Aryl hydrocarbon hydroxylase induction in human leukocytes.

A method for determining aryl hydrocarbon hydroxylase induction in human leukocytes is described. Leukocytes from healthy volunteers were cultured in the presence of phytohemagglutinin, a mitogen. Addition of 3-methylcholanthrene to 72-hour cultures induced a fourfold increase in aryl hydroxylase activity. In the absence of a mitogenic agent, 3-methylcholanthrene stimulation of increased enzymatic activity did not occur.

Induction of aryl hydrocarbon hydroxylase in human pulmonary alveolar macrophages by cigarette smoking.

Pulmonary alveolar macrophages were obtained from healthy volunteers by saline pulmonary lavage, and aryl hydrocarbon hydroxylase was measured in the cells. Enzyme activity was low in cells from five nonsmokers with a mean of 0.008+/-0.004 U/10(6) cells. Cells obtained from nine cigarette smokers contained higher enzyme levels, with a mean of 0.095+/-0.024 U/10(6) cells. A former cigarette smoker was lavaged on five occasions. Enzyme activity during two lavages 4 mo apart were 0.010 and 0.009 U/10(6) cells, respectively. 1 wk after smoking was resumed, the enzyme activity rose slightly to 0.013, and reached 0.041 U/10(6) cells by 1 mo. Upon cessation of smoking, the enzyme activity returned to control levels by the next lavage, 2 mo later. These data indicate that aryl hydrocarbon hydroxylase may be induced in pulmonary alveolar macrophages of subjects chronically exposed to cigarette smoke.

Comparison of aryl hydrocarbon hydroxylase induction in cultured blood lymphocytes and pulmonary macrophages.

Aryl hydrocarbon hydroxylase induction was studied in cultured peripheral blood lymphocytes and pulmonary alveolar macrophages from 15 smokers and 8 nonsmokers with a variety of pulmonary diseases. Enzyme levels in lymphocytes from cigarette smokers cultured in medium without an inducing agent were 57+/-6 mU/10(6) cells (mean+/-SEM), while enzyme levels in lymphocytes from nonsmokers were 20+/-2 mU/10(6) cells (P < 0.001). When lymphocytes were cultured in the presence of the inducing agent, benzo-(a)anthracene, enzyme activity was increased to 168+/-23 mU/10(6) cells in smokers' cells and 99+/-22 mU/10(6) cells in lymphocytes from nonsmokers (P < 0.04). When noninduced enzyme values in cultured macrophages were compared, smokers' cells had enzyme levels of 45+/-5 mU/10(6) cells, whereas nonsmokers had enzyme activity of 24+/-2 mU/10(6) cells (P < 0.002). However, pulmonary macrophages from smokers or nonsmokers, cultured in the presence of benzo(a)-anthracene, had similar levels of induced enzyme activity (P > 0.1). A positive correlation was observed for nonsmokers (r = 0.596, P > 0.1 <0.2) or smokers (r = 0.640, P < 0.04), when enzyme values for noninduced cultures of macrophages and lymphocytes from individual patients were simultaneously compared. Enzyme values for macrophages and lymphocytes cultured in the presence of an inducer also revealed a positive correlation for individual smokers (r = 0.801, P < 0.001) or nonsmokers (r = 0.785, P < 0.01). Inducibility (expressed as fold-induction) for macrophages and lymphocytes from individual patients was also positively correlated (r = 0.889, P < 0.001 for nonsmokers and r = 0.942, P < 0.001 for smokers). These results indicate that the capacity for aryl hydrocarbon hydroxylase induction is similar whether tested in lymphocytes or pulmonary macrophages from this group of pulmonary disease patients.

Cytogenetic analysis of a gossypol-induced murine myxosarcoma.

Cytogenetic analysis of gossypol acetate-induced murine myxosarcoma demonstrated a stemline of 78 chromosomes and the presence of three marker (M) chromosomes produced by robertsonian translocation. Tumor cells at passage 1 that contain chromosomes M1 and M2 were nontumorigenic, whereas cells at passage 3 were tumorigenic in syngeneic mice and showed M1, M2, and M3. The presence of M3 has been implicated to be responsible for the tumorigenic phenotype.

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.

Reduction of nicotinamide adenine dinucleotide levels by ultimate carcinogens in human lymphocytes.

The effect of several classes of DNA-damaging chemicals and closely related compounds on cellular nicotinamide adenine dinucleotide (NAD) levels was studied in freshly isolated peripheral human lymphocytes. Of the 21 compounds examined, 7 were direct DNA-damaging agents and 14 were non-DNA-damaging compounds or required metabolic activation to casue DNA damage. Rapid lowering of cellular NAD levels was caused by each of the direct DNA-damaging chemicals examined in this study including N-methyl-N'-nitr-N-nitrosoguanidine, methyl methanesulfonate, N-acetoxy-2-acetylaminofluorene, 7-bromomethylbenz(a)anthracene, and the benzo(a)pyrene derivatives, r-7,t-8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene and benzo(a)pyrene-4,5-epoxide. The indirect-acting carcinogen 2-acetylaminofluorene, 13 polycyclic aromatic hydrocarbons, and derivatives that were non-DNA-damaging did not cause lowering of NAD. The results suggest a general correlation between DNA damage and acute lowering of cellular NAD pools.

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.

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.

Correlation of carcinogen-induced unscheduled DNA synthesis and NAD reduction in fresh human lymphocytes.

Incorporation of [3H]thymidine into the DNA of fresh human lymphocytes, treated with various chemical mutagens, was measured and correlated with cellular NAD levels before and after treatment. NAD levels in lymphocytes were significantly reduced following treatment with mutagenic chemicals. Reduction of cellular NAD pools was directly correlated with [3H]thymidine incorporation. As NAD levels decreased, [3H]thymidine incorporation increased. Theophylline, a known inhibitor of poly(ADP-ribose)polymerase, inhibited both the NAD reduction in cells treated with DNA damaging agents and the incorporation of [3H]thymidine into DNA. The inhibitory effect of theophylline on NAD depletion and on [3H]thymidine incorporation was dose and cell number dependent. Near normal responses to carcinogen exposure could be restored to theophylline-treated cells following the removal of theophylline. These data suggest that conversion of NAD to poly(ADP-ribose) may be necessary, or at least closely associated with, DNA repair in human lymphocytes.

Metabolism of benzo(a)pyrene in animals with high aryl hydrocarbon hydroxylase levels and high rates of spontaneous cancer.

Ambystoma tigrinum found in a sewage polluted pond had high levels of aryl hydrocarbon hydroxylase (AHH) activity that decreased to the basal level of control animals after being held several months in clean water. The qualitative formation of benzo(a)pyrene (BP) metabolites by salamander hepatic microsomes was similar to those seen for other species. Inhibition of epoxide hydrase activity did not alter the total metabolite production but did change the ratio of metabolites. A correlation appears to exist between high AHH induction, the presence of polycyclic hydrocarbon pollutants, and the high rate of spontaneous cancer in salamanders.

Induction of aryl hydrocarbon hydroxylase in human peripheral blood lymphocytes by chrysene.

Many of the polycyclic aromatic hydrocarbons (e.g., benzo[a]pyrene (B[a]P), benzanthracene (BA), 3-methylcholanthrene (3-MC)) are not only carcinogenic, but also induce AHH in human tissues. Recently, chrysene has been implicated as an etiologic determinant of chemical carcinogenesis. Here we describe the ability of chrysene to induce AHH in cultured human lymphocytes. Lymphocytes were obtained from 9 healthy subjects, divided into 2 sets, and cultured in duplicate, triplicate, or quadruplicate for 48 h. Chrysene (25 microM final concentration) in acetone was then added to the induced culture set and the control set received acetone alone. Lymphocytes were then cultured an additional 24 h before harvesting. AHH was quantitated by a fluorometric analysis of the phenolic metabolites produced by incubating the lymphocytes with B[a]P for 35 min. A significant increase in enzyme induction occurred in the chrysene-induced cultures compared with control (non-induced) cells (one-tailed student t-test; P less than 0.001). It was also observed that the interindividual variation in AHH inducibility seen with other PAHs is also observed with chrysene.

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.

Steroid hydroxylase induction in cultured human lymphocytes: effects of the menstrual cycle.

Steroid hydroxylases (SAH) are inducible in cultured human lymphocytes following treatment with estradiol-17beta. The enzyme systems induced are carbon monoxide sensitive and convert estradiol-17beta to a metabolite chromatographically indistinguishable from estriol. The level of inducibility of SAH varies drastically over a normal menstrual cycle with maximum induction in the late follicular phase and minimum induction during the luteal phase. The use of an oral contraceptive containing both a synthetic progestogen and ethynyl estradiol reduced SAH induction levels to those typically seen during the luteal phase of the menstrual cycle.

Alkylation of deoxyguanosine by the sesquiterpene lactone hymenoxon.

Hymenoxon, a toxic sesquiterpene lactone found in the ruminant forage plant Hymenoxys odorata, binds deoxyguanosine in a cell-free system, and forms adducted guanine residues in sheep lymphocyte DNA. Mitogen-stimulated DNA synthesis in lymphocytes was inhibited by hymenoxon at concentrations greater than 100 microM. Unscheduled DNA synthesis in lymphocytes was initiated by hymenoxon concentrations exceeding 50 microM, and inhibited by concentrations above 100 microM. We describe an HPLC method which separates unmodified hymenoxon and deoxyguanosine from the hymenoxon-deoxyguanosine adduct, and allows the quantitation of adducts in hymenoxon-treated cells.

High-density lipoproteins decrease both binding of a polynuclear aromatic hydrocarbon carcinogen to DNA and carcinogen-initiated cell transformation.

Lipophilic carcinogens partition into the three major classes of lipoproteins potentially present in serum used as a medium supplement for cell culture. Different serum lots sequester differing quantities of the procarcinogen benzo[a]pyrene, dependent on the serum lipoprotein concentrations. In the presence of high-density lipoproteins a mutagenic benzo[a]pyrene metabolite was bound to cellular DNA at decreased levels when compared to cells exposed to the mutagen in the absence of high-density lipoproteins. Fetal calf serum with low levels of lipoproteins, specifically, high-density lipoproteins, is associated with efficient methylcholanthrene-initiated transformation of C3H/10T1/2 cells, while calf serum with a significant concentration of high-density lipoproteins requires up to a 500% increase in methylcholanthrene concentration to achieve similar levels of transformation in this mouse embryo cell line. When concentrated serum lipoproteins or purified HDL were added to fetal calf serum containing MCA at 1 microgram/ml, the C3H/10T1/2 transformation frequency was decreased compared to the transformation frequency achieved in the presence of 1 microgram/ml of MCA in fetal calf serum without supplementation. The results suggest that high-density lipoprotein partitioning of lipophilic polynuclear aromatic hydrocarbon mutagens from the cell culture medium may effectively reduce the concentration of carcinogen available for interaction with cellular DNA in vitro, which, in turn, may be associated with decreased carcinogen-induced transformation of cells.

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.

Inhibition of DNA polymerase activity by methyl methanesulfonate.

Methyl methanesulfonate (MMS) inhibits both thymidine incorporation into DNA in mitogen-activated human lymphocytes and deoxythymidine triphosphate incorporation into template DNA by DNA polymerase-alpha in a cell-free system. When MMS-modified DNA was used as the template for DNA synthesis utilizing unmodified DNA polymerase-alpha, nucleotide incorporation into template DNA was not inhibited. When unmodified DNA was used as the template for DNA synthesis utilizing MMS-modified DNA polymerase-alpha, nucleotide incorporation was differentially inhibited dependent on the MMS concentration. An analysis of the kinetics of DNA polymerase-alpha inhibition showed that incorporation of all 4 deoxynucleoside triphosphates into DNA template was noncompetitively inhibited by MMS, which is consistent with nonspecific MMS modification of the enzyme. These data indicate that MMS modification of DNA polymerase-alpha alone is sufficient to inhibit the incorporation of deoxynucleoside triphosphates into template DNA in vitro. The data further indicate that alkylation of both DNA polymerase-alpha and DNA template synergistically increases inhibition of DNA synthesis.

Human lymphocytes treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene require low-density lipoproteins for DNA excision repair.

Human lymphocytes which were non-mitogen-stimulated, and which were depleted of lipoproteins, were found to be deficient in DNA excision repair typically initiated in these cells in response to treatment with a direct-acting polynuclear aromatic hydrocarbon carcinogen. Lymphocytes either depleted of lipoproteins or supplemented with human low-density lipoproteins formed DNA-carcinogen adducts which were not chromatographically distinguishable. The state of lipoprotein depletion did not alter lymphocyte uptake of thymidine from the medium. Lymphocytes which were depleted of lipoproteins, treated with carcinogen, and subsequently supplemented with low-density lipoproteins, regained the ability to engage in DNA excision repair. The data suggest that either low-density lipoprotein(s), or a component(s) of low-density lipoprotein(s), is required by human lymphocytes in order to initiate excision repair of carcinogen-damaged DNA.

High-density lipoproteins decrease both DNA binding and mutagenicity of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in V79 Chinese hamster cells.

The effects of separate lipoproteins or of serum with high or low lipoprotein concentrations on formation of lipophilic carcinogen adducts with DNA and on mutagenicity of the carcinogen was investigated using V79 Chinese hamster lung cells. Binding of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) to DNA and BPDE induction of 6-thioguanine (6-TG)-resistant mutants in V79 cells was significantly lower after 1 or 4 h when the medium was supplemented with purified HDL, and was lower after 1 h but not 4 h when the medium was supplemented with serum containing a high concentration of mixed lipoproteins (LP). Cells grown in medium without serum or LP supplementation exhibited the highest levels of both BPDE-DNA adduct formation and mutagenesis after 1 h. At 1 h, cells exposed to BPDE in LDL-supplemented medium showed decreased adduct formation and mutagenesis when compared to cells treated with BPDE in PBS-supplemented medium. After 4 h, cells treated with BPDE in LDL-supplemented medium gave the highest levels of adduct formation and the highest mutation frequency. These results suggest that both LDL and HDL effectively decrease the concentration of BPDE available to V79 cells exposed to the mutagen for short periods of time, resulting in decreased interaction of BPDE with DNA and decreased BPDE-associated mutagenesis, but that both BPDE-DNA adduct formation and mutagenesis increased as a function of increased exposure time in the presence of LDL. The results suggest that LDL, but not HDL, uptake by adsorptive endocytosis may be associated with potentiated entry of BPDE into V79 cells as a function of time.

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.