DEHP, bis(2)-ethylhexyl phthalate, alters gene expression in human cells: possible correlation with initiation of fetal developmental abnormalities.

Diethylhexylphthalate (DEHP) is a widely distributed phthalate, to which humans are exposed to due to its variety of commercial and manufacturing uses. As a plasticiser, it is found in a wide number of products, and metabolites of DEHP have been detected in urine samples from a high percentage of the people screened for phthalates. We utilised DNA microarray analysis to evaluate DEHP for gene expression disrupting activity using the human cell line MCF-7, and found that DEHP significantly dysregulated approximately 34% of the 2400 genes spotted on the NEN2400 chip we used. The results suggest that DEHP, a known estrogen agonist and probable androgen antagonist, alters the expression of a number of genes, many of which are critical for fetal development. Down-regulation of two genes, FGD1 and PAFAH1B1, related in that both are essential for fetal brain development, was corroborated using quantitative real time PCR. These studies show DEHP to be a highly effective human gene expression-altering chemical, and that, at appropriate concentrations, it has the possibility of altering fetal central nervous system development, resulting in the birth defects lissencephaly and/or faciodigitogenital dysplasia.

Development of an in vitro model of excess intracellular reactive oxygen species.

These investigations characterize an in vitro model for generating excess intracellular reactive oxygen species (ROS). This novel model may be useful in the identification and delineation of cellular mechanisms associated with aging due to the link between age and excess oxidative events. The human cell line, MCF7, was stably transfected using the pSV3.neo plasmid housing a gene encoding the Aequorea victoria green fluorescent protein (GFP). Transfected cells were analyzed for maintenance of GFP over time, showing stability of the GFP gene. These studies demonstrate that the presence of fluorescing GFP significantly increases intracellular ROS, creating oxidative stress in these cells. Antioxidant supplementation was evaluated to determine the effectiveness of intracellular H2O2 reduction. The results demonstrate that supplementation with a potent antioxidant, such as reduced glutathione, protects cells from oxidative damage by decreasing intracellular concentrations of H2O2. This model for intracellular generation of excess ROS establishes a clear method by which the utility of antioxidant supplementation to protect against intracellularly generated reactive oxygen species may be evaluated.

Aging and DNA polymerase alpha: modulation by dietary restriction.

Aging is an inevitable characteristic of biological processes in living organisms. For the last several years, investigators have proposed numerous mechanisms to explain the basic understanding of aging and its intervention and have provided many insights into the molecular bases and the biological events that contribute to the progressive decline in function observed during cellular aging. It is probable that a number of interacting factors, such as increased somatic mutations, changes in genetic expression, and decreased efficiency of protein synthesis, may contribute to the age-dependent deterioration of physiological processes. One cellular function involved in all of the above factors is that of normal DNA synthesis required for maintaining genomic integrity. This suggests that changes in function of DNA replicative enzymes are almost certain to be a factor in one or more of the negative cellular phenomena associated with aging. This is a particularly attractive hypothesis, since the accumulation of inactive or error-prone DNA polymerases during aging would be expected to initiate a sequence of events leading to synthesis of altered proteins and the general dysfunction of a wide range of cellular processes. Dietary restriction is the only anti-aging regimen uniquely suited to identifying these cellular processes and could play a significant role in maintaining cellular mechanisms necessary to reduce the rate at which mutations accumulate during aging. The observation that dietary restriction may impede the age-related decline in the activity and fidelity of DNA polymerases and in the decline of repair DNA synthesis, suggests potential mechanisms by which dietary restriction could extend the lifespan of animals, including humans.

Homologies between human and dolphin chromosomes detected by heterologous chromosome painting.

Human chromosome-specific probes for the entire karyotype were hybridized to metaphase spreads of the Atlantic bottlenose dolphin, Tursiops truncatus, to directly compare the evolutionary conservation of chromosomal segments between these two distantly related species. All human chromosomal paints, except the Y probe, hybridized to Tursiops counterparts, and every dolphin chromosome was painted except for the smallest submetacentric pair. In our analysis, 36 segments of conserved synteny common to the human and dolphin genomes were identified. The distribution of conserved chromosomal segments and the specific rearrangement patterns found between the two genomes are presented and discussed.

An accessory protein of DNA polymerase alpha declines in function with increasing age.

Isoforms of DNA polymerase alpha (pol alpha/primase; pol alpha) were isolated from the livers of C57BL/6 mice either 3 months old (young) or 13 months old (mature). The 13-month-old mice were from two groups, one in which food was available ad libitum (AL), and one in which calories had been restricted to 60% of the AL intake (CR). The polymerases from young vs. mature and CR vs. AL mice differed in total and specific pol alpha activity, with the highest values exhibited by enzymes from 3-month-old mice. A more active isoform of pol alpha was typically isolated from CR animals than from AL animals. Differences in charge were used to chromatographically separate pol alpha into elution peaks exhibiting differing degrees of enzyme activity. DNA pol alpha isolated from tissues of mature mice exhibited a decline in activity which was not associated with decreased recoverable levels of pol alpha protein, but with a decline in the tendency of pol alpha to co-purify with an accessory protein, alpha AP, that binds double-stranded DNA (dsDNA). Low activity pol alpha isoforms which did not co-purify with alpha AP were stimulated by interaction with exogenous alpha AP. Pol alpha isoforms which co-purified with the dsDNA-binding accessory protein exhibited higher specific activity and less enhancement of activity upon interaction with exogenous alpha AP. Calorie restricted animals exhibited a pol alpha isoform that was more like pol alpha from younger animals in that it typically copurified with alpha AP, the DNA-binding accessory protein.

A high resolution GBG-banded karyotype of the Atlantic bottlenose dolphin, Tursiops truncatus: generation of an ideogram, and NOR localization by fluorescence in situ hybridization.

A high resolution karyotype was prepared using GBG-banded chromosomes from kidney epithelial cell lines of the Atlantic bottlenose dolphin, Tursiops truncatus. The karyotype was used to generate a banded ideogram of T. truncatus that will facilitate cytogenetic analyses essential for comparison of dolphin chromosomes with those of potentially related terrestrial vertebrates. Fluorescence in situ hybridization analysis of the karytype using a rodent (Geomys bursarius) 28S ribosomal DNA (rDNA) probe allowed localization of four nucleolar organizer (NOR) regions to the short arms of two chromosome pairs. FISH mapping, using DNA probes, is dependent on a pre-existing banded ideogram, and provides the means to initiate physical mapping of the dolphin genome.

Differential expression of DNA polymerase alpha in normal and transformed human fibroblasts.

The expression of DNA polymerase alpha (pol alpha) was studied in human fibroblast lines W138 (fetal lung) and GM3529 (skin, established from a 66 yr old donor), and their Simian virus 40 (SV40) large tumor antigen (TAg)-transformed corollaries, 2RA and 2-1 respectively. Both SV40-transformed and pSV3.neo (SV40-derived plasmid)-transformed cells express TAg, a virally encoded protein not expressed by the normal parent cell lines. Northern blot hybridization studies showed increased recovery of pol alpha mRNA from transformed cells compared with normal cells. This increase was correlated with increased pol alpha mRNA transcription as determined by nuclear run-on assays. Northern blot analyses also showed an increase in the instability of translationally active pol alpha mRNA in transformed cells. The results suggest that TAg, in addition to its dsDNA binding, pol alpha binding, retinoblastoma protein binding and helicase activities, may be involved either directly or indirectly in regulation of the steady state mRNA levels of pol alpha at the transcriptional level in both fetal and aged donor-derived transformed fibroblasts.

Effects of benzo(a)pyrene and tetrachlorodibenzo(p)dioxin on fetal dolphin kidney cells: inhibition of proliferation and initiation of DNA damage.

Dolphin kidney cells (CDK) were exposed in vitro to benzo(a)pyrene (BaP) in the presence or absence of 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD), a cytochrome P450-inducing agent, and/or alpha-naphthoflavone (alpha NF), an inhibitor of cytochrome P450 induction. BaP inhibited mitosis in CDK cells in a dose-dependent manner. TCDD, while inhibiting cell proliferation, did not show a strict dose-dependent mode of action. BaP inhibition of mitosis was decreased by alpha NF, which also decreased the inhibitory effects of TCDD on CDK proliferation. BaP treatment initiated both 3H-thymidine incorporation and the increased alkali lability of DNA functions of the initiation of excision repair. Cells pre-treated with TCDD and then exposed to BaP exhibited increased BaP-DNA adduct levels and increased DNA excision repair. These data indicate that dolphin cells metabolized BaP in vitro as a function of cytochrome P450-associated activities, that BaP metabolites covalently bound to cellular DNA and initiated excision repair. Inhibition of the cytochrome P450-mediated metabolism of BaP decreased the BaP-associated inhibition of mitosis in dolphin cells.

Dose-dependent induction of the microsomal monooxygenase system by phenobarbital and 3-methylcholanthrene in the ad libitum and calorie-restricted female rat.

1. We characterized the dose-dependent induction of the microsomal monooxygenase system by phenobarbital (PB) and 3-methylcholanthrene (MC) in the female Fischer 344 rat, which was either calorie restricted (CR) or fed ad libitum (AL). 2. Maximal induction of the major inducible isozymes (2B1/2B2 or 1A1) in rat was achieved at the lowest of the inducer doses employed (10 mg/kg body weight) in both feeding groups. 3. The patterns of dose-dependent PB induction and its magnitude differed between total P450 induction and induction of catalytic activities in AL and CR groups, whereas no differences between CR and AL rat were found in either spectrally detected P450 or EROD activity patterns of dose-dependent MC induction. 4. Calorie restriction increased the inducibility of some hepatic drug-metabolizing enzyme activities. 5. Monoclonal antibody-directed inhibition of MC-induced ethoxyresorufin O-deethylation (EROD) was 55-60% at all induction levels in AL rat and 65-70% in CR rat, while MAb inhibition of PB-induced pentoxyresorufin O-depenthylation (PROD) averaged about 55% in AL and 60% in CR rat.

Ethoxyresorufin and pentoxyresorufin O-dealkylation by hepatic microsomes from female Fischer 344 rats: effects of age and diet.

Ethoxyresorufin (EROD) and pentoxyresorufin (PROD) O-dealkylase activities, and contributions of the P450 cytochromes CYP1A1, CYP2B1 and 2, CYP2C6 and CYP2C12 to these metabolic activities, were evaluated in hepatic microsomes from ad libitum and calorie restricted female Fischer 344 rats across an age continuum from 1 to 26 months. The presence of CYP1A in microsome preparations was confirmed by western blot analysis. Uninduced levels of EROD and PROD peak in very young animals, decline to about 3 months of age, and do not exhibit an additional substantive decline after about 3 months of age. Monoclonal antibody (MAb) 1-7-1 (anti-CYP1A) strongly inhibited EROD activity in all microsome preparations, with the highest levels of inhibition in microsomes from 1- and 3-month-old AL animals. PROD activity in 1-month uninduced animals was apparently not attributable solely to CYP2B1 and 2, since it was inhibited by about 30% in both 1- and 26-month-old AL rats by an MAb specific for CYP2C12. However, in CR rats, CYP2C12 did not contribute to PROD activity. Approximately 40% of PROD activity in old AL rats and 20% of PROD activity in old CR rats was inhibited by an MAb specific for CYP2C6. These data indicate that long-term calorie restriction modulates either the expression or post-translational modification patterns of constitutive P450 isozymes in rats as they age, with P450 patterns in calorie restricted rats more closely resembling those found in young animals.

Gastrointestinal uptake and vascular transport of 2,4'-dichlorobiphenyl.

Absorption from the gastrointestinal tract and subsequent vascular transport of [3H]-2,4'-dichlorobiphenyl (Aroclor 1232; DCB) was investigated in an ovine model system. Rapid uptake of DCB and transport as a component of blood plasma without prior occurrence in thoracic duct lymph indicates that DCB was absorbed directly via the gastric mucosa with water soluble compounds. [3H]-DCB did not circulate associated with plasma lipid fractions in vivo, and did not bind to or sequester within plasma lipids in vitro. HPLC analysis of plasma fractions treated in vitro showed DCB to elute within a molecular weight range consistent with unbound product. Further, [3H]-DCB-derived label was associated with low molecular weight plasma components in vivo. Essentially the same elution profile was seen for [3H]-DCB-derived label found in urine. Metabolism of DCB as a function of time resulted in the apparent formation of a biotransformed product(s) that circulated with a plasma fraction(s) at the low end of the albumin molecular size range. These data suggest that DCB was not absorbed and transported in a manner typical of polychlorinated biphenyls with a higher chlorine content; rather, that it was absorbed, transported within the vascular system, and excreted in a pattern typical of a water soluble compound.

A comparison of DNA polymerase alpha from untransformed and SV40-transformed human fibroblasts.

1. DNA polymerase alpha (pol alpha) isolated from Simian virus 40 (SV40)-transformed cells showed more than 3-fold higher specific activity than pol alpha from normal cells. The enzymes from untransformed and transformed cells also differed in molecular size, thermolability, sensitivity to inhibitors and specificity of template-primer utilization. 2. Western analysis using anti-Tag to probe both a crude cell homogenate and partially purified pol alpha from SV40 transformed cells showed multiple immunoreactive bands with different molecular sizes. 3. While alpha polymerases from both normal and transformed cells exhibited tightly associated primase activity, they showed different DNA binding affinities. 4. These data suggest that T antigen binding to pol alpha alters the initiation of DNA replication and/or the function of pol alpha in SV40-transformed cells, and that pol alpha from SV40-transformed human fibroblasts have different catalytic subunit characteristics than pol alpha from untransformed cells.

Characterization of DNA polymerase alpha from untransformed and pSV3.neo-transformed human fibroblasts.

1. The specific activity of DNA-polymerase alpha isolated from pSV3.neo-transformed cells was more than 9-fold higher than that of polymerase alpha from untransformed cells. 2. Western blot analysis, using anti-SV40 large T antigen, of both a crude cellular extract and of partially purified polymerase alpha from pSV3.neo-transformed cells revealed a single 76 kDa immunoreactive band not found in either crude extracts or partially purified enzyme from untransformed cells. 3. The alpha polymerases from untransformed and transformed cells differed in molecular size, sensitivity to various inhibitors, specificity of template-primer utilization, and binding affinity for DNA cellulose, but showed essentially no differences in Km or Vmax. 4. These data suggest that polymerase alpha isolated from pSV3.neo-transformed cells exhibits altered physical and catalytic characteristics compared with its untransformed cell counterpart, and that those alterations may be associated with increased replication of the genome in plasmid-transformed cells.

Inhibition of DNA synthesis and decreased reproductive capacity in cattle maintained on semi-arid rangeland with Acacia species as a major component of available browse.

The ability of bovine lymphocytes to initiate in vitro blastogenesis in response to mitogen stimulation or to initiate DNA excision repair after treatment with a mutagen was evaluated as a function of the differing environmental conditions under which donor animals were maintained. Crossbred Brahman-Hereford F1 females were held on either a humid, coastal bermudagrass, improved pasture at Overton, TX, or in low or high grazing pressure herds on a semi-arid rangeland (Acacia-dominated shrubland) at Uvalde, Tx. Peripheral blood lymphocytes (PBL) from these animals were examined to determine their in vitro ability to engage in phytohemagglutinin (PHA)-stimulated blastogenesis and to initiate excision repair of DNA damage after exposure of the cells to the model polynuclear hydrocarbon carcinogen 7,8-dihydrodiol-9, 10-epoxy-benzo(a) pyrene (BPDE). PBL from cattle at both locations were compared, with significantly decreased blastogenesis and DNA excision repair observed in PBL from Uvalde high grazing pressure cattle. Cattle in the Uvalde high grazing pressure herd also exhibited significantly decreased reproductive efficiency. The data indicate that ingestion of sufficient quantities of palatable, but toxic, forage species available at the Uvalde test site is sufficient to decrease DNA synthesis associated with either mitogen-stimulated blastogenesis or DNA excision repair in bovine PBL, and suggest that the reduction in PBL DNA synthesis may be correlated with the changed reproductive efficiency in animals ingesting an increased ratio of forage from Acacia species.

Decreased fidelity of DNA polymerases and decreased DNA excision repair in aging mice: effects of caloric restriction.

Hepatic DNA polymerases from calorie restricted and ad libitum 26 month old C57BL/6 mice showed a decline in fidelity of nucleotide incorporation compared with weanling animals. Both alpha and beta polymerases from calorie restricted aged mice exhibited a higher level of fidelity than polymerases from ad libitum aged mice. UV-initiated unscheduled DNA synthesis was significantly higher in hepatocytes from weanling and 18 month old calorie restricted animals compared with cells from 18 month old ad libitum animals, while MMS-initiated unscheduled DNA synthesis did not differ significantly between cells from young and old or ad libitum and calorie restricted animals. These data suggest that calorie restriction could play a significant role in decreasing the age-related decline of cellular mechanisms expected to reduce the rate at which mutations accumulate during aging, and could potentially prolong the onset age of mutation-associated diseases of the elderly.

Effect of dietary restriction on the fidelity of DNA polymerases in aging mice.

DNA polymerases purified from hepatic tissues of C57BL/6 mice showed an age-related decrease in both specific activity and fidelity of the various enzyme forms. Polymerases from dietary restricted mice exhibited less of a decline in specific activity and copied synthetic DNA templates with relatively higher fidelity than did enzymes from animals fed ad libitum. Polymerases treated with inositol-1,4-bisphosphate [I(1,4)P2] showed varying levels of increased activity, with fidelity increases up to 3-fold. These data indicate that aging is associated with decreases in both specific activity and fidelity of DNA polymerases isolated from a nondividing tissue, and that dietary restriction impedes the age-related decline in both specific activity and fidelity of these polymerases. The data further indicate that DNA polymerases may interact with phosphoinositide hydrolysis products resulting in increased specific activity and fidelity of the enzymes. Phosphoinositide interactions with polymerases could constitute an important mechanism moderating the age-related decrease in function and accuracy of DNA polymerases.

Comparative uptake, vascular transport, and cellular internalization of aflatoxin-B1 and benzo(a)pyrene.

Studies of the uptake of benzo(a)pyrene (BaP) and aflatoxin-B1 (AFB1) after gastric instillation showed that BaP was absorbed via the intestinal lymphatic drainage and transported to the vascular circulation sequestered within lipoproteins in thoracic duct lymph, while AFB1 was absorbed with water soluble compounds into the gastrointestinal venous drainage and was not transported in association with lipoproteins. BaP was taken up into plasma lipoproteins over a broad concentration range, while AFB1 was not sequestered within lipoproteins over the same concentration range. Low density lipoproteins (LDL) facilitated BaP uptake into fibroblasts and impeded BaP uptake into hepatocytes. High density lipoproteins (HDL) facilitated BaP uptake into hepatocytes and impeded BaP uptake into fibroblasts. The uptake of AFB1 into either fibroblasts or hepatocytes was not affected by lipoproteins.

Interaction of phosphatidylinositol-4-monophosphate with a low activity form of DNA polymerase alpha: a potential mechanism for enzyme activation.

1. DNA polymerase alpha isolated from Norman murine myxosarcoma exhibited two isozyme forms, one with low specific activity and low DNA binding affinity (A1), and one with high specific activity and high DNA binding affinity (A2). 2. DNA polymerase alpha A1, but not A2, showed a significant increase in specific activity after treatment with phosphatidylinositol, ATP and phosphatidylinositol kinase, or with phosphatidylinositol-4-monophosphate. 3. Treatment of DNA polymerase alpha A1 with the phospholipase C hydrolysis product of phosphatidylinositol-4-monophosphate, inositol-1,4-bisphosphate, was sufficient to effect the transient increase in activity of polymerase A1 to a form not chromatographically distinguishable from isozyme form A2.

Purification of Norman Murine Sarcoma DNA polymerase alpha forms with different DNA template primer binding affinity and different specific activity.

1. DNA polymerase alpha was isolated from Norman Murine Myxosarcoma cells using ion exchange, immunoaffinity, and DNA affinity chromatography, showing two distinct enzyme forms designated A1 and A2. 2. Chromatographic analysis of polymerase alpha forms A1 and A2 indicate a charge difference and a difference in affinity of binding to DNA between polymerase alpha forms which were equally reactive to anti-DNA polymerase alpha monoclonal IgG. 3. Polymerase A1 specific activity was about 3600 U/mg while A2 specific activity was about 40,000 U/mg.

Repair of benzo[a]pyrene-initiated DNA damage in human cells requires activation of DNA polymerase alpha.

Normal human fibroblasts treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) yielded DNA polymerase alpha with elevated levels of activity, incorporated [3H]thymidine as a function of unscheduled DNA synthesis, and exhibited restoration of normal DNA-strand length as a function of unscheduled DNA synthesis. Lipoprotein-deficient fibroblasts treated with BPDE did not show elevated levels of DNA polymerase alpha activity, exhibited minimal [3H]thymidine incorporation, and had fragmented DNA after 24 h of repair in the absence of lipoprotein or phosphatidylinositol supplementation. When DNA polymerase beta activity was inhibited, cells with normal lipoprotein uptake exhibited [3H]thymidine incorporation into BPDE-damaged DNA but did not show an increase in DNA-strand length. DNA polymerase alpha activity and [3H]thymidine incorporation in lipoprotein-deficient fibroblasts increased to normal levels when the cells were permeabilized and low-density lipoproteins or phosphatidylinositol were introduced into the cells. DNA polymerase alpha isolated from normal human fibroblasts, but not from lipoprotein-deficient fibroblasts, showed increased specific activity after the cells were treated with BPDE. When BPDE-treated lipoprotein-deficient fibroblasts were permeabilized and 32P-ATP was introduced into the cells along with lipoproteins, 32P-labeled DNA polymerase alpha with significantly increased specific activity was isolated from the cells. These data suggest that treatment of human fibroblasts with BPDE initiates unscheduled DNA synthesis, as a function of DNA excision repair, which is correlated with increased activity of DNA polymerase alpha, and that increased DNA polymerase alpha activity may be correlated with phosphorylation of the enzyme in a reaction that is stimulated by low-density lipoprotein or by the lipoprotein component, phosphatidylinositol.