Biomedical subjects

J Nath

Publications and source records attributed to J Nath.

At least 55 records · Page 3Linked to original sources

PubMed1996

Inhibitory effect of folinic acid on radiation-induced micronuclei and chromosomal aberrations in V79 cells.

Folinic acid (FA), clinically called leucovorin, has been widely used as a nutrient supplement in dietary intake and is capable of inhibiting cytotoxicity and chromosomal damage induced by chemicals. However, data on its antigenotoxic effect on radiation-induced chromosomal damage are limited. The present study was, therefore, performed to investigate the effect of FA on radiation-induced (X-rays and UV radiation) micronuclei (MN) and structural chromosomal aberrations (SCA) concurrently in V79 Chinese hamster lung cells. Exponentially growing cells were exposed to five doses of X-rays (1-12 Gy) and UV radiation (50-800 microJ x 10(2)/cm2) and post-treated with 5 or 50 micrograms FA/ml of culture medium for 16 h. The slides were analyzed for the presence of MN and SCA using standard procedures. The results showed that X-ray treatment alone produced dose-related cytotoxicity as measured by nuclear division index (NDI) and mitotic index (MI). X-rays produced a clear dose-related clastogenicity as measured by percent of micronucleated binucleated cells (MNBN) (5-79%) and percent of aberrant cells (11-92%). FA at 5 micrograms/ml slightly decreased X-ray induced chromosomal damage in both assays; however, the inhibition was significant (12-46% of MNBN, 14-48% in aberrant cells) only when X-ray-treated cultures were post-treated with 50 micrograms FA/ml. Post-treatment of FA had no effect on X-ray induced cytotoxicity as measured by NDI and MI. A similar a dose-related increase in % MNBN (0.5-10.3%) and percent aberrant cells (6-35%) was produced by UV radiation treatment alone. There were significant percentages of MNBN and aberrant cell inhibitions at both 5 and 50 micrograms/ml in both assays. As in the case of X-ray-treated cells, there was a clear dose-related cytotoxicity in UV-treated cells alone. No reduction in NDI or MI was found when UV-exposed cells were post-treated with 5 or 50 micrograms of FA. These data demonstrate the beneficial effect of FA in decreasing radiation-induced chromosomal damage.

Animals↗

PubMed1996

Cytogenetic analysis of mice chronically fed the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine.

The cytogenetic effects in mice chronically fed the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP) were evaluated by chromosome painting, micronucleated normochromatic erythrocytes (MN NCEs) and sister chromatid exchanges (SCEs). PhIP and numerous other heterocyclic amines have been isolated from cooked foods, and many have been found to be carcinogenic in laboratory rodents. Female C57BL/6N mice were chronically fed a diet containing 0, 100, 250 or 400 ppm of PhIP beginning at 8 weeks of age. Peripheral blood and bone marrow were taken from 5 mice per treatment group at 1, 4 and 6 months from the start of exposure. PhIP was removed from the diet for a final month of the experiment, at which time blood was taken from the remaining animals. Chromosome-specific composite DNA probes for mouse chromosomes 2 and 8 were hybridized to metaphase cells from each tissue. The 1- and 4-month time points showed no statistically significant difference between the control and exposed mice for either tissue in chromosome aberration frequencies. Both MN NCEs and SCEs were analyzed at a single time point during exposure (4 months for MN NCEs and 6 months for SCEs) and again 1 month after removing PhIP from the diet. MN NCEs in the peripheral blood showed a statistically significant dose response, with all values decreasing significantly 1 month after removing PhIP from the diet. SCE frequencies in the peripheral blood showed an approximate doubling compared to control mice, and decreased to control levels 1 month after removing PhIP from the diet. SCE frequencies in the bone marrow of exposed mice showed no difference from the control animals. These results show that chronic ingestion of PhIP by female C57BL/6 mice does not produce persistent cytogenetic damage as visualized by chromosome aberrations, MN NCEs or SCEs.

Animals↗

PubMed1996

Induction of chromosomal aberrations by 2,4-dichloro-6-aminophenol in cultured V79 cells.

2,4-Dichloro-6-aminophenol (DCAP) is a major metabolite of the herbicide 2,4-dichloro-6-nitrophenol ammonium (DCNPA). In our previous studies this metabolite, but not the parent compound, was found to induce sister chromatid exchanges (SCEs) and micronucleus (MN) formation in Chinese hamster lung fibroblasts (V79 cells). Further studies were carried out to determine whether DCAP can also induce structural chromosomal aberrations (CAs) in cultured V79 cells. The assay was performed under three different treatment conditions: 1) 3 h treatment followed by 17 h post-treatment incubation. 2) 12 h treatment, and 3) 20 h treatment with multiple sampling times. Results showed that CAs were induced by DCAP in V79 cells. The aberrations were mainly chromatid types (gaps, breaks, and deletions), triradials, quadriradials, and complex rearrangements. However, induction of CAs appears to be dependent on the treatment condition. The 3 h treatment following a 17 h post-treatment incubation gave rise to 24% aberrant cells (excluding gaps) and a total of 44 aberrations per 100 metaphases, while the 12 h treatment only gave a borderline response and the 20 h treatment did not yield a significant increase of CAs at any concentration tested. It seems that a short-term treatment with higher concentrations is a better treatment protocol for genotoxicity studies of certain cytotoxic chemicals such as DCAP. Results of this study further indicate that the herbicide DCNPA is potentially hazardous to the exposed population.

Aminophenols↗

PubMed1996

Activation status of the X chromosome in human micronucleated lymphocytes.

The frequency of X chromosome aneuploidy in human female peripheral blood lymphocytes has been reported by several investigators to be significantly higher than expected based upon chance alone. Studies in our laboratory showed that 72% of the micronuclei in the peripheral blood of human females contained the X chromosome. Such a high frequency of X chromosome loss suggests that some unique mechanism may be responsible for this phenomenon. The present study was carried out to test the hypothesis that the lost or micronucleated chromosome is the inactive and not the active X. Blood samples were obtained from two unrelated females, 36 and 33 years of age, each with a different X; 9 reciprocal translocation. In each, the normal X chromosome is inactive and the translocated X is active. isolated lymphocytes were cultured according to standard techniques and blocked with cytochalasin B. Using a modified micronucleus assay, we scored 10,000 binucleated cells from the 36 year old, while 9,500 binucleated cells were scored from the 33 year old. The slides were first labeled and the kinetochore status of each micronucleus was determined. This was followed by simultaneous hybridization with a 2.0 kilobase centromeric X chromosome-specific probe and a chromosome 9 specific whole chromosome painting probe. All micronucleated cells were relocated and scored for their probe status. A total of 217 micronuclei were scored from the two subjects, of which 96 (44.2%) contained the X chromosome. Of these 96 micronuclei, 80 (83.3%) contained the inactive X, based on the absence of chromosome 9 material in the micronucleus. These results support our hypothesis that the inactive X chromosome is preferentially included in the micronuclei, and suggest that the X chromosome hypoploidy observed at metaphase in aging women is a related phenomenon.

Adult↗

PubMed1995

Y chromosome aneuploidy, micronuclei, kinetochores and aging in men.

This investigation was conducted to determine the relationship between Y chromosome loss and increased micronucleus formation with age. We also investigated the status of kinetochore proteins in the micronuclei. Umbilical cord blood samples were obtained from 18 newborn males, and peripheral blood was obtained from 35 adult males ranging in age from 22 to 79 years. Isolated lymphocytes from all 53 donors were cultured and blocked with cytochalasin B. Two thousand binucleate cells per donor were scored using a modified micronucleus assay to determine the kinetochore status of each micronucleus. This assay showed 23.8% of the micronuclei to be kinetochore-positive, while 76.2% of the micronuclei were kinetochore-negative. Cells were then hybridized with a 3.56-kb biotinylated Y chromosome-specific probe. All micronucleate cells were relocated and their Y probe status was determined. A significant increase in Y-bearing micronuclei with age was observed. Metaphase cells from the same samples were analyzed for the presence or absence of Y chromosome. The relationship between Y chromosome-positive micronuclei and Y chromosome-negative metaphase cells was highly significant, suggesting that Y chromosome-deficient metaphase cells result from cells which had previously lost a Y chromosome due to micronucleation. The cause of micronucleus formation from a lagging Y chromosome appears probably to be either a faulty or a diminished amount of kinetochore protein.

Adult↗

PubMed1995

Comparative studies on radiation-induced micronuclei and chromosomal aberrations in V79 cells.

Induction of micronuclei (MN) and structural chromosomal aberrations (SCA) by physical agents such as X-rays and UV radiation has been studied extensively in a variety of cell lines for genotoxicity assessment. However, comparative data on the relationship between these two cytogenetic endpoints are limited. This study compares SCA and MN formation in V79 Chinese hamster lung cells treated with X-rays and UV radiation. Four replicate cultures of exponentially growing cells were exposed to four doses of X-rays (100-800 rads). For two replicate cultures, cytochalasin B (3 micrograms/ml) was added and cells were harvested 16 h later for MN and nuclear division index (NDI) assessment. For the remaining two replicate cultures, colcemid (0.025 micrograms/ml) was added 16 h post-treatment and cells were harvested 2 h later for SCA and mitotic index (MI) analyses. This experiment was duplicated using four doses of UV radiation (100-800 microJ x 10(2)/cm2). In the X-ray experiment, generally, a decrease in the NDI and MI was noted with increasing dose. Also, there was a clear dose-related increase in percent micronucleated binucleated (MNBN) and aberrant cells. A similar dose response, but with lower frequencies, was observed in the UV radiation treatment. These data suggest a good correlation between chromosome damage as measured by percent MNBN and aberrant cells and cytotoxicity as measured by NDI and MI.

Animals↗

PubMed1995

Silica-induced micronuclei and chromosomal aberrations in Chinese hamster lung (V79) and human lung (Hel 299) cells.

Silica is one of the most abundant and widely used mineral groups. A large number of workers are potentially exposed to one or more forms of silica. Therefore, the potential carcinogenic hazard of silica to the exposed workers is of great concern. This study examines the genotoxic potential of silica with the micronucleus and chromosomal aberration assays using cultured Chinese hamster lung fibroblasts (V79) and human embryonic lung (Hel 299) cells. One-day-old cultures were treated with two types of silica, Min-U-Sil 5 and Min-U-Sil 10, for 24 h at concentrations of 40, 80, 160 and 320 micrograms/cm2. Both Min-U-Sils at 160 and 320 micrograms/cm2 induced micronucleus formation in V79 and Hel 299 cells. In V79 cells, a significant increase in the micronucleus frequency was also found with 40 and 80 micrograms/cm2. However, the chromosomal aberration frequency was unaffected by either Min-U-Sil 5 or 10 treatment of V79 or Hel 299 cells. Results indicated that silica, in different particle sizes, was capable of inducing micronuclei but not chromosomal aberrations in cultured animal and human lung cells and suggested that V79 cells were relatively more sensitive to silica than Hel 299 cells.

Animals↗

PubMed1994

Involvement of a botulinum toxin-sensitive 22-kDa G protein in stimulated exocytosis of human neutrophils.

Studies of human peripheral blood neutrophils (PMNs) demonstrated that botulinum neurotoxin D (BT-D) ADP-ribosylates a 22-kDa PMN G protein (G22k) and inhibits the exocytosis of both specific and azurophilic granules stimulated by FMLP. Furthermore, this inhibition of PMN exocytosis by BT-D was found to be correlated with the degree of irreversible ADP-ribosylation of G22k by BT-D and to require modification of at least 85% of PMN G22k before significant inhibition of secretion is observed. Although both pertussis toxin and BT-D inhibited exocytosis in FMLP-stimulated PMNs, the inhibitory effects of the two toxins were found to be additive. Pertussis toxin and BT-D also inhibited Ca2+/GTP/GTP gamma S-induced secretion in digitonin-permeabilized PMNs, but there were distinct differences between the inhibitory effects of the two toxins. In contrast to BT-D, the exotoxin botulinum C3 was found to ADP-ribosylate primarily a 24- to 25-kDa PMN protein, and it was not found to inhibit Ca(2+)- and GTP-induced secretion in permeabilized PMNs. Ultrastructural studies of BT-D-treated PMNs showed an accumulation of distinct membrane-bound organelles in the periphery of the cells after FMLP stimulation, suggestive of a toxin-induced block in organelle-plasma membrane fusion. Taken together, these findings indicate that BT-D-sensitive G22k has a functional role in stimulated exocytosis of PMNs.

ADP Ribose Transferases↗

PubMed1994

Sex chromosomes, micronuclei and aging in women.

Several studies on aneuploidy and aging have shown a significant increase in the loss of chromosomes in both males and females with age. Others have observed a significant increase in micronucleus formation in lymphocytes with age. The objectives of this investigation were to determine the relationship between sex chromosome loss and increased micronucleus frequencies with age, to establish sex chromosome loss frequencies unbiased by cellular survival factors or slide preparation, and to determine the effect of smoking on sex chromosome loss. Blood samples were obtained from 8 newborn females and 38 adult females ranging in age from 19 to 77. Isolated lymphocytes were cultured according to standard techniques and blocked with cytochalasin B. Two thousand binucleated cells per donor were scored using a modified micronucleus assay to determine the kinetochore status of each micronucleus. Slides were then hybridized with a 2.0 kb centromeric X chromosome-specific probe labeled with biotinylated dUTP, and detected with fluorescein-conjugated avidin. All micronucleated cells were relocated and their X chromosome status was determined. We found the X chromosome to be present in 72.2% of the micronuclei scored; additionally our results show a significant increase with age in the number of micronuclei containing an X chromosome.

Adult↗

PubMed1993

Rapid and reversible tubulin tyrosination in human neutrophils stimulated by the chemotactic peptide, fMet-Leu-Phe.

Neutrophil activation by specific stimuli, such as the oligopeptide chemotactic factor fMet-Leu-Phe (fMLF), is associated with an increased enzymatic addition of tyrosine to tubulin alpha-subunits, as measured by 14C tyrosine uptake. In studies using immunoblots we have found that this increased tyrosine uptake into tubulin in activated neutrophils reflects an increase in the proportion of cellular tubulin that is tyrosinated rather than simply an increase in the turnover of tyrosinated subunits. However, the increased accumulation of tyrosinated tubulin was also found to follow an initial depletion of tyrosinated tubulin and concomitant increase in detyrosinated tubulin between 0 and 60 sec following stimulation of neutrophils with fMLF. Immunogold electron microscopy studies of intact microtubules recovered from activated neutrophils demonstrated that these rapid changes in the relative content of tubulin isoforms in the cells were not associated with the formation or disappearance of microtubule microdomains composed of only one form of tubulin. Previously, we have shown that under conditions of fMLF-stimulated exocytosis there is an increased binding of neutrophil granules to endogenous microtubules. Since neutrophil activation by fMLF is associated with increased tyrosination of alpha-tubulin subunits, we speculated that rapid changes in the levels of tyrosinated tubulin in the microtubules of activated neutrophils might have a role in the regulation of granule-microtubule interactions. When the binding of purified neutrophil granules to reconstituted rat brain microtubules containing approximately 50% tyrosinated tubulin was measured by electron microscopy and compared with granule binding to microtubules that contained no detectable tyrosinated tubulin, granule-microtubule associations were found to be significantly favored by detyrosinated vs. tyrosinated tubulin. These findings indicate that interactions between cytoplasmic granules and microtubules in activated neutrophils may be modulated by rapid changes in the relative content of detyrosinated and tyrosinated tubulin in the microtubule network of the cells.

Amino Acid Sequence↗

PubMed1992

A novel post-translational incorporation of tyrosine into multiple proteins in activated human neutrophils. Correlation with phagocytosis and activation of the NADPH oxidase-mediated respiratory burst.

Activation of human neutrophils by PMA causes a post-translational incorporation of 14C-labeled tyrosine into multiple neutrophil (PMN) proteins, that is distinctly different from the enzymatic tyrosinolation of tubulin in FMLP-stimulated PMN. Post-translational incorporation of other radiolabeled amino acids, including the structurally similar amino acid phenylalanine, does not occur under identical conditions of neutrophil activation, suggesting an involvement of the phenolic hydroxyl group of tyrosine in the PMA-mediated reaction. Similar to the stimulation of PMN tubulin tyrosinolation by FMLP, the PMA-induced incorporation of tyrosine into multiple PMN proteins is closely associated with activation of the NADPH oxidase-mediated respiratory burst in stimulated PMN and can be inhibited by a variety of reducing agents, inhibitors of peroxidase-mediated reactions, and intracellular scavengers of oxygen radicals. Moreover, the PMA-induced post-translational incorporation of tyrosine does not occur in PMN from patients with chronic granulomatous disease and is significantly reduced (50%) in PMN of an individual with myeloperoxidase deficiency. A similar stimulus-induced incorporation of tyrosine into multiple PMN proteins is also observed in PMN exposed to various phagocytic stimuli, and the incorporated radioactivity in cells undergoing phagocytosis is substantially enriched (40- to 50-fold) in isolated PMN phagolysosomes. Consistent with this latter observation, HPLC fractionation of stimulated PMN proteins and analysis of the incorporated radioactivity reveal that the 14C label is primarily associated with PMN membrane proteins. Furthermore, this post-translational incorporation of tyrosine, like that associated with PMA stimulation, is associated with production of oxygen radicals and the generation of protein carbonyl derivatives, which are indicative of oxidative protein modifications via mixed function oxidases. Our findings indicate that tyrosine incorporation into membrane proteins of stimulated PMN is functionally relevant to the physiologic host-defense responses of human neutrophils undergoing phagocytosis.

Blood Proteins↗

PubMed1992

Cytogenetic effects of vincristine sulfate and ethylene dibromide in human peripheral lymphocytes: micronucleus analysis.

Micronuclei kinetics and persistence in mononucleated and binucleated human peripheral lymphocytes following short-term (4 hr) and continuous (until harvest) in vitro exposure to vincristine sulfate (VS) and ethylene dibromide (EDB) were studied. Lymphocytes were exposed to chemicals for various doses and harvested at different culture times. Micronucleus frequencies were scored in both mononucleated and binucleated cells on the same slide. VS-treated cells showed a significantly higher incidence of micronucleus in both mononucleated and binucleated cells than controls (P less than 0.01). The cells treated continuously with VS produced comparatively higher frequencies of micronucleated cells than those treated for 4 hr. Highest micronuclei frequencies were observed 24 hr after chemical treatment in both mononucleated and binucleated cells and decreased later with time. However, the micronucleus frequencies remained significantly higher than the controls even in the cells harvested at 144 hr. VS induced a large number of micronucleated cells with multiple micronuclei. VS also caused a severe decrease in nuclear division due to cytotoxic effect. Lymphocytes treated with EDB for 4 hr and continuously showed a statistically higher incidence of micronuclei in binucleated cells compared to the controls (P less than 0.05), whereas in mononucleated cells higher micronucleus frequencies were observed only in cultures treated continuously. Continuous presence of EDB induced both dose- and time-dependent increase of micronuclei in both mono- and binucleated cells (P less than 0.05). EDB induced relatively few multiple micronucleated cells in comparison with VS. EDB did not affect nuclear divisions even with continuous treatment. High micronucleus frequencies observed at 144 hr harvest following 4 hr treatment of both EDB and VS suggest the persistence of DNA damage in cells. These studies suggest that micronuclei kinetics in human peripheral lymphocytes depends on the genotoxic potentially and cytotoxicity of a genotoxicant.

Cell Division↗

PubMed1992

Clastogenic and aneuploidogenic effects of cigarette smoke condensate, mitomycin C and vincristine sulfate.

Immunofluorescent staining of kinetochores in micronuclei (MN) of cytochalasin-B-blocked cells was used to distinguish between clastogenic and aneuploidogenic effects of cigarette smoke condensate (CSC), mitomycin C (MMC) and vincristine sulfate (VS) in Chinese hamster lung V79 cells by using an anti-kinetochore antibody. Within the base-line micronucleated cells (2%), 50 to 58% contained kinetochore-positive (KC+) MN. MMC induced a significantly higher number of MN compared to the controls (P < 0.01), and the majority of micronucleated cells (77% at 0.08 microgram/ml) contained kinetochore-negative (KC-)MN. In contrast, VS induced a large number of MN (P < 0.01) but most of the micronucleated cells contained KC+ MN (93% at 0.08 microgram/ml). Cigarette smoke condensate (CSC) induced both KC+ (P < 0.01) and KC-(P < 0.05) MN suggesting the presence of both clastogenic and aneuploidogenic agents. However, the higher frequency of KC+ than KC- micronucleated cells suggests a stronger aneuploidogenic effect of CSC.

Aneuploidy↗

PubMed1991

Studies on three structurally related phenylenediamines with the mouse micronucleus assay system.

Three structurally related compounds, 4-chloro-o-phenylenediamine (COP), 4-nitro-o-phenylenediamine (NOP) and p-phenylenediamine dihydrochloride (PPD), are used in fur dyes, inks and hair coloring formulations. COP has been reported to be carcinogenic in both rats and mice. NOP and PPD are non-carcinogens, but have consistently tested positive in short-term in vitro genotoxicity assays. Studies were undertaken to evaluate their genotoxicity with the in vivo mouse bone-marrow micronucleus assay. Five CD-1 male mice per dose were injected i.p. with the compounds and sacrificed at intervals of 24, 48 and 72 h. 2000 cells were scored per animal to determine the frequency of micronucleated-polychromatic erythrocytes (MPCE). COP induced significant dose-related increases in MPCE over the 3 doses tested at each of the sampling intervals. The peak response occurred at 24 h. No response was observed in animals treated with PPD or NOP.

Animals↗

PubMed1991

Antimutagenicity studies of chlorophyllin using the Salmonella arabinose-resistant assay system.

Studies with the arabinose-resistant Salmonella forward mutation assay system were performed to determine the antimutagenic activity of chlorophyllin against the mutagenic activity of aflatoxin B1 (AFB1), 2-aminoanthracene (2AA), benzo[a]pyrene (BaP), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and solvent extracts of coal dust (CD), diesel emission particles (DE), airborne particles (AP), tobacco snuff (TS), black pepper (BP) and red wine (RW). Various concentrations of each chemical and complex mixture extract were assayed for mutagenic activity with and/or without S9 in a preincubation test. One concentration of each chemical and complex mixture extract was then tested with various concentrations of chlorophyllin. Results showed that chlorophyllin, at concentrations of 2.5 mg/plate or less, completely or almost completely inhibited the mutagenicity of 2AA, AFB1, BaP, MNNG and solvent extracts of CD, DE and RW. With concentrations from 1.25 to 5 mg/plate, chlorophyllin inhibited over 50% of the mutagenicity of AP, TS and BP extracts. These results further substantiate the antimutagenic efficacy of chlorophyllin against chemicals and complex mixtures.

Aflatoxin B1↗

PubMed1990

Antibodies to actin in autoimmune neutropenia.

In an effort to characterize the cellular antigens recognized by anti-neutrophil antibodies in autoimmune neutropenia, we studied sera, purified immunoglobulin G (IgG) and isolated F(ab')2 from 70 neutropenic patients suspected of this diagnosis. Anti-neutrophil antibodies were found in the sera of 36 of these patients by either 125I-staph A binding or immunofluorescence cytometric techniques that detected increased binding of patients' IgG to normal neutrophils. Anti-neutrophil antibody positive sera were then evaluated for specific binding to electrophoretically separated neutrophil membrane-associated proteins by immunoblotting. A 43-Kd protein was consistently identified by eight anti-neutrophil antibody positive sera. The specificity of binding to this protein was confirmed with affinity purified IgG and F(ab')2 fragments prepared from these sera. Sera from 20 healthy normal controls and from 22 non-neutropenic, anti-neutrophil antibody negative rheumatoid arthritis patients failed to bind this protein. Separate studies identified the 43-Kd protein as actin. Purified Acanthamoeba actin comigrated with the protein and was specifically bound by anti-neutrophil antibody positive IgG. Moreover, two actin-specific monoclonal antibodies bound to the 43-Kd membrane-associated protein in immunoblots. In addition, a rabbit anti-actin antiserum not only bound to this same 43-Kd protein but also expressed anti-neutrophil antibody activity against normal human neutrophils, as did purified human anti-actin IgG prepared by affinity chromatography from the serum of one of the index patients. These studies indicate that the anti-neutrophil antibodies of certain patients with autoimmune neutropenia include autoantibodies specific for actin. The molecules on the surface of neutrophils, which have actin-like antigenic epitopes and are recognized by these anti-actin antibodies, remain to be characterized.

Actins↗

PubMed1989

Studies of signal transduction in the respiratory burst-associated stimulation of fMet-Leu-Phe-induced tubulin tyrosinolation and phorbol 12-myristate 13-acetate-induced posttranslational incorporation of tyrosine into multiple proteins in activated neutrophils and HL-60 cells.

A specific stimulation of tubulin tyrosinolation in human neutrophils (PMNs) is induced by the synthetic peptide chemoattractant N-formylmethionylleucylphenylalanine (fMet-Leu-Phe), and this stimulation is closely associated with activation of the NADPH oxidase-mediated respiratory burst (Nath, J., and Gallin, J. I. (1983) J. Clin. Invest. 71, 1273-1281). In contrast, along with tubulin tyrosinolation, a distinctly different respiratory burst-associated random posttranslational incorporation of tyrosine into multiple PMN proteins is observed in PMNs stimulated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) or sn-1,2-dioctanoylglycerol (DAG). In studies exploring the mechanism(s) of signal transduction for these distinct neutrophil responses, we found that the fMet-Leu-Phe-induced stimulation of tubulin tyrosinolation in PMNs and in differentiated HL-60 cells is completely blocked by pertussis toxin, while the PMA-induced random incorporation of tyrosine is not inhibited. We also found that expression of the fMet-Leu-Phe-mediated stimulation of tubulin tyrosinolation in HL-60 cells is correlated with increases in the specific activity of protein kinase C and with the acquisition of respiratory burst activity which occur during induced myeloid maturation of these cells. Furthermore, both the fMet-Leu-Phe-induced stimulation of tubulin tyrosinolation and the PMA or DAG-induced random posttranslational incorporation of tyrosine into multiple proteins in activated neutrophils, were found to be reversibly inhibited (greater than 70%) by the protein kinase inhibitors 1-(5-isoquinolinesulfonyl)piperazine (C-I) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), in parallel with inhibition of superoxide (O2-) generation. In related studies, we also found that fMet-Leu-Phe-stimulated O2- production is comparably inhibited by C-I and H-7, but in a highly temperature-dependent manner. Inhibition was observed only when C-I or H-7 is added to PMNs at physiologic temperature, i.e. 37 degrees C. Interestingly, inhibition of the PMA-induced O2- generation by C-I or H-7 was not found to be similarly temperature-dependent. Considered together, these findings argue against the suggestion that there is a protein kinase C-independent pathway for activation of the respiratory burst in neutrophils stimulated with N-formyl peptides.

Cell Line↗

PubMed1989

Effects of quinoline and 8-hydroxyquinoline on mouse bone marrow erythrocytes as measured by the micronucleus assay.

Both quinoline and 8-hydroxyquinoline (HOQ) were tested for their genotoxicity in CD1 male mice by using a bone marrow micronucleus assay. Mice were intraperitoneally treated in single injections with three dose levels (25, 50, and 100 mg/kg) of each chemical with corn oil as solvent vehicle. Bone marrow was sampled at 24, 48, and 72 h postinjection. Quinoline resulted in a significant dose-related increase in the number of micronucleated polychromatic erythrocytes (MPCE) at the 24 h sampling time for all doses tested. The high dose (100 mg/kg) and the medium dose (50 mg/kg) also induced statistically significant increases (P less than .05) in the number of MPCEs at 48 h interval. The ratios of polychromatic to normochromatic erythrocytes at the 24 h sampling time were lower for the treated than the control animals. Although HOQ resulted in some increases in the number of MPCEs over the control, this compound induced a statistically significant increase in the number of micronucleated normochromatic erythrocytes (MNCEs) at all three doses following 24 h treatment. Both low and medium doses also induced a higher incidence of MNCEs at the 48 and 72 h sampling times. No data were available for the high dose at these times. The cytotoxic effect of this compound was expressed as low PCE/NCE ratios with all doses at 24 h after injection and as a high mortality rate in animals treated with the high dose (100 mg/kg).

Animals↗