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.

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.

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.

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.

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.

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.

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.

Profiles of antioxidant/electrophile response element (ARE/EpRE) nuclear protein binding and c-Ha-ras transactivation in vascular smooth muscle cells treated with oxidative metabolites of benzo[a]pyrene.

Activation of nuclear protein binding to the antioxidant/electrophile response element (ARE/EpRE) by benzo[a]pyrene (BaP) in vascular smooth muscle cells (vSMCs) is associated with transcriptional deregulation of c-Ha-ras. This response may be mediated by oxidative intermediates of BaP generated during the course of cellular metabolism. To test this hypothesis, the profile of ARE/EpRE protein binding and transactivation elicited by BaP was compared with that of 3-hydroxy BaP (3-OH BaP) (0.03 to 3.0 microM), BaP 7,8-dihydrodiol (BaP 7,8-diol) (0.03 to 3.0 microM), BaP 3,6-quinone (BaP 3,6-Q) (0.0003 to 3.0 microM), and H(2)O(2) (25 to 100 microM). Specific protein binding to the consensus c-Ha-ras ARE/EpRE was observed in vSMCs treated with all BaP metabolites at concentrations considerably lower than those required for the parent compound. H(2)O(2), a by-product of BaP 3,6-Q redox cycling, also increased binding to the ARE/EpRE. Treatment of vSMCs with oxidative BaP metabolites or H(2)O(2) transactivated the c-Ha-ras promoter in all instances, but the response was consistently half of the maximal induction elicited by BaP. Similar proteins cross-linked specifically to the consensus c-Ha-ras ARE/EpRE sequence in cells treated with BaP or its oxidative intermediates. The protein binding profile in the c-Ha-ras promoter was similar to that in the NADPH:quinone reductase gene (NQO(1)) and the glutathione S-transferase Ya gene (GSTYa) promoters, but the relative abundance of individual complexes was promoter-specific. We conclude that oxidative intermediates of BaP mediate activation of nuclear protein binding to ARE/EpRE and contribute to transcriptional de-regulation of c-Ha-ras in vSMCs.

Atypical cytochrome P450 induction profiles in glomerular mesangial cells at the mRNA and enzyme level. Evidence for CYP1A1 and CYP1B1 expression and their involvement in benzo[a]pyrene metabolism.

Recent studies in this laboratory have shown that benzo[a]pyrene (BaP) modulates growth factor-related gene expression and proliferation of renal glomerular mesangial cells (GMCs) in vitro. Because many of the toxic and biochemical effects of this polycyclic aromatic hydrocarbon are mediated through oxidative metabolism, the present studies were conducted to examine the patterns of cytochrome P450IA1 (CYP1A1) and P4501B1 (CYP1B1) inducibility in mesangial cells and the molecular consequences of this response. Exposure of cultured GMCs to BaP (30 microM) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 10 nM) for 24 hr induced CYP1A1 mRNA levels, a response abolished by cotreatment with 10 microM cycloheximide. The pattern of hydrocarbon inducibility was atypical in that BaP was a more effective inducer of CYP1A1 gene expression than TCDD, and both hydrocarbons induced aryl hydrocarbon hydroxylase (AHH) activity, but not ethoxyresorufin-O-deethylase activity. Cotreatment with alpha-naphthoflavone (alpha NF, 1 microM) or ellipticine (ELLIP, 0.1 nM) only partially inhibited the induction of AHH activity by BaP (30 microM). BaP and TCDD also induced expression of the CYP1B1 protein and the pattern of induction was comparable to that observed for CYP1A1. Treatment of GMCs with 30 microM BaP was associated with the formation of eight DNA adducts, and their occurrence could be inhibited by pretreatment with alpha NF (1 microM), but not ELLIP (0.1 nM). These results demonstrate that CYP1A1 and CYP1B1-related activities are induced in GMCs by BaP and TCDD and this induction is associated with metabolism of BaP to reactive intermediates that bind covalently to DNA.

Analysis of benzo[a]pyrene partitioning and cellular homeostasis in a rat liver cell line.

The uptake and subcellular partitioning of benzo[a]pyrene (BaP) were examined in a rat-liver cell line (Clone 9) using confocal and multiphoton microscopy. Following a 16-h treatment, intracellular accumulation of BaP increased with increasing concentration, and cytoplasmic BaP fluorescence reached saturation at 10 microM. Analysis of the kinetics of BaP uptake at this concentration indicated that BaP is rapidly partitioned into all cytoplasmic membranes within several min, although saturation was not reached until 4 h. Based upon the rapid uptake of BaP into membranes, the chronology of changes in gap junction-mediated intercellular communication (GJIC), plasma membrane potential (PMP), and steady state levels of intracellular Ca2+ in relation to the time-course for induction of microsomal ethoxyresorufin-0-deethylase (EROD) activity were examined. EROD activity in Clone 9 cells treated for 16 h increased with increasing concentrations of BaP and reached the highest levels at 40 microM BaP. In addition, kinetic analysis of EROD activity in Clone 9 cells treated with 10 microM BaP indicated that significant induction of EROD activity was not detected before 3 h, and it reached maximal levels by 16 h of treatment at this concentration. Both GJIC and PMP were directly affected by the partitioning of BaP into cellular membranes. The most sensitive index of BaP-induced changes in membrane function was GJIC which revealed a 25% suppression in cells exposed to 0.4 microM BaP for 16 h. Kinetic analysis revealed that suppression of GJIC occurred within 15 min of exposure of cells to 10 microM BaP, whereas significant suppression of PMP was not detected prior to 30-min exposure at this concentration. Elevation of basal Ca2+ level was also detected simultaneously with PMP at this dose. These data suggest that early changes in cellular membrane functions occur prior to detectable induction of EROD activity, although basal metabolic activation of BaP may contribute to these changes.

Cell cycle related inhibition of mouse vascular smooth muscle cell proliferation by prostaglandin E1: relationship between prostaglandin E1 and intracellular cAMP levels.

Elucidation of the cellular and molecular mechanisms which regulate vascular smooth muscle cell proliferation is critical to the understanding of atherogenesis. The present studies were conducted to evaluate the relationship between prostaglandin E1 (PGE1) and cAMP in the regulation of DNA synthesis in mouse vascular smooth muscle cells (SMCs). Quiescent cultures of SMCs were challenged with 10% fetal bovine serum to initiate cell cycle transit and PGE1 (10 microM) or dibutyryl cAMP (1, 10, 100 microM) added at 0, 8, 16, 24, and 32 h. DNA synthesis as measured by [3H] thymidine incorporation and intracellular cAMP levels were measured 24 h following individual treatments. PGE1 modulated DNA synthesis in a cell cycle related fashion, with inhibition only observed in cells challenged 16 h or longer following initiation of cell cycle transit. The decrease in DNA synthesis induced by PGE1 was associated with increased intracellular cAMP levels at 16 and 24 h, but not 32 h. Exposure of SMCs to dibutyryl-cAMP also inhibited DNA synthesis in a cell cycle related fashion, with the most pronounced effect seen at 16 h. These results demonstrate that the effects of PGE1 are restricted to a defined period within the cell cycle following S phase entry and implicate modulation of intracellular cAMP levels in the inhibitory response.

Proliferative responses of quail aortic smooth muscle cells to benzo[a]pyrene: implications in PAH-induced atherogenesis.

Repeated exposure of avian and rodent species to benzo(a)pyrene (BaP) has been associated with the development of aortic lesions of atherosclerotic etiology. Because the occurrence of these lesions may involve alterations in the regulation of smooth muscle cell (SMC) growth, the present studies were conducted to evaluate the proliferative responses of quail aortic SMCs to BaP treatment in vivo and in vitro. Measurements of [3H]thymidine incorporation and cell growth were conducted in cultured aortic SMCs isolated from male Japanese quail treated with 10 mg/kg BaP or vehicle weekly for 10 weeks or in naive aortic SMCs exposed in vitro to BaP (0.003-30 microM). Inhibition of DNA synthesis was observed in primary and early passage cultures of aortic SMCs isolated from BaP-treated quail relative to controls. Continued propagation of these cultures yielded a population of BaP cells which proliferated at faster rates than controls. The proliferative phenotype induced by BaP was first observed after the tenth passage and preserved in all subsequent passages tested. In vitro growth of SMCs from BaP-treated animals was serum- and anchorage-dependent. A 24-h exposure of cycling SMC cultures to BaP (0.003-30 microM) was associated with a dose-dependent decrease in DNA synthesis and significant delay in the progression of SMCs through the cell cycle. A time-course study revealed that maximal inhibition of DNA synthesis occurred 10 h after addition of 3 microM BaP to cycling cultures of SMCs. As seen in SMCs isolated from BaP-treated quail, serial subculture of SMCs exposed to 0.3 microM BaP in vitro for 24 h yielded a fast-growing population of cells. In these cultures, expression of the proliferative phenotype was observed after the fifth passage. These data suggest that BaP induces the expression of a proliferative phenotype in aortic SMCs characterized by enhanced serum responsiveness. This phenotypic modulation may contribute to the initiation and/or progression of vascular lesions of atherosclerotic etiology induced by BaP.

Inhibition of DNA synthesis in primary cultures of adult rat hepatocytes by benzo[a]pyrene and related aromatic hydrocarbons: role of Ah receptor-dependent events.

Studies were conducted to examine the effects of benzo[a]pyrene (BaP) and related-aromatic hydrocarbons (AHs) on the DNA synthetic profiles of adult rat hepatocytes in primary culture. Scheduled DNA synthesis in control cultures peaked at 64 h and was negligible by 72 h after initial seeding of freshly isolated hepatocytes. A concentration-dependent inhibition of DNA synthesis was observed in 1-day old hepatocyte cultures treated with BaP (0.3-30 microM) for up to 28 h. Comparable inhibitory responses were observed in cultures treated for 24 h with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 0.01-1 nM) or 2,3,7,8-tetrachlorodibenzofuran (TCDF, 0.01-1 nM), but not in cultures treated with perylene (0.01-100 nM) or benzo[e])pyrene (1-1000 nM). Ethoxyresorufin-O-deethylase (EROD) activity was highly inducible in hepatocytes challenged for 24 h with BaP (0.3-3 microM) or TCDD (0.1-100 nM) with peak induction observed at 12 or 36 h after chemical challenge, respectively. To determine if DNA synthesis inhibition by these agents involved aryl hydrocarbon receptor (AhR)-related events, subsequent experiments were conducted to examine the interactions of alpha-naphthoflavone (alpha-NF) and ellipticine (ET) with BaP and TCDD in this cell system. Pretreatment with alpha-NF (10 nM) for 24 h prevented the inhibitory effects of both BaP (3 microM) and TCDD (1 nM), while ET (0.01 nM) pretreatment selectively antagonized the effects of BaP (3 microM). Pretreatment of hepatocytes with TCDD or TCDF (1 nM) for 24 h before the onset of DNA synthesis followed by challenge with BaP (3 microM) partially antagonized the inhibitory response to BaP. These data implicate AhR-related signal transduction in the inhibition of hepatocyte DNA synthesis by BaP and related AHs and suggest that in the case of BaP, metabolism by cytochrome P450 to toxic intermediates contributes to the inhibitory response.

Growth-related signaling as a target of toxic insult in vascular smooth muscle cells: implications in atherogenesis.

Aberrant smooth muscle cell proliferation is a focal point in the genesis and progression of atherosclerosis. To date, limited information is available on the molecular and cellular basis of the atherogenic response and the potential contribution of environmental chemicals to the overall process. This review highlights major findings in this laboratory on the mechanism(s) responsible for the acquisition of a proliferative phenotype in vascular smooth muscle cells following repeated cycles of treatment with allylamine and benzo(a)pyrene, known atherogenic chemicals. These agents share the ability to induce and promote aberrant proliferative behavior in smooth muscle cells, but appear to interfere with distinct molecular targets.

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 inhibits protein kinase C activity in subcultured rat aortic smooth muscle cells.

Recent studies in this laboratory have shown that benzo[a]pyrene (BaP) interferes with protein kinase C (PKC)-mediated phosphorylation of aortic smooth muscle cell (SMC) proteins in vivo. To evaluate the biochemical basis of this response, the present studies have been conducted to examine the time- and concentration-dependent effects of BaP on PKC activity in vitro. Growth-arrested subcultures of rat aortic SMCs were exposed to 0.3, 3, or 30 microM BaP in the presence of fetal bovine serum for various times and then processed for measurements of exogenous histone Type III-S phosphorylation under PKC-activating conditions. Challenge of SMCs with BaP for 8 h was associated with a concentration-dependent inhibition of PKC activity in both cytosolic and particulate fractions. While no changes of enzymatic activity were observed in either fraction following exposure of SMCs to 0.3 microM BaP, higher concentrations of BaP inhibited PKC in both cytosolic and particulate fractions. A 49% and 68% reduction of cytosolic PKC activity was observed in SMCs treated with 3 and 30 microM BaP, respectively. The inhibitory response elicited by BaP was more pronounced in the particulate fraction where 61% and 89% decreases in PKC activity were observed in cultures treated with 3 and 30 microM BaP, respectively. Time course studies revealed that inhibition of PKC activity by 30 microM BaP occurred as early as 30 min and was sustained for up to 24 h in both fractions. Benzo[a]pyrene (30 microM) did not interfere with the ability of phorbol-12-myristate-13-acetate to induce PKC translocation from the cytosolic to particulate compartment since maximal translocation occurred by 5 min and lasted for up to 60 min in both control and BaP-treated cultures. The inhibitory effects of BaP were independent of new protein or RNA synthesis, but appear to involve oxidative metabolism of the parent compound since 3-hydroxy-BaP, the major P450-derived BaP metabolite in SMCs, also inhibited cytosolic and particulate PKC activity. Collectively, these data demonstrate that BaP and its 3-hydroxy metabolite inhibit PKC activity in rat aortic SMCs and raise the possibility that interference with PKC-mediated protein phosphorylation participates in the deregulation of SMC growth and differentiation induced by BaP.

Differential phospholipid metabolism in rat aortic smooth muscle cells of varying proliferative potential upon long term exposure to phorbol 12-myristate 13-acetate.

Subchronic exposure of rats to allylamine (AAM) modulates aortic smooth muscle cells (SMCs) from a quiescent to a proliferative phenotype. This response is associated with alterations in phospholipid metabolism and protein kinase C (PKC) activity. The present studies were conducted to evaluate the effects of long-term exposure to phorbol 12-myristate 13-acetate (PMA) on phospholipid metabolism in SMCs derived from control and AAM-treated animals, cells of varying proliferative potential. Measurements of 32P/[3H]myristic acid incorporation into parent phospholipids and phosphatidic acid (PA) and the extent of PKC-mediated histone phosphorylation were conducted following exposure of pre- and postconfluent subcultures of SMCs to PMA for 3 h. Increased 32P incorporation into phosphatidylcholine (PC) was observed in both pre- and postconfluent cultures of control and AAM cells treated with PMA relative to vehicle. This response was attenuated in pre- and postconfluent AAM cells relative to control counterparts. PMA enhanced 32P incorporation into phosphatidylinositol (PI) in preconfluent cultures of control cells, but decreased 32P incorporation in cultures of AAM cells relative to vehicle. A similar relationship was observed in the PI profile of postconfluent cultures. The alterations in primary phospholipid profiles induced by PMA correlated with the loss of PKC-mediated histone phosphorylation in the cytosolic and particulate fractions of both cell types. The pattern of 32P incorporation into PA, a phospholipid metabolite, paralleled that of PC in cultures of both cell types. In the presence of ethanol, vehicle-treated control and AAM cells exhibited a modest increase in phosphatidylethanol (PEt) formation, as measured by [3H]myristic acid incorporation. PMA enhanced PEt formation in control and AAM cultures, but selectively decreased [3H]myristic acid incorporation into PA in AAM cells. These data demonstrate that long-term PMA treatment differentially modulates phospholipid metabolism in aortic SMCs of varying proliferative potential. These alterations are associated with modulation of PLD-mediated hydrolysis of membrane phospholipids.

Protein kinase C (PKC)-mediated protein phosphorylation in rat aortic smooth muscle cells is enhanced by allylamine.

The profile of endogenous protein phosphorylation mediated by protein kinase C (PKC) was examined in cell fractions prepared from subcultured aortic smooth muscle cells (SMCs) isolated from rats treated with 70 mg/kg allylamine (AAM) or tap water for 20 days. Increased phosphorylation of endogenous proteins was observed under unstimulated conditions in the particulate, but not cytosolic, fraction of cells from AAM-treated animals (i.e. AAM cells) relative to control cells. Although the same phosphorylation bands were observed in the particulate or cytosolic fraction of control and AAM cells following phorbol ester stimulation of the enzyme, enhanced PKC-mediated phosphorylation was observed in both fractions of AAM cells relative to control cells. Measurements of exogenous histone Type III-S phosphorylation by PKC following in vitro exposure of naive SMCs to 100 microM AAM for up to 60 min revealed that AAM selectively increased histone phosphorylation in the cytosolic fraction of SMCs. These results demonstrate that AAM treatment enhances PKC-mediated protein phosphorylation in rat aortic SMCs and raise the possibility that such alterations participate in the angiotoxic response to AAM.

Allylamine enhances c-Ha-ras protooncogene expression in rat aortic smooth muscle cells.

Studies were conducted to evaluate the mechanisms involved in the deregulation of proliferative control induced by allylamine (AAM) in rat aortic smooth muscle cells (SMCs). Subcultured SMCs from animals treated with AAM (70 mg/kg) or tap water for 20 days were processed for measurements of [3H]thymidine incorporation and c-Ha-ras mRNA levels. Pre-confluent AAM cells stimulated with 10% fetal bovine serum exhibited enhanced mitogenic responsiveness relative to control cells. Decreased [3H]thymidine incorporation was observed in post-confluent cultures of both cell types relative to pre-confluent counterparts. A 5-fold increase in c-Ha-ras transcript levels was observed in pre-confluent/cycling cultures of AAM cells relative to controls. C-Ha-ras expression was markedly reduced in post-confluent cultures of both cell types as compared to pre-confluent counterparts. No difference between control and AAM cells was observed during G1-synchronization of pre- or post-confluent cultures. These results suggest that the enhanced proliferative capacity induced by AAM is associated with alterations in cell cycle-related expression of the c-Ha-ras protooncogene.