, the metal cofactor levels among metalloproteins, and the identification of stroke-related metalloproteins. The study included 14 case-control pairs of AIS and 23 case-control pairs of ICH. Controls were matched to cases based on gender, ethnicity, and age (±5 years). AIS cases were statistically higher from their respective controls for protein bound co-factors Se and Cd, while unique correlations of metal cofactor concentrations among metalloproteins were identified between Pb-W, Sr-W, Pb-V, and Cu-V. ICH cases were statistically higher from their respective controls for Se and Co cofactors, whereas Cd and Pb were statistically lower. Unique correlations between metal cofactors for ICH cases were identified between Pb-W, Sr-W, Pb-V, and Cu-V. Stroke-related metalloproteins were identified, including calpain-15, protein-activated inward rectifier potassium channel 1, tau-tubulin kinase 1, and voltage-dependent L-type calcium channel subunit beta-3. Linear discriminant analysis (LDA) was able to classify patients between stroke cases or controls with 93% accuracy as well as classify patients with one of the four stroke groups with 85% accuracy. Additionally, this study found utmost importance in vanadium (V) and tungsten (W) correlations for both bound and total metal concentrations, suggestive of binding to transferrin or inhibition of oxidoreductases. Future work in stroke patients will seek to quantify varying selenoproteins, including selenoprotein P and glutathione peroxidase and identified zinc finger tissue leakage proteins, and further explore the role of trace metal fluctuations with transferrin.

Self‐cyclizing Antioxidants to Prevent DNA Damage Caused by Hydroxyl Radical

Antioxidant therapy is a promising treatment strategy for protecting DNA from the damage caused by reactive oxygen species (ROS). Here, we report new self‐cyclizing antioxidant reagents that are selective for the hydroxyl radical. Our mechanistic investigation revealed that the reagents react with three equivalents of oxidant in a cascade reaction to form a bicyclic final product. Among the reagents synthesized, 1 c showed favorable properties in vitro and in cellular studies. Using As2O3, which triggers ROS production, we showed that 1 c prevents formation of the guanine oxidation product 2,2,4‐triamino‐2H‐oxazol‐5‐one‐2′‐deoxyribonucleoside and lowers cellular levels of reactive oxygen. The described self‐cyclizing antioxidants are efficient, flexible, and tunable reagents with the potential to limit toxic oxidative stress.

Prenatal and early postnatal lead exposure in mice: neuroimaging findings

BACKGROUND Childhood lead exposure has been linked to adult gray matter loss accompanied by changes in myelination and neurochemistry noninvasively revealed by magnetic resonance imaging (MRI) methods. However, the extent, duration and timing of lead exposure required to produce such imaging changes in humans are difficult to ascertain. METHODS To determine if such changes are related to early exposure to low levels of lead, we treated mouse dams with 0, 3, or 30 ppm of lead acetate in drinking water for 2 months prior to mating through gestation until weaning of the offspring at post-natal day 21. Two male and two female pups from each litter were imaged at post-natal day 60. Volumetric, diffusion tensor imaging and magnetic resonance spectroscopy (MRS) measurements were obtained using a seven Tesla Bruker animal MRI scanner. RESULTS Postnatal blood lead levels were identical between groups at the time of imaging. No effects of lead exposure were detected in the volumetric or MRS data. Mean diffusivity in the hippocampus showed significant effects of lead exposure and gender. CONCLUSIONS These data suggest that low-level, gestational lead exposure in a mouse model produces minimal changes observed by MRI.

A preliminary method development study to identify potential stroke biomarkers in plasma using multiple chromatographies with nanoLC-ESIMS detection

Stroke is the leading cause of disability in the USA. Rapid, reliable diagnosis via a point-of-care blood test may facilitate earlier treatment to reduce disability. We have recently reported detailed methods of chromatographic separations of plasma samples coupled with nanoESI-ion trap a list of proteins which are viable candidates for further investigation as stroke biomarkers.