Lung genotoxicity of benzo(a)pyrene in vivo involves reactivation of LINE-1 retrotransposon and early reprogramming of oncogenic regulatory networks.

Several lines of evidence have implicated long interspersed nuclear element-1 (LINE-1) retroelement in the onset and progression of lung cancer. Retrotransposition-dependent mechanisms leading to DNA mobilization give rise to insertion mutations and DNA deletions, whereas retrotransposition-independent mechanisms disrupt epithelial programming and differentiation. Previous work by our group established that tobacco carcinogens such as benzo(a)pyrene (BaP) reactivate LINE-1 in bronchial epithelial cells through displacement of nucleosome remodeling and deacetylase (NuRD) corepressor complexes and interference with retinoblastoma-regulated epigenetic signaling. Whether LINE-1 in coordination with other genes within its regulatory network contributes to the in vivo genotoxic response to BaP remains largely unknown. Evidence is presented here that intratracheal instillation of ORFeusLSL mice with BaP alone or in combination with adenovirus (adeno)-CRE recombinase is genotoxic to the lung and associated with activation of the human LINE-1 transgene present in these mice. LINE-1 reactivation modulated the expression of genes involved in oncogenic signaling, and these responses were most pronounced in female mice compared with males and synergized by adeno-CRE recombinase. This is the first report linking LINE-1 and genes within its oncogenic regulatory network with early sexually dimorphic responses of the lung in vivo.

A computerized scoring system to improve assessment of heparin-induced thrombocytopenia risk.

Essentials Current risk scores for heparin-induced thrombocytopenia (HIT) are not computer-friendly. We compared a new computerized risk score with the 4Ts score in a large healthcare system. The computerized risk score agrees with the 4Ts score 85% of the time. The new score could potentially improve HIT diagnosis via incorporation into decision support. SUMMARY: Background (HIT) is an immune-mediated adverse drug event associated with life-threatening thrombotic complications. The 4Ts score is widely used to estimate the risk for HIT and guide diagnostic testing, but it is not easily amenable to computerized clinical decision support (CDS) implementation. Objectives Our main objective was to develop an HIT computerized risk (HIT-CR) scoring system that provides platelet count surveillance for timing and degree of thrombocytopenia to identify those for whom diagnostic testing should be considered. Our secondary objective was to evaluate clinical management and subsequent outcomes in those identified as being at risk for HIT. Methods We retrospectively analyzed data from a stratified sample of 150 inpatients treated with heparin to compare the performance of the HIT-CR scoring system with that of a clinically calculated 4Ts score. We took a 4Ts score of ≥ 4 as the gold standard to determine whether HIT diagnostic testing should be performed. Results The best cutoff point of the HIT-CR score was a score of 3, which yielded 85% raw agreement with the 4Ts score and a kappa of 0.69 (95% confidence interval 0.57-0.81). Ninety per cent of patients with 4Ts score of ≥ 4 failed to undergo conventionally recommended diagnostic testing; 38% of these experienced persistent, unexplained thrombocytopenia, and 4% suffered life-threatening thrombotic complications suggestive of undiagnosed HIT. Conclusion The HIT-CR scoring system is practical for computerized CDS, agrees well with the 4Ts score, and should be prospectively evaluated for its ability to identify patients who should be tested for HIT.

Line-1: Implications in the etiology of cancer, clinical applications, and pharmacologic targets.

Long interspersed nuclear elements-1 (Line-1 or L1) accounts for approximately 17% of the human genome. The majority of L1s are inactive, but ∼100 remain retrotransposon competent (RC-L1) and able to retrotranspose through RNA intermediates to different locations of the genome. L1 is involved in both disease initiation and progression via retrotransposition dependent and independent mechanisms. Retrotransposed L1 sequences disrupt genetic loci at sites of insertion, while the activities of L1 si/piRNAs, mRNAs, and ORF1 and ORF2 proteins, and have been implicated in the etiology and progression of several human diseases. Despite these relationships, little is known about the clinical utility of L1 as a biomarker of disease initiation and progression, or the utility of small molecules to inhibit and reverse the harmful effects of L1. In this review, we discuss the life cycle of L1, somatic and germline inhibitions, the mechanisms of L1 retrotransposition dependent and independent disease initiation and progression, clinical utilities, and potential of L1s as pharmacologic targets for the treatment of cancer.

Negative regulation of the tumor suppressor p53 gene by microRNAs.

The tumor suppressor p53, encoded by the TP53 gene, is recognized as the guardian of the human genome because it regulates many downstream genes to exercise its function in cell cycle and cell death. Recent studies have revealed that several microRNAs (miRNAs) are important components of the p53 tumor suppressor network with miR-125b and miR-504 directly targeting TP53. In this study, we use a screening method to identify that two miRNAs (miR-25 and miR-30d) directly target the 3'UTR of TP53 to downregulate p53 protein levels and reduce the expression of genes that are transcriptionally activated by p53. Correspondingly, both miR-25 and miR-30d adversely affect apoptotic cell death, cell cycle arrest and cellular senescence. Inhibition of either miR-25 or miR-30d expression increases endogenous p53 expression and elevates cellular apoptosis in several cell lines, including one from multiple myeloma that has little TP53 mutations. Thus, beyond miR-125b and miR-504, the human TP53 gene is negatively regulated by two more miRNAs: miR-25 and miR-30d.

L1 retrotransposon and retinoblastoma: molecular linkages between epigenetics and cancer.

Long interspersed nuclear elements (LINEs) are mobile sequences shown to play a fundamental role in eukaryotic genome evolution. Recently, increasing interest has been directed at unveiling molecular mechanisms by which LINE-1 (L1), a ubiquitous member of this family, regulates gene expression and mammalian cell development, differentiation, and cancer. This mini review summarizes recent studies conducted to examine stress-induced L1 reactivation, with special attention given to the role of E2F/Rb transcription factors in epigenetic silencing of L1 and its potential role as a global modifier of chromatin structure and function. The last section focuses on the impact of histone deacetylase inhibitors in the regulation of gene function, chromatin structure, and cancer treatment through alterations in epigenetic signaling.

Genetic networks of cooperative redox regulation of osteopontin.

Osteopontin is a primary cytokine and matrix-associated protein involved in medial thickening and neointima formation. Osteopontin binds integrin receptors, activates cell migration and matrix metalloproteinases, and mediates arteriosclerotic lesion formation and vessel calcification. To understand the complex biology of osteopontin, computational methodology was employed to identify sets of genes whose transcriptional states were predictive of osteopontin gene expression based on the transcriptional states of 12,400 genes and ESTs across 235 independent Affymetrix Murine Genome Array MG_U74Av2 hybridizations. Arginase [GenBank: U51805] and Mac-2 antigen [GenBank: X16834] were identified as primary attractors within the gene-gene interaction network of osteopontin. Resolution of molecular interactions among these genes indicated that the majority of predictor genes could be linked through redox regulated transcription by nuclear factor kappa-B and transforming growth factor beta inducible early gene 1 regulatory elements. Subsequent molecular analyses established redox sensitivity of a 200 bp region within the 5' UTR of opn promoter and implicated nuclear factor kappa-B and transforming growth factor beta inducible early gene 1 cis-acting elements in the regulation of osteopontin.

MicroRNA-21 promotes cell transformation by targeting the programmed cell death 4 gene.

MicroRNAs (miRNAs) are small noncoding RNA molecules that negatively control expression of target genes in animals and plants. The microRNA-21 gene (mir-21) has been identified as the only miRNA commonly overexpressed in solid tumors of the lung, breast, stomach, prostate, colon, brain, head and neck, esophagus and pancreas. We initiated a screen to identify miR-21 target genes using a reporter assay and identified a potential miR-21 target in the 3'-UTR of the programmed cell death 4 (PDCD4) gene. We cloned the full-length 3'-UTR of human PDCD4 downstream of a reporter and found that mir-21 downregulated, whereas a modified antisense RNA to miR-21 upregulated reporter activity. Moreover, deletion of the putative miR-21-binding site (miRNA regulatory element, MRE) from the 3'-UTR of PDCD4, or mutations in the MRE abolished the ability of miR-21 to inhibit reporter activity, indicating that this MRE is a critical regulatory region. Western blotting showed that Pdcd4 protein levels were reduced by miR-21 in human and mouse cells, whereas quantitative real-time PCR revealed little difference at the mRNA level, suggesting translational regulation. Finally, overexpression of mir-21 in MCF-7 human breast cancer cells and mouse epidermal JB6 cells promoted soft agar colony formation by downregulating Pdcd4 protein levels. The demonstration that miR-21 promotes cell transformation supports the concept that mir-21 functions as an oncogene by a mechanism that involves translational repression of the tumor suppressor Pdcd4.

Toxicogenomic profile of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the murine fetal heart: modulation of cell cycle and extracellular matrix genes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and similar environmental contaminants have been demonstrated to be potent cardiovascular teratogens in developing piscine and avian species. In the present study, we investigated the effects of TCDD on gene expression during murine cardiovascular development. C57Bl6N pregnant mice were dosed with 1.5, 3.0, or 6.0 microg TCDD/kg on gestational day (GD) 14.5, and microarray analysis was used to characterize the global changes in fetal cardiac gene expression on GD 17.5. TCDD significantly altered expression of a number of genes involved in xenobiotic metabolism, cardiac homeostasis, extracellular matrix production/remodeling, and cell cycle regulation. Interestingly, while the AhR-responsive genes Cyp1A1, Cyp1B1, Ugt1a6, and Ahrr, were all induced by TCDD in the fetal murine heart, other AhR-responsive genes, Cyp1a2, Nqo1, and Gsta1, were not. Quantitative real-time polymerase chain reactions confirmed the changes in expression of several G1/S-type cyclins and extracellular matrix-related genes. These results demonstrate the global changes in cardiac gene expression that result from TCDD exposure of the fetal murine heart and implicate genes involved in cell cycle and extracellular matrix regulation in TCDD-induced cardiac teratogenicity and functional deficits.

Novel genomic targets in oxidant-induced vascular injury.

To study the complex interaction between oxidative injury and the pathogenesis of vascular disease, vascular gene expression was examined in male Sprague-Dawley rats given 35 or 70 mg/kg allylamine, a synthetic amine converted to acrolein and hydrogen peroxide within the vascular wall. Vascular lesions and extensive vascular remodeling, coupled to increased production of 8-epi-PGF2alpha, nuclear localization of NFkappaB, and alterations in glutathione homeostasis, were observed in animals treated with allylamine for up to 20 days. Transcriptional profiling, immunohistochemistry, and in situ hybridization showed that genes involved in adhesion and extracellular matrix (ECM) (alpha(1) integrin, collagen), cytoskeletal rearrangements (alpha-smooth muscle actin, alpha-tropomyosin), and signal transduction (NFkappaB, osteopontin, and LINE) were altered by oxidant treatment. To evaluate mechanisms of gene dysregulation, cultured aortic smooth muscle cells were challenged with allylamine or its metabolites and processed for molecular analysis. These agents increased formation of reactive oxygen species and elicited changes in gene expression similar to those observed in vivo. Oxidative stress and changes in gene expression were inhibited by N-acetyl cysteine, a precursor of glutathione. These results indicate that genes along the ECM-integrin-cytoskeletal axis, in addition to LINE, are molecular targets in oxidant-induced vascular injury.

2,3,7,8-Tetrachlorodibenzo-p-dioxin elicits aryl hydrocarbon receptor-mediated apoptosis in the avian DT40 pre-B-cell line through activation of caspases 9 and 3.

The halogenated aromatic hydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to induce immunotoxicity, but relatively little is known regarding its effects on B-lymphocytes, and on avian B-cells in particular. In this study, the avian bursal pre-B-cell line DT40 was exposed to TCDD ranging from 1 to 500 nM for 1 and 6 h. At 100 nM, TCDD caused a significant increase in the number of apoptotic cells, as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assay, and induced the expression of the chicken cytochrome P450 1A4 (CYP1A4) mRNA, a hallmark of TCDD exposure. TCDD induced transient upregulation of aryl hydrocarbon receptor (AhR) mRNA. At 100 nM, both caspase 3 and caspase 9 were transiently upregulated after 1 h, but returned to normal levels after 6 h of exposure. Challenge with TCDD after AhR blockade with resveratrol, a competitive AhR antagonist, prevented changes in caspases 3 and 9 and in the AhR message itself, suggesting that the effects of TCDD were mediated via the AhR. TCDD did not cause significant changes in the relative gene expression of caspase 8, Bcl-2 and Bcl-xL. We conclude that avian DT40 pre-B-cells exposed to TCDD are susceptible to apoptosis, likely through activation of executioner caspase 3.

Genomic profiles and predictive biological networks in oxidant-induced atherogenesis.

Atherogenic stimuli trigger complex responses in vascular smooth muscle cells (VSMCs) that culminate in activation/repression of overlapping signal transduction cascades involving oxidative stress. In the case of benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon present in tobacco smoke, the atherogenic response involves interference with redox homeostasis by oxidative intermediates of BaP metabolism. The present studies were conducted to define genomic profiles and predictive gene biological networks associated with the atherogenic response of murine (aortic) VSMCs to BaP. A combined oxidant-antioxidant treatment regimen was used to identify redox-sensitive targets during the early course of the atherogenic response. Gene expression profiles were defined using cDNA microarrays coupled to analysis of variance and several clustering methodologies. A predictor algorithm was then applied to gain insight into critical gene-gene interactions during atherogenesis. Supervised and nonsupervised analyses identified clones highly regulated by BaP, unaffected by antioxidant, and neutralized by combined chemical treatments. Lymphocyte antigen-6 complex, histocompatibility class I component factors, secreted phosphoprotein, and several interferon-inducible proteins were identified as novel redox-regulated targets of BaP. Predictor analysis confirmed these relationships and identified immune-related genes as critical molecular targets of BaP. Redox-dependent patterns of gene deregulation indicate that oxidative stress plays a prominent role during the early stages of BaP-induced atherogenesis.

Identification of albumin precursor protein, Phi AP3, and alpha-smooth muscle actin as novel components of redox sensing machinery in vascular smooth muscle cells.

Aerobic organisms are continually subjected to environmental stressors that compromise redox homeostasis and induce cellular injury. In vascular smooth muscle cells (vSMCs), the activation/repression of redox-regulated genes after environmental stress often involves protein binding to cis-acting antioxidant response elements (AREs). The present study was conducted to identify proteins that participate in redox-regulated protein binding to human c-Ha-ras and mouse glutathione S-transferase A1 AREs in vSMCs after oxidant injury. Challenge of vSMCs with 0.3 or 3 microM hydrogen peroxide, 3-methylcholanthrene, benzo[a]pyrene-7,8-diol, 3-hydroxy benzo[a]pyrene, and benzo[a]pyrene-3,6-quinone induced concentration-related increases in ARE protein binding. The profiles of ARE complex assembly were comparable, but exhibited chemical specificity. Pretreatment with 0.5 mM N-acetylcysteine inhibited activation of ARE protein binding in hydrogen peroxide-treated cells. Preparative electrophoretic mobility shift assays coupled to Western analysis identified NF-E2-related proteins 1 and 2 and JunD in complexes assembled on AREs. Polyethylenimine affinity and sequence-specific serial immobilized DNA affinity chromatography followed by N-terminal sequencing identified albumin precursor protein, phi AP3, and alpha-smooth muscle actin as members of the ARE signaling pathway. Sequence analysis of albumin protein revealed homology to the redox-regulated transcription factors Bach1 and 2, as well as cytoskeletal and molecular motor proteins. These results implicate albumin precursor protein, phi AP3, and alpha-smooth muscle actin as participants in redox sensing in vSMCs, and suggest that protein complex assembly involves interactions between leucine zipper and zinc finger transcription factors with cytoskeletal proteins.

Differential expression of ribosomal L31, Zis, gas-5 and mitochondrial mRNAs following oxidant induction of proliferative vascular smooth muscle cell phenotypes.

Treatment of cultured vascular smooth muscle cells (vSMCs) with benzo(a)pyrene (BaP), a prooxidant present in the particulate phase of tobacco smoke, induces highly proliferative (i.e. atherogenic) phenotypes. Critical early target genes in vSMCs have been identified, but patterns of gene expression following repeated cycles of carcinogen treatment in vivo have yet to be evaluated. In the present study, male Sprague-Dawley rats (175-200 g) were given weekly injections of BaP (10 mg/kg) for 8 weeks to induce atherogenic phenotypes. At the end of this atherogenic regimen, vSMCs were established in serial culture and monitored for patterns of proliferative activity and gene expression. vSMCs isolated from BaP-treated animals (hence forth referred to as BaP cells) exhibited constitutively increased growth rates, and marked enhancement of proliferation in response to serum mitogens. Differential display polymerase chain reaction (DD-PCR) and Northern blot analyses revealed that mRNAs for ribosomal protein L31 and Zis genes were suppressed, while gas-5 and mitochondrial mRNAs were overexpressed in BaP cells relative to control mRNA populations. In situ hybridization experiments in vascular tissue confirmed these alterations in vivo. This is the first report linking expression of these genes to proliferative dysregulation during the course of experimentally-induced atherogenesis.

Constitutive and inducible expression of Cyp1a1 and Cyp1b1 in vascular smooth muscle cells: role of the Ahr bHLH/PAS transcription factor.

Ahr is a ligand-activated bHLH/PAS transcription factor involved in cytochrome P450 (CYP) gene regulation and murine susceptibility to atherogenic stimuli. The present studies were conducted to examine constitutive and inducible expression of Cyp1a1 and Cyp1b1 in vascular smooth muscle cells (VSMCs) from Ahr(+/+) and Ahr(-/-) mice. Cyp1a1 mRNA was not expressed constitutively in VSMCs irrespective of Ahr phenotype. Although Cyp1a1 was inducible in Ahr(+/+) by 3 micromol/L benzo(a)pyrene, a known hydrocarbon inducer, the protein was uninducible. In contrast, Cyp1b1 mRNA and protein were expressed under constitutive and inducible conditions irrespective of Ahr phenotype or growth status. CYP-encoded aryl hydrocarbon hydroxylase activity was higher in Ahr(-/-) VSMCs under constitutive conditions and induced by benzo(a)pyrene in Ahr(+/+) and Ahr(-/-) VSMCs. CYP expression was influenced by mitogenic status, because randomly cycling cells consistently exhibited higher levels than growth-arrested counterparts. Actinomycin D (2 microgram/mL) or cycloheximide (10 micromol/L) did not inhibit constitutive or hydrocarbon-inducible aryl hydrocarbon hydroxylase activity in VSMCs. These data indicate that in murine VSMCs, expression of Cyp1al and Cyp1b1 is differentially influenced by Ahr phenotype and mitogenic status, with patterns that may dictate inherent susceptibility to atherogenic stimuli.

Dietary gamma-linolenic acid suppresses aortic smooth muscle cell proliferation and modifies atherosclerotic lesions in apolipoprotein E knockout mice.

The present study was conducted to evaluate the antiatherogenic effects of dietary gamma-linolenic acid (GLA) (primrose oil) in apolipoprotein E (apoE) genetic knockout mice. Five-wk-old male mice were fed cholesterol-free diets containing 10 g/100 g lipid as corn oil (CO) [control diet, 0 mol/100 mol GLA and (n-3) polyunsaturated fatty acids (PUFA)], primrose oil (PO, 10 mol/100 mol GLA), fish oil-CO mix [FC; 9:1 wt/wt, 0 mol/100 mol GLA and 17 mol/100 mol (n-3) PUFA] or fish oil-PO mix [FP, 1:3 wt/wt, 8 mol/100 mol GLA and 5 mol/100 mol (n-3) PUFA] for 15 wk. Subsequently, diets were supplemented with cholesterol (1.25 g/100 g) and sodium cholate (0.5 g/100 g) and fed for an additional 10 and 16 wk. Plasma cholesterol and triglyceride levels generally did not differ among groups at 20, 30 and 36 wk of age. Mice fed GLA-containing diets (PO and FP) had significantly (P < 0.05) higher liver phospholipid levels of dihomo-gamma-linolenic acid, the elongated product of GLA, relative to CO and FC groups. Consumption of GLA (PO and FP diets) significantly reduced (P < 0.05) aortic vessel wall medial layer thickness at 20 and 30 wk. A parallel GLA-dependent suppression in the number of proliferating (proliferating cell nuclear antigen positive) aortic smooth muscle cells was also observed. Diets containing either GLA or (n-3) PUFA reduced (P < 0.05) atherosclerotic lesion size in 30-wk-old mice. These results indicate that dietary GLA can suppress smooth muscle cell proliferation in vivo and retard the development of diet-induced atherosclerosis in apoE knockout mice.

Antagonistic interactions among nephrotoxic polycyclic aromatic hydrocarbons.

Although the liver and pulmonary toxicity of polycyclic aromatic hydrocarbons (PAHs) has been extensively characterized, limited data concerning the nephrotoxic potential of these chemicals are available. The present studies were conducted to define the kidney cell-specific toxic responses to anthracene (ANTH), benzo[a]pyrene (BaP), and chrysene (CHRY). Given that exposure to environmental chemicals from a specific source is rarely limited to a single compound, a second goal was to evaluate the nephrotoxic potential of binary and ternary mixtures of these chemicals. Cultured rat glomerular mesangial cells (rGMCs) and porcine cortico-tubular epithelial kidney cells (LLCPK-1) were challenged with hydrocarbon concentrations ranging from 0.03 to 30 microM for up to 24 h and were processed for measurements of mitochondrial membrane permeability, trypan blue dye exclusion, cytoplasmic enzyme leakage, and protein synthesis. BaP induced a threefold increase in mitochondrial fragility, a modest increase in cellular death, and 40% decrease in the rate of protein synthesis in rGMCs. Anthracene was also cytotoxic to rGMCs, inducing a twofold increase in mitochondrial fragility and a 40% decrease in the rate of protein synthesis, but no changes in cellular viability. Although CHRY was devoid of toxicity to rGMCs, a 40% decrease in the rate of protein synthesis was observed in LLCPK-1 cells treated with this hydrocarbon. BaP and ANTH were not overtly cytotoxic to LLCPK-1 cells at any of the concentrations tested. Binary and ternary mixtures of BaP with ANTH and CHRY in rGMCs, and mixtures of CHRY with ANTH and BaP in LLCPK-1 cells, yielded antagonistic interactions. Based on these data, it is concluded that PAHs exhibit chemical- and cell-specific nephrotoxicity, but that toxicological outcomes are influenced by the presence of multiple hydrocarbons in complex mixtures.

Environmental health training of promotoras in colonias along the Texas-Mexico border.

Poverty, overpopulation, and a lack of environmental controls have combined with cultural and linguistic division to produce a looming public health threat in unincorporated communities on the US-Mexico border. These rapidly multiplying colonias, from a Spanish term for neighborhoods, are settlements of varying size located along the border. Along the American side of the Texas-Mexico border alone, there are approximately 1800 colonias--the largest number of any border state--most of which lack basic water and sewer systems, paved roads, and safe and sanitary housing. Promotoras, from a Spanish term for lay community educators, are community leaders who live in the colonias and build important bridges between residents and the federal and state bureaucracies. These women have been trained to introduce their neighbors to state "systems" of government, education, and medical and social services that otherwise may lie out of reach. Promotoras are able to "translate" this training into culturally meaningful instruction that empowers community self-development. When neighbors teach neighbors, the message is received with greater trust and readiness to act.

Impact of cellular metabolism on the biological effects of benzo[a]pyrene and related hydrocarbons.

Polycyclic aromatic hydrocarbons are ubiquitous contaminants in the environment. Benzo[a]pyrene (BaP), a prototypical member of this class of chemicals, has been extensively studied for its toxic effects in laboratory animals and human populations. BaP toxicity is often mediated by oxidative metabolism to reactive intermediates that interact with macromolecules leading to alterations in target cell structure and function. More recent evidence suggests that disruption of cellular signaling pathways involved in the regulation of growth and differentiation contribute significantly to the toxicity of BaP and its metabolites. This review summarizes recent advances in our understanding of biological mechanisms of BaP toxicity at the molecular level, and the role of metabolic intermediates in carcinogenesis, atherogenesis, and teratogenesis.

Induction of 78 kD glucose-regulated protein (GRP78) expression and redox-regulated transcription factor activity by lead and mercury in C6 rat glioma cells.

Lead (Pb) and mercury (Hg) are widespread environmental contaminants that induce prominent neural toxicity. Although the brain is not the major Pb and Hg depot in the body, these metals preferentially accumulate in astroglia to exert toxic effects. In this study, we examined the effects of Pb acetate and HgCl(2) on the expression of GRP78, a molecular chaperone in the endoplasmic reticulum (ER) that may provide cytoprotection in response to cellular stresses in the C6 rat glioma cell line. We also evaluated the DNA binding activities of several redox-regulated transcription factors in metal-treated cells. Our results showed that mRNA levels of GRP78 were up-regulated by Pb and Hg at 0.1 and 1 micro M, but down-regulated at higher concentrations (10 micro M). GRP78 protein levels increased in a concentration- and time-dependent manner in Pb and/or Hg-treated cells. Pb increased protein binding to the GST- Upsilon a antioxidant/electrophile response element (ARE/EpRE) and to the NF- kappaB consensus binding sequence of the cytomegalovirus 2 (CMB2) promoter, but decreased protein binding to the Ha-ras ARE/EpRE or to the c-fos 12-O-tetradecanoyl-phorbol-13-acetate (TPA) response element (TRE). In contrast, Hg activated DNA binding by all redox-regulated transcription factors. These studies shed some light on the molecular mechanisms of Pb and Hg toxicity in C6 rat glioma cells and suggest that GRP78 and oxidative stress may participate in the neurotoxic response to these metals.

Benzo(a)pyrene activates L1Md retrotransposon and inhibits DNA repair in vascular smooth muscle cells.

Benzo(a)pyrene (BaP) modulates vascular smooth muscle cells (vSMCs) from a quiescent to proliferative phenotype, a shift associated with activation of L1Md retrotransposon [K.P. Lu, K.S. Ramos, Biochem. Biophys. Res. Commun. 253 (1998) 828-833]. The present studies were conducted to evaluate L1Md activation profiles in murine vSMCs treated with BaP or its oxidative metabolites, and to screen for possible insertional mutations into p53 and retinoblastoma (RB) genes. We also sought to examine the profile of DNA damage and repair in BaP-treated vSMCs. Northern analysis revealed that BaP (0. 03-3microM), and its major reactive 7,8-diol metabolite (0. 03-3microM), activate L1Md gene in a concentration-dependent manner. Two other metabolites, 3-OH BaP and 3,6-BaP quinone (0.03-3microM), as well as hydrogen peroxide (25-75microM) also activated L1Md. No insertional mutations into either p53 or RB genes were observed in vSMCs treated with BaP in vitro, although a slight elevation of p53 mRNA was observed as early as 4h after chemical challenge. Treatment of vSMCs with 3 or 30microM BaP for 4h increased unscheduled DNA synthesis (UDS) 1.4- and 2.5-fold, respectively. Challenge with 0. 3microM BaP for 24h inhibited DNA repair capacity in vSMCs for up to 48h. These results demonstrate that BaP and its oxidative metabolites activate L1Md retrotransposon in vSMCs, which coupled to DNA damage and inhibition of DNA repair are part of the atherogenic response elicited by BaP and related hydrocarbons.