Effect of immobilization and concurrent exposure to a pulse-modulated microwave field on core body temperature, plasma ACTH and corticosteroid, and brain ornithine decarboxylase, Fos and Jun mRNA.

Exposure of humans and rodents to radiofrequency (RF) cell phone fields has been reported to alter a number of stress- related parameters. To study this potential relationship in more detail, tube-restrained immobilized Fischer 344 rats were exposed in the near field in a dose-dependent manner to pulse-modulated (11 packets/s) digital cell phone microwave fields at 1.6 GHz in accordance with the Iridium protocol. Core body temperatures, plasma levels of the stress-induced hormones adrenocorticotrophic hormone (ACTH) and corticosterone, and brain levels of ornithine decarboxylase (Odc), Fos and Jun mRNAs were measured as potential markers of stress responses mediated by RF radiation. We tested the effects of the loose-tube immobilization with and without prior conditioning throughout a 2-h period (required for near-field head exposure to RF fields), on core body temperature, plasma ACTH and corticosteroids. Core body temperature increased transiently (+/-0.3 degrees C) during the initial 30 min of loose-tube restraint in conditioned animals. When conditioned/tube-trained animals were followed as a function of time after immobilization, both the ACTH and corticosterone levels were increased by nearly 10-fold. For example, within 2-3 min, ACTH increased to 83.2 +/- 31.0 pg/dl, compared to 28.1 +/- 7.7 pg/dl for cage controls, reaching a maximum at 15-30 min (254.6 +/- 46.8 pg/dl) before returning to near resting levels by 120 min (31.2 +/- 10.2 pg/dl). However, when non-tube-trained animals were submitted to loose-tube immobilization, these animals demonstrated significantly higher (3-10-fold greater) hormone levels at 120 min than their tube-trained counterparts (313.5 +/- 54.8 compared to 31.2 +/- 10.2 pg/dl; corticosterone, 12.2 +/- 6.2 microg/dl compared to 37.1 +/- 6.4 microg/dl). Hormone levels in exposed animals were also compared to those in swim-stressed animals. Swimming stress also resulted in marked elevation in both ACTH and corticosterone levels, which were 10-20 fold higher (541.8 compared to 27.2-59.1 pg/dl for ACTH) and 2-5 fold higher (45.7 compared to 8.4- 20.0 microg/dl for corticosteroids) than the cage control animals. Three time-averaged brain SAR levels of 0.16, 1.6 and 5 W/ kg were tested in a single 2-h RF-field exposure to the Iridium cell phone field. When RF-exposed and sham-exposed (immobilized) animals were compared, no differences were seen in core body temperature, corticosterone or ACTH that could be attributed to near-field RF radiation. Levels of Odc, Fos and Jun mRNA were also monitored in brains of animals exposed to the RF field for 2 h, and they showed no differences from sham-exposed (loose-tube immobilized) animals that were due to RF-field exposure. These data suggest that a significant stress response, indicated by a transient increase in core body temperature, ACTH and corticosterone, occurred in animals placed in even the mild loose-tube immobilization required for near-field RF exposure employed here and in our other studies. Failure to adequately characterize and control this immobilization response with appropriate cage control animals, as described previously, could significantly mask any potential effects mediated by the RF field on these and other stress-related parameters. We conclude that the pulse-modulated digital Iridium RF field at SARs up to 5 W/kg is incapable of altering these stress-related responses. This conclusion is further supported by our use of an RF-field exposure apparatus that minimized immobilization stress; the use of conditioned/tube-trained animals and the measurement of hormonal and molecular markers after 2 h RF-field exposure when the stress-mediated effects were complete further support our conclusion.

Recent advances in research on radiofrequency fields and health.

Since the Royal Society of Canada report on potential health risks of radiofrequency (RF) fields from wireless telecommunications in the spring of 1999, there have been several newly published reports on risks associated with the use of mobile phones. This article provides a summary of scientific research on the potential health effects of radiofrequency fields that has been reported since the original Royal Society report was published. This update also discusses several earlier results not included in the original report.

Optimization of cDNA representational difference analysis for the identification of differentially expressed mRNAs.

Representational difference analysis (RDA) is a powerful and sensitive tool for identification of differentially expressed genes (M. Hubank and D. G. Schatz, 1999, Methods Enzymol. 303, 325-349; 1994, Nucleic Acids Res. 22, 5640-5648) that will identify both up- and downregulated genes differentially expressed between two cDNA populations. This manuscript provides a thorough description of an optimized RDA method. This procedure while still based on the traditional RDA originally developed by Lisitsyn and co-workers(N. A. Lisitsyn, 1995, Trends Genet. 11, 303-307; N. A. Lisitsyn, F. S. Leach, B. Vogelstein, and M. H. Wigler, 1994, Cold Spring Harbor Symp. Quant. BioL 59, 585-587; N. Lisitsyn, N. Lisitsyn, and M. Wigler, 1993, 259, 946-951) and modified by Hubank and Schatz for RNA (1994, Nucleic Acids Res. 22, 5640-5648) is improved and requires less starting material than many existing methods. Several key modifications are included (1). Size-exclusion gel-filtration microspin columns are used throughout the procedure to remove the primers and low molecular weight cDNAs. This results in reducing the number of ethanol precipitations required and in improving the yield of desirable amplification products (2). Elimination of the mung bean nuclease treatment in favor of a simple dilution of PCR serves as a means of markedly reducing the single-stranded cDNAs that can interfere with the amplification of differentially expressed products (3). The use of up to six unique noninteracting primers ensures that no anomalous amplification occurs due to carryover of primers or incomplete digestion from the ends of the cDNAs (4). A set of cDNA standards was developed and various concentrations were used to better characterize the ability of representational difference analysis to identify rare messages in a complex cDNA population (5). Integral to this manuscript, a detailed laboratory protocol is available from the authors (craig.byus@ucr.edu) and provides a step-by-step description of the modified procedure.

Spontaneous and nitrosourea-induced primary tumors of the central nervous system in Fischer 344 rats exposed to frequency-modulated microwave fields.

In a 2-year bioassay, we exposed Fischer 344 rats to a frequency-modulated (FM) signal (836.55 MHz +/- 12.5 KHz deviation) simulating radiofrequency exposures in the head of users of hand-held mobile phones. We tested for effects on spontaneous tumorigenicity of central nervous system (CNS) tumors in the offspring of pregnant rats and also for modified incidence of primary CNS tumors in rats treated with a single dose of the neurocarcinogen ethylnitrosourea (ENU) in utero. ENU dosage (4 mg/kg) was selected to give an expected brain tumor incidence of 10-15% over the mean life span of 26 months. Pregnant dams (n = 102) were randomly assigned to six groups. Their offspring were treated as cohorts in each of the six groups (n = 90 per group; total, n = 540): Sham ENU/Sham Field, Sham ENU/Field Exposed, ENU/Sham Field, ENU/Field Exposed, ENU/Cage Control, and Sham ENU/Cage Control. Intermittent field exposures began on gestation day 19 and continued until weaning at 21 days, resuming thereafter at 31 days and continuing until experiment termination at 731-734 days. Energy absorption rates (SARs) in the rats' brains were similar to localized peak brain exposures of a phone user (female, 236 g, 1.0 W/kg; male, 450 g, 1.2 W/kg). Of the original 540 rats, 168 died before the termination of the experiment. In these rats, ENU significantly reduced survival from a mean of 708 days in three groups without ENU treatment to 645 days in three groups treated with ENU (P < 0.0005). There were no effects on survival attributable to FM field exposure in either ENU-treated or in sham-treated groups. Spontaneous CNS tumor incidence in control groups was 1.1-4.4% but sharply higher in rats receiving ENU (14.4-22.2%; P < 0.0001). No FM field-mediated changes were observed in number, incidence, or histological type of either spontaneous or ENU-induced brain tumors, nor were gender differences detected in tumor numbers. These negative findings with FM fields contrast with our study using standard digital phone fields pulsed on and off at 50/se, where a trend was noted toward reduced incidence of both spontaneous and ENU-induced CNS tumors (W. R. Adey et al., Radiat. Res., 152: 293-302, 1999). Although consistent but not attaining significance in the experiment overall (spontaneous CNS tumors, P < 0.08 one-tailed; P < 0.16 two-tailed; ENU-induced CNS tumors, P < 0.08 one-tailed, P < 0.16 two-tailed), the trend was significant (P < 0.015 one-tailed, P < 0.03, two-tailed) in rats that received ENU and died prior to experiment termination, with a primary brain tumor as the cause of death. We discuss differences in the signaling structure of digital and FM fields. Certain bioeffects induced by either amplitude-modulated or pulsed radiofrequency fields at athermal levels have not been seen with fields of similar average power but unvarying in intensity (continuous wave or frequency-modulated fields).

Spontaneous and nitrosourea-induced primary tumors of the central nervous system in Fischer 344 rats chronically exposed to 836 MHz modulated microwaves.

We have tested an 836.55 MHz field with North American Digital Cellular (NADC) modulation in a 2-year animal bioassay that included fetal exposure. In offspring of pregnant Fischer 344 rats, we tested both spontaneous tumorigenicity and the incidence of induced central nervous system (CNS) tumors after a single dose of the carcinogen ethylnitrosourea (ENU) in utero, followed by intermittent digital-phone field exposure for 24 months. Far-field exposures began on gestational day 19 and continued until weaning at age 21 days. Near-field exposures began at 35 days and continued for the next 22 months, 4 consecutive days weekly, 2 h/day. SAR levels simulated localized peak brain exposures of a cell phone user. Of the 236 original rats, 182 (77%) survived to the termination of the whole experiment and were sacrificed at age 709-712 days. The 54 rats (23%) that died during the study ("preterm rats") formed a separate group for some statistical analyses. There was no evidence of tumorigenic effects in the CNS from exposure to the TDMA field. However, some evidence of tumor-inhibiting effects of TDMA exposure was apparent. Overall, the TDMA field-exposed animals exhibited trends toward a reduced incidence of spontaneous CNS tumors (P < 0. 16, two-tailed) and ENU-induced CNS tumors (P < 0.16, two-tailed). In preterm rats, where primary neural tumors were determined to be the cause of death, fields decreased the incidence of ENU-induced tumors (P < 0.03, two-tailed). We discuss a possible approach to evaluating with greater certainty the possible inhibitory effects of TDMA-field exposure on tumorigenesis in the CNS.

Putrescine export in Xenopus laevis oocytes occurs against a concentration gradient: evidence for a non-diffusional export process.

Putrescine export was found to occur by a non-diffusional highly regulated process using Xenopus oocytes as a model system of polyamine transport. Untreated oocytes were observed to possess high endogenous intracellular putrescine and spermidine levels with no detectable polyamine interconversion or biosynthesis over the assay intervals. The putrescine uptake process demonstrated a rapid saturation within a 5 min interval. Spermidine demonstrated a relatively larger uptake capacity with only a minimal ability to export. A kinetic analysis of the concentration-dependence of the putrescine and spermidine uptake processes indicated that the putrescine uptake process may possess two concurrent uptake components while spermidine uptake may possess a two-component process with an allosteric regulation. Elevated intracellular putrescine levels were observed to decrease against a 10-fold higher extracellular concentration gradient in a rapid and specific manner. No noticeable changes in the intracellular levels of other polyamines were observed over the same time interval. The uptake and export rates of putrescine transport also showed a concurrent, rapid and cyclical regulation. These findings support a non-diffusional putrescine export process which is highly regulated.

Tolerance to putrescine toxicity in Chinese hamster ovary cells is associated with altered uptake and export.

When Chinese hamster ovary (CHO) cells were cultured with low concentrations of putrescine (< 5 mM) their cell cycle time increased significantly and a fraction of the cells died. A cell line tolerant to the cytotoxic and growth inhibitory effects of millimolar concentrations of putrescine was developed by growing CHO cells over many months in increasing concentrations of the polyamine. A putrescine-tolerant cell line was obtained which was capable of growing in concentrations up to 25 mM putrescine and displayed growth and cell division rates similar to the original untreated/parental CHO cells. The tolerant cells grown in putrescine displayed relatively high intracellular putrescine yet the cell-associated putrescine concentration was estimated to be 10-fold less than the culture medium level. This high concentration of cellular putrescine diminished within 60 min when the cells were changed to non-putrescine-containing media. The putrescine-tolerant phenotype was further characterized in regards to the mechanisms involved in putrescine uptake, efflux, and biosynthesis. The parental and tolerant cell lines had similar or identical levels of cellular spermidine and spermine and no differences in the acetylated polyamine pools or diamine oxidase activity. The activity of ornithine decarboxylase was also similar in the two cell lines in both the presence and the absence of ornithine. The tolerant cells, however, had a decreased uptake rate for putrescine. The tolerant cell line also showed a greatly enhanced ability to export putrescine, especially when treated with ornithine, suggesting that an upregulated polyamine export system may be present in the tolerant cells which could be responsible for the increased cell survival in high putrescine concentrations. The data are discussed in regard to the potential for identifying the transport protein(s) responsible for the maintenance of nontoxic intracellular concentrations of putrescine in a tolerant cell line grown in putrescine.

A kinetic characterization of putrescine and spermidine uptake and export in human erythrocytes.

Using human erythrocytes as a model system for the study of mammalian polyamine transport, detailed kinetic parameters regarding the uptake and export of putrescine and spermidine were determined. The putrescine uptake data indicated a multi-component uptake system comprised of a low-capacity saturable component and a non-saturable component. The saturable putrescine uptake component demonstrated a calculated Km of 21.0 microM and a V(max) of only 6.52 x 10(-13) M/s. The non-saturable linear putrescine uptake rate was defined by a significant pH dependence, a lack of uptake inhibition by related polyamines, and a permeability pi of 3.19 x 10(-8) s-1. These findings suggested that non-saturable putrescine uptake involved a process of simple diffusion. Spermidine uptake exhibited Michaelis-Menten kinetics with a Km and Vmax of 12.5 microM and 1.36 x 10(-12) M/s, respectively. Spermidine uptake did not demonstrate pH dependence and was not significantly inhibited by any of the tested polyamines. The Arrhenius plot of spermidine uptake was determined to be biphasic with calculated activation energies of spermidine uptake of 135.2 kJ/mol for 19-21 degrees C and 59.3 kJ/mol for 21-35 degrees C. These data suggest the possibility of multiple spermidine uptake processes which are not mediated by simple diffusion across the cell membrane. The putrescine export process demonstrated both saturable and non-saturable components. The calculated Km, V(max) and pi for putrescine export were 33.8 microM, 1.19 x 10(-11) M/s and 2.81 x 10(-7) s-1, respectively. The spermidine export process was non-saturable up to intracellular spermidine concentrations of 4 microM. At similar intracellular and extracellular concentrations of putrescine and spermidine, however, export processes displayed rates which were an order of magnitude greater than their respective uptake rates. This finding supports the possible presence of mediated putrescine and spermidine export processes different than simple diffusion.

Regulation of the efflux of putrescine and cadaverine from rapidly growing cultured RAW 264 cells by extracellular putrescine.

Cultures of the macrophage-like RAW 264 cells were adapted to divide normally in a synthetic serum-supplemented culture medium lacking any polyamines and diamine oxidase activity. These rapidly dividing cells actively effluxed large amounts of putrescine and cadaverine, compared with the intracellular levels, into the culture medium. The efflux of putrescine was stimulated by the amino acid ornithine, whereas efflux of cadaverine was inhibited. Relatively low levels of spermidine and N1-acetyl-spermidine, compared with those of exported putrescine, were observed to accumulate in the culture medium. A careful analysis of the changes in the intracellular concentration of putrescine relative to the steady-state net rate of putrescine export, as the doubling time of the cultures increased from 16 h to 22 h, indicated that an inverse relationship existed between these two parameters. As the intracellular putrescine concentrations increased, the net rate of putrescine export decreased markedly. Determination of the rate of putrescine uptake indicated that putrescine uptake also decreased significantly as the cultures neared confluency, and at no time during the growth of the culture did the rate of putrescine uptake approximate to the high rate of putrescine efflux. The decrease in the putrescine export rate seen as the cells grew toward confluency was determined to be primarily due to the inhibitory effect of the effluxed putrescine in the medium (Ki = 2 microM), and not to contact inhibition. The data suggested that the efflux of putrescine and cadaverine is not mediated to a significant degree by a process involving simple diffusion.

Characterization of putrescine and cadaverine export in mammalian cells. A pharmacological approach.

We characterized the mechanism(s) involved in the efflux of putrescine/cadaverine from cultured mammalian cells using various pharmacological agents. Verapamil and quinine inhibited putrescine and cadaverine export in monocytic-leukemic RAW 264 and H35 hepatoma cells in a concentration-dependent manner with an IC50 in the micromolar range. Verapamil, which inhibits L-type calcium channels, inhibited putrescine export, regardless of whether calcium was present in the extracellular medium or not. Furthermore, the export of putrescine in the absence of verapamil did not appear to depend upon extracellular calcium. Neither intracellular calcium, external sodium, changes in intracellular pH nor phosphorylation affected the levels of putrescine export independently from changes in intracellular putrescine levels. The data suggest that verapamil and quinine inhibit putrescine/cadaverine efflux from the cell by binding directly to an integral membrane protein.

Selective putrescine export is regulated by insulin and ornithine in Reuber H35 hepatoma cells.

Cultured Reuber H35 rat hepatoma cells under highly viable serum-free conditions were found to selectively export putrescine from inside the cell into the culture medium, but not spermidine, spermine, or their acetylated derivatives. Even untreated cells, with very low intracellular putrescine levels, constitutively exported significant amounts of only putrescine for a 12 h period. Administration of the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) which markedly elevates ornithine decarboxylase (ODC), did not potentiate putrescine export over what was measured in the unstimulated cultures. However, addition of 1 mM ornithine to the cultures resulted in increased intracellular putrescine (maximum at 4 h) with a marked concomitant increase in putrescine export between 0 and 8 h, after which putrescine export again stopped. Treatment with 10(-7) M insulin yielded intracellular putrescine levels that remained elevated for 36 along with a continuous and more rapid export of putrescine over the same 36 h time period. When insulin and ornithine were administered together, even higher levels of intracellular putrescine and putrescine export were observed, with putrescine efflux proceeding over the 36 h time-course at the highest observed rates of 1.5 (0-12 h) and 1.0 (12-36 h) nmol/mg total protein per h. Exposure to DFMO, an inhibitor of ODC, depleted intracellular putrescine stores and effectively suppressed putrescine export. There was not a positive correlation between the time-dependent decreases in the intracellular putrescine concentrations and the respective alterations in the rate of putrescine export under a variety of conditions. Furthermore, the drug verapamil was capable of completely inhibiting putrescine export (IC50 approx. 1 microM) without any change in the level of intracellular putrescine. This data was not consistent with the involvement of simple diffusion of putrescine through the membrane as the major mechanism for putrescine export. The potential mechanisms involved in putrescine export and the role of this process in regulating intracellular polyamine levels, as well as, possible functions of extracellular putrescine are discussed.

Regulation of putrescine export in lipopolysaccharide or IFN-gamma-activated murine monocytic-leukemic RAW 264 cells.

The regulation of putrescine/polyamine export out of the cell was investigated during activation of monocytic-leukemic RAW 264 cells with LPS and IFN-gamma. The RAW 264 cells exported putrescine constitutively at a significant rate into the culture medium. This export process appeared to be selective for putrescine in that only a small amount of other polyamines (spermidine and N1-acetylspermidine) was found in the culture medium. LPS and IFN-gamma alone and in combination markedly stimulated putrescine export and nitrite production throughout a 24-h period. The efflux of putrescine but not nitrite was further increased by the addition of ornithine (the amino acid precursor of putrescine) to the culture medium. LPS and ornithine also stimulated the intracellular accumulation of putrescine in primary inflammatory macrophages and the export of putrescine into the peritoneal exudate of the mouse. A detailed comparison of the steady state rates of accumulation of intracellular putrescine/polyamines and the rate of putrescine efflux from the cells constitutively and after LPS, IFN-gamma, and ornithine indicated that a surprisingly large fraction of total polyamine biosynthesis is comprised of exported putrescine. The observed dose-dependent inhibition of putrescine export with the drug verapamil implicated the involvement of a specific membrane transport system sensitive to calcium influx in this process. The data are discussed in regard to the potential involvement of putrescine export in the regulation of intracellular polyamine levels, cell differentiation, and macrophage-mediated cytotoxicity.

Biosynthesis and selective export of 1,5-diaminopentane (cadaverine) in mycoplasma-free cultured mammalian cells.

Macrophage-like RAW 264 and H35 hepatoma cells grown under serum-free conditions exported putrescine and an unidentified diamine into the culture medium. Unlike putrescine, the unknown compound could be detected only extracellularly. Analyses of dansylated polyamine standards and mass spectroscopy confirmed that the unknown compound was cadaverine (1,5-diaminopentane). The cells were free of mycoplasma as evidenced by a negative result using a probe specific for prokaryotic rRNA. After prophylactic treatments with two different mycoplasmacidal agents, the cells continued to export cadaverine. Attempts to "infect" a noncadaverine-exporting cell line with culture medium and cell-free lysates proved unsuccessful, establishing that cadaverine was in fact a bona fide product of these mammalian cells. Cadaverine export by RAW 264 and H35 cells was stimulated by lipopolysaccharide and insulin, respectively. However, administration of exogenous ornithine caused cadaverine export to decrease significantly with concomitant increases in putrescine export. alpha-Difluoromethylornithine, a selective inhibitor of ornithine decarboxylase, inhibited both cadaverine and putrescine export. When cells were labeled with [3H]lysine, the great majority of the radioactivity recovered in exported polyamines was found in cadaverine. The cumulative data suggested that cadaverine formation may be caused by the action of intracellular ornithine decarboxylase upon lysine to produce cadaverine, which is then effluxed from the cell with a high degree of efficiency.

Acute hypoxia increases ornithine decarboxylase activity and polyamine concentrations in fetal rat brain.

The cellular responses to hypoxia are poorly understood. To test the hypothesis that ornithine decarboxylase (ODC; L-ornithine carboxy-lyase; EC 4.1.1.17) activity and polyamine concentrations change in response to acute hypoxia, we performed the following studies. Pregnant Sprague-Dawley rats inspired various O2 concentrations (9-21%) for various time periods (0.5-48 h) from days 15 to 21 of gestation. In fetal brains we measured the activity of ODC, ODC mRNA, and polyamines. In response to 4-h acute mild hypoxia, ODC activity in fetal rat brain (cerebrum, cerebellum, and hippocampus) increased to 330-450% from control values (P < 0.001), after which it declined to control levels in 6-8 h. The 4-h ODC response varied inversely with inspired O2 concentration and was not mimicked by beta 2 agonist or blocked by beta 2-antagonist administration. The ODC response was associated with an increase in fetal brain putrescine concentration to 190% above control at 4-6 h (P < 0.01) and an increase in the polyamines spermidine and spermine to about 115% above control at 6-8 h. We also observed that ODC mRNA increased significantly after 2-4 h of hypoxia. ODC activity and polyamine concentrations appear to be useful enzymatic markers for fetal brain hypoxia. The magnitude and time course of the acute hypoxic ODC increase were similar to responses to extracellular signals that result in differentiation or cell growth. Thus, the well-defined and regulated ODC activity response may represent a protective mechanism in brain to hypoxia.

An ion-exchange chromatography procedure for the isolation and concentration of basic amino acids and polyamines from complex biological samples prior to high-performance liquid chromatography.

The original objective of this study was to develop a selective and sensitive method for the analysis and quantification of basic amino acids from biological samples via reversed-phase high-performance liquid chromatography. Using various previously described techniques for the separation of amino acids, we were unsuccessful in measuring levels of histidine, arginine, ornithine, and lysine in biological samples due to the presence of interfering compounds. A "cleanup" procedure for the isolation of the basic amino acids using a weakly acidic cation exchange resin, Biorex-70 (Bio-Rad), is described in detail. Upon separation from the bulk of the neutral and acidic amino acids, the basic amino acids were subjected to precolumn fluorescence derivatization using 9-fluorenylmethyl chloroformate (FMOC) and the fluorescent derivatives were separated by RP-HPLC. The advantages of this method over previously described amino acid analysis techniques are (i) isolation and stable recovery (greater than 95%) of the desired basic amino acids, (ii) sensitivity of detection (low pmol range), (iii) complete resolution of derivatized amino acids via HPLC, (iv) limited amount of sample required for analysis, and (v) samples readily concentrated by lyophilization or rotoevaporating. This ion-exchange cleanup procedure was also adapted for the analysis of polyamines in concentrated culture media samples and proved additionally advantageous by eliminating the use of costly C-18 extraction columns required by previously described techniques.

The level of substrate ornithine can alter polyamine-dependent DNA synthesis following phorbolester stimulation of cultured hepatoma cells.

Although the precise intracellular function(s) of the polyamines remain incompletely defined, a myraid of evidence now shows that the polyamines must accumulate or be maintained at a specific intracellular concentration in order for all mammalian cells to grow or divide. The initial step in polyamine biosynthesis normally involves the decarboxylation of ornithine by the enzyme ornithine decarboxylase (ODCase E.C. 4.1.1.17) to yield putrescine. Increases in the steady-state level of intracellular ornithine have been reported to markedly alter the accumulation of the polyamines following stimulation of Reuber H35 Hepatoma cells with 12-O-tetradecanoylphorbol-beta-acetate (TPA) in the presence of serum (Wu and Byus: (Biochem. Biophys. Acta 804:89-99, 1984); Wu et al.: (Cancer Res. 41:3384-3391, 1981). We wished to determine whether or not incubation of H35 hepatoma cells with exogenous ornithine would result in a stimulation of DNA synthesis following treatment with the mitogens TPA and insulin. For these studies, H35 cells were maintained under serum-free conditions for 2-3 days in order to obtain synchronous cultures suitable for analysis of the level of DNA synthesis. Cultures treated in this manner were highly viable, maintained similar growth rates, and possessed the equivalent levels of intracellular ornithine and polyamines as the serum-containing cultures. Arginine levels, however, were approximately twofold higher following culture under serum-restricted conditions for 3 days. The addition of exogenous ornithine (0.5 mM) was accompanied by a 4-5-fold increase in intracellular steady-state ornithine levels and by a 6-8-fold increase in the presence of TPA and ornithine. In a manner identical to the serum-containing cultures (Wu and Byus (1984] the addition of TPA and exogenous ornithine to the serum-free cells caused a dose-dependent increase in intracellular putrescine (up to 5-fold) and a concomitant decrease in ODC activity in comparison to stimulation with TPA alone. The addition of TPA led to a 3-5-fold increase in the incorporation of tritiated thymidine into DNA. In the presence of exogenous ornithine, TPA-induced DNA synthesis was further stimulated more than twofold in a dose-dependent manner. Insulin (10(-10)-10(-8) M) proved to be more efficacious as a mitogen in the H35 cells and led to greater stimulation of DNA synthesis than TPA. Insulin alone also resulted in a higher steady-state level of ornithine and putrescine in comparison with TPA alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dietary arginine restriction upon ornithine and polyamine metabolism during two-stage epidermal carcinogenesis in the mouse.

Polyamine synthesis is required in normal or neoplastic tissues if they are to continue to grow or divide. The highly inducible enzyme ornithine decarboxylase (ODC) catalyzes the conversion of ornithine to putrescine as the initial step in polyamine biosynthesis. The level of substrate pools of ornithine in cultured cells has been reported to markedly alter mitogen-induced ODC activity, putrescine accumulation, and DNA synthesis (V. Wu and C. V. Byus, Biochim. Biophys. Acta, 804: 89-99, 1984; V. Wu et al., Cancer Res., 41: 3384-3391, 1981). We attempted to limit the amount of ornithine available for polyamine biosynthesis in an animal by using a dietary approach. Since arginine serves as one of the intermediate biosynthetic precursors of ornithine, female CD-1 mice were placed on a special synthetic amino acid diet deficient in arginine. The ability of this arginine-free diet to alter epidermal ornithine and polyamine metabolism and tumorigenesis was assessed in the mouse two-stage model of skin carcinogenesis. The basal level of ornithine in the epidermis in control animals receiving the amino acid complete diet was very high compared to other tissues (155 nmol/mg protein). However, when the mice were fed the isocaloric arginine-free diet for a 2-week period, the levels of epidermal ornithine and arginine decreased by 40% (P less than 0.01). This reduction was blocked by the addition of 2% ornithine to the drinking water of the arginine-restricted animals. Acute administration of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to the epidermis caused a transient (4 and 8 h) reduction in ornithine and arginine but not lysine in the animals receiving the control, and ornithine-supplemented diets. The animals fed the special arginine-free diet exhibited a 40-50% reduction in tumor multiplicity or papillomas/mouse (P less than 0.05) and had a significantly lower tumor incidence or percentage of animals with tumour throughout a 19-week promotion period (P less than 0.02). However, the major effect of arginine restriction was consistent with an increase in tumor latency. The addition of ornithine completely reversed the reduction in the rate and extent of tumorigenesis in the arginine-free animals. The accumulation of putrescine (but not spermidine or spermine) in the epidermis following a single administration of TPA was significantly reduced in the animals receiving the arginine-free diet. The papillomas or tumors from the animals deprived of arginine had markedly reduced (less than 35%) levels of putrescine compared to the tumors from control animals, and appeared to be more sensitive to dietary arginine restriction than was the chronically promoted but untransformed epidermis.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased ornithine decarboxylase activity in cultured cells exposed to low energy modulated microwave fields and phorbol ester tumor promoters.

Ornithine decarboxylase (ODC) is present in all nucleated cells and is the rate-limiting enzyme for synthesis of polyamines. In turn, the polyamines are required for DNA synthesis and cell growth. In Reuber H35 hepatoma cells, we show that ODC activity is increased by about 50% during exposure to a 1-h "athermal" (less than 0.1 degree C temperature rise) (450 MHz, 1.0 mW/cm2 peak-envelope-power) microwave field sinusoidally amplitude-modulated at 16 Hz. The increased activity of ODC persisted for several hours following the 1-h exposure to the field. A similar field amplitude-modulated at 60 and 100 Hz did not alter the hepatoma cell ODC activity. The stimulated ODC activity in the cultured cells that followed treatment with a phorbol ester tumor promoter (12-O-tetradecanoylphorbol-13-acetate) was further potentiated by prior exposure to the same low energy electromagnetic field. This field did not alter either basal or 12-O-tetradecanoylphorbol-13-acetate-stimulated DNA synthesis. We observed a similar increase in the basal ODC activity of cultures of two additional cell lines (Chinese hamster ovary; and 294T melanoma) exposed for 1 h to the amplitude-modulated field. Chinese hamster ovary cells exposed to the radio frequency field for 1 h also responded to subsequent treatment with 12-O-tetradecanoylphorbol-13-acetate by exhibiting a further increase in ODC activity. We have observed previously that the activity of this enzyme is increased in cultured cells following a transient exposure to a 60-Hz electric field. Altered ODC activity may serve as a sensitive and specific molecular marker of the transductive coupling of weak pericellular electromagnetic fields to biological systems.