ODC Activity Research Articles

Bifidobacterium longum, a lactic acid-producing intestinal bacterium inhibits colon cancer and modulates the intermediate biomarkers of colon carcinogenesis.

The human colon can be described as a complex microbial ecosystem, comprising several hundred bacterial species. Some of these enteric bacteria are beneficial to the host and have been shown to exert antimutagenic and anticarcinogenic properties. We have investigated the colon tumor inhibitory activity of Bifidobacterium longum, a lactic acid-producing enterobacterium. The modifying effects of this lactic culture on colonic mucosal and/or tumor cell proliferation, ODC activity and ras-p21 oncoprotein expression in colon carcinogenesis were also analyzed. Male F344 rats were fed a modified AIN-76A diet containing 0 or 2% lyophilized cultures of B. longum and s.c. administered azoxymethane (AOM) dissolved in normal saline at a dose of 15 mg/kg body wt, once weekly for 2 weeks. Vehicle controls received an equal volume of normal saline s.c. Animals were maintained on control or experimental diets until termination of the study. Animals intended for analysis of cell proliferation were killed 20 weeks after the second AOM injection, whereas animals intended for colon tumor analysis and measurement of ODC activity and ras-p21 expression were killed 40 weeks after the last AOM injection. The data demonstrate that dietary administration of lyophilized cultures of B. longum resulted in significant suppression of colon tumor incidence and tumor multiplicity and also reduced tumor volume. Results also revealed that ingestion of B. longum significantly inhibited AOM-induced cell proliferation, ODC activity and expression of ras-p21 oncoprotein. Data suggest that oral administration of probiotic B. longum exerts strong antitumor activity, as indicated by modulation of the intermediate biomarkers of colon cancer, and consequently reduced tumor outcome.

An HPLC-linked non-radiochemical assay of OPRT and ODC activity in erythrocytes: One case of orotic aciduria

An HPLC-linked non-radiochemical assay of OPRT and ODC activity in erythrocytes: One case of orotic aciduria

Polyaminen and brain injury

The cerebral ODC/polyamine system is disturbed by brain injury. The main modifications are important increases in ODC activity and putrescine concentration, with minor variations in spermidine and spermine concentrations. A great diversity of stimuli such as cerebral ischemia or overstimulation of the central nervous system by chemical or non-chemical agents can induce polyamine disturbances. Both the contribution of polyamines to the brain damage and their involvement in the repair mechanisms triggered after brain insults have been proposed.

The inhibitory effect of vitamin E on pulmonary polyamine biosynthesis, cell proliferation and carcinogenesis in mice

We investigated the modulating effect of vitamin E on pulmonary polyamine biosynthesis, cell proliferation and carcinogenesis in mice treated with urethane. Pulmonary ornithine decarboxylase induction and subsequent polyamine accumulation were observed during the initiation and promotion phases of the urethane-induced lung carcinogenesis in mice. The increases of ODC activity and polyamine level during both phases were almost inhibited when a high vitamin E diet was provided. The urethane-increased level of pulmonary proliferating cell nuclear antigen as a marker of cell proliferation during the carcinogenesis was inhibited by vitamin E treatment. Also, vitamin E suppressed the urethane-induced elevation of pulmonary cyclooxygenase activity as a marker of tumor promotion. In conjugation with these events, vitamin E reduced the development of lung tumors in mice treated with urethane. These results indicated that vitamin E could act as a useful chemopreventive agent against lung carcinogenesis in mice due to the regulation of cell proliferation.

Involvement of Polyamines in the Action of Transforming Growth Factor β and Interleukin-1 on Cultured Chick Embryo Fibroblasts

In this study we have examined the relationship between growth factor-induced proliferation and ODC/polyamine levels. TGFβ promotes cell growth and enhances [3H]-thymidine incorporation in chick embryo fibroblasts maintained in a serum-depleted medium. The action on DNA synthesis declines in the second day of treatment. IL-1 does not affect proliferation or [3H]-thymidine incorporation either when it is added alone or in combination with TGFβ. The response of the cells to TGFβ is associated with a significant stimulation of ODC activity and Put, Spd levels together with an enhancement of the Spd/polyamines ratio. IL-1, which does not act on cell proliferation, fails to activate ODC and to increase polyamine levels, thus indicating that the ODC/polyamine system is most likely to be an important link in the chain of events that leads to growth factor-induced proliferation. © 1997 John Wiley & Sons, Ltd.

Involvement of polyamines in the action of transforming growth factor beta and interleukin-1 on cultured chick embryo fibroblasts.

In this study we have examined the relationship between growth factor-induced proliferation and ODC/polyamine levels. TGF beta promotes cell growth and enhances [3H]-thymidine incorporation in chick embryo fibroblasts maintained in a serum-depleted medium. The action on DNA synthesis declines in the second day of treatment. IL-1 does not affect proliferation or [3H]-thymidine incorporation either when it is added alone or in combination with TGF beta. The response of the cells to TGF beta is associated with a significant stimulation of ODC activity and Put, Spd levels together with an enhancement of the Spd/polyamines ratio. IL-1, which does not act on cell proliferation, fails to activate ODC and to increase polyamine levels, thus indicating that the ODC/polyamine system is most likely to be an important link in the chain of events that leads to growth factor-induced proliferation.

Polyamine levels and ODC activity in intestinal-type and diffuse-type gastric carcinoma.

Gastric carcinomas are divided into two types according to Lauren's classification: intestinal and diffuse types. Polyamines (putrescine, spermidine, and spermine) are polycations involved in neoplastic growth of gastrointestinal mucosa. A key role is also played by ornithine decarboxylase, the rate limiting enzyme in polyamine metabolism. Our aim was to investigate whether there were differences between the two types of tumor in polyamine metabolism. Twenty-seven patients with gastric carcinoma entered the study. Seventeen carcinomas were classified as diffuse type and 10 as intestinal type. Polyamine levels were evaluated by high-performance liquid chromatography. Ornithine decarboxylase activity was measured by a radiometric technique. Polyamine levels and ornithine decarboxylase activity were significantly higher in intestinal type samples than diffuse type samples. A similarity of polyamine levels in intestinal type samples with levels previously observed in patients with colorectal adenocarcinoma was also found. These findings show a different proliferative behavior of these two types of tumor, and therefore different therapeutic strategies can be hypothesized.

Porphyrin-mediated photosensitization has a weak tumor promoting activity in mouse skin: possible role of in situ-generated reactive oxygen species.

Reactive oxygen species (ROS) have been implicated in skin tumor promotion. Earlier, we showed that porphyrin-mediated cutaneous photosensitization results in the in situ generation of ROS. Recently, we have provided the first in situ evidence for the involvement of ROS in stage I tumor promotion. In this study we further show that in situ-generated ROS act as weak complete tumor promoters in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin. Papillomas were induced in Swiss albino mice by a single topical application of DMBA as initiator. The promotion was achieved in these mice by the sustained generation of ROS through dihematoporphyrin ether (DHE)-mediated cutaneous photosensitization, which was done once every day (six times a week) for 24 weeks. The first appearance of visible papillomas could be recorded 24 weeks after the initiation. The highest tumor incidence of 60% occurred at a dose of 2.5 mg/kg body wt DHE. Increasing the dose of DHE produced a decrease in the incidence as well as in the number of papillomas. In contrast, the number of carcinomas/mouse increased with increasing dose of DHE. Histopathology of the tumor samples indicated the formation of in situ carcinoma also in skin. ROS generated through DHE-mediated photosensitization resulted in a approximately 3 fold induction of ODC activity 9 h after photosensitization. DHE-mediated photosensitization enhanced [3H]thymidine incorporation in cutaneous DNA in a dose-dependent manner. A maximum 5-fold induction of [3H]thymidine incorporation was observed at a dose of 10 mg/kg body wt DHE. The longer latency period, low incidence of tumor induction, low tumor yield and low induction of ODC activity as compared with TPA represent the weak but complete tumor promoting potential of in situ-generated ROS. The low tumor incidence and tumor yield observed at higher doses of DHE may be due to the ablation of tumors at early stages due to the strong photodynamic action of DHE.Our data indicate that porphyrin-mediated photosensitization has a weak tumor promoting effect in mouse skin and in situ-generated ROS may play an important role in the development of this response.

Ornithine decarboxylase activity in rat intestinal mucosa and liver is stimulated by central administration of 2-deoxy- d-glucose but not of 2,5-anhydro- d-mannitol

It has been shown that 2-deoxy- d-glucose (2-DG) inhibits glucose utilization and elicits feeding through the lateral hypothalamus. In contrast, 2,5-anhydro- d-mannitol (2,5-AM), blocking glycogenolysis and/or gluconeogenesis, elicits feeding through the ventromedial hypothalamus. The aim of the present study was to determine whether ornithine decarboxylase (0130 activity in the rat small intestine is stimulated by infusion into the third ventricle of 2-DG or of 2,5-AM. Under anesthesia, a cannula was implanted into the third ventricle one week before the experiment. Each rat was infused with 6, 12, and 24 μmol 2-DG or 2,5-AM into the third ventricle without disturbing the behavior. Ingestive behavior was observed for one h after the infusion. ODC activity in the intestinal mucosa and the liver was measured 2 h after the infusion. Additionally, ODC activity was measured in vagotomized rats. Both test solutions elicit feeding at 24 μmol/rat. Infusion of 2-DG into the cerebroventricle significantly increased ODC activity in the duodenal and jejunal mucosa and the liver. In contrast to 2-DG, infusion of 2,5-AM did not increase ODC activity in the intestinal mucosa or liver. Truncal vagotomy attenuated the increase of ODC activity in the intestinal mucosa and liver induced by 2-DG. The present study showed that 2-DG, but not 2,5-AM, increased ODC activity in the peripheral organs, indicating that glucose-metabolism at specific sites of the central nervous system, including the lateral hypothalamus, is important for stimulatory signals to ODC activity. It is also indicated that the stimulatory signals from the central nervous system are mediated, at least in part, via the efferent vagal nerve.

Inhibition of the expression of ornithine decarboxylase by some kappa-opioidergic receptor ligands in difluoromethylornithine-resistant L1210 cells.

In difluoromethylornithine resistant L1210 cells stimulated to growth from quiescence, the selective kappa-opioidergic agonist trans-(+/-)-3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneaceta mid e (U-50488H) caused a dose dependent inhibition of the induction of ODC activity, with a half-maximal effect at about 1 microM. U-50488H also provoked reduction of ODC mRNA level and increase of ODC turnover, as well as inhibition of cell growth. U-69593, another kappa-selective agonist, was only slightly effective. The action of U-50488H on ODC induction was not blocked by naloxone, beta-chlornaltrexamine or by the kappa-selective opioid antagonists Mr1452 and nor-binaltorphimine (nBNI). Actually Mr1452 and nBNI exerted some inhibitory effect. Furthermore, the separated enantiomers (+) and (-) of U-50488H were similarly effective. The (-)cis-(1S,2R)-U50488 stereoisomer, exhibiting low affinity for kappa and high affinity for sigma receptors and carbetapentane, another sigma ligand, also inhibited ODC induction, although less effectively than U-50488H. None of several other opioid ligands tested had significant effects on ODC induction. In conclusion, the inhibition of ODC expression by U-50488H does not involve classical, enantiospecific opioid receptors; rather, these results suggest the involvement of a distinct site of action linked to inhibition of lymphoid cell proliferation.

Hypoosmotic stimulation of ornithine decarboxylase activity in the brine shrimpArtemia franciscana

In the brine shrimp, long-term (several days) osmotic and ionic regulation is controlled principally by the activity of Na+K+ATPase. However, short-term cell volume regulation in Artemia and most euryhaline crustaceans is not understood. In this study, Artemia stage 1 nauplii reared at 32 ppt salinity and exposed to hypoosmotic conditions (12 or 4 ppt) exhibited an immediate increase in the activity of ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis. ODC activity increased within 15 min relative to control and reached a peak within 2 hr of hypoosmotic exposure. The level of increased enzyme activity was directly related to the degree of hypoosmotic exposure (a three- and sevenfold increase in activity at 12 and 4 ppt salinity, respectively). This increase in activity was apparently due to the synthesis of a new enzyme, based on cycloheximide administration. Activity decreased significantly by 4 hr but remained above that of the 32 ppt control throughout the 24-hr exposure period. Levels of putrescine increased significantly within 2 hr in organisms exposed to 4 ppt; spermidine and spermine levels were not affected. When nauplii were exposed to altered ion and/or osmotic concentrations it was determined that decreased total osmotic concentration was the proximate factor responsible for ODC induction. However, reduced ion concentration resulted in the induction/activation of DFMO-insensitive ODC activity, suggesting the production of a novel enzyme in polyamine metabolism during hypotonic exposure. Changes in ODC activity and polyamines preceded acclimatory changes normally associated with salinity acclimation, further suggesting that modulation of ODC activity and subsequent polyamines synthesis, degradation, and/or transport are mechanistically related to short-term regulation of osmotic and ionic balance. © 1996 Wiley-Liss, Inc.

Control of ornithine decarboxylase activity by polyamines and absence of antizyme in Tetrahymena

1. In cells of Tetrahymena pyriformis and thermophila, ODC activity was significantly suppressed but ODC decay was not stimulated by putrescine. 2. Free antizyme and ODC-antizyme complex were both not detected in extracts of cells of T. pyriformis treated with putrescine. 3. It was concluded that in Tetrahymena, unlike vertebrate cells, ODC is not subject to polyamine-induced destabilization mediated by antizyme.

Conjugated Polyamine Levels and Putrescine Synthesis in Cherimoya Fruit during Storage at Different Temperatures

In a previous study U. Plant Physiol. 143, 207-212, 1994) we reported a rise in free putrescine levels in cherimoya fruit. ( Annona cberimola Mill.) during ripening. In an attempt to explain such an increase, conjugated polyamine levels, arginine decarboxylase. (ADC, EC 4.1.1.19) and ornithine decarboxylase. (ODC, EC 4.1.1.17) activities were determined in cherimoya fruit stored at 20, 10, 8 and 6 °C. No PCAinsoluble conjugated spermine and PCA-soluble conjugated putrescine titers were detected at any temperature or time interval. PCA-soluble conjugated spermine titers were observed to decline early on in the low-temperature storage period. While PCA-insoluble conjugated spermidine content grew steadily in fruit kept at 20 °C, it was found to decrease throughout the storage period in fruit at lower temperatures. While PCA-insoluble conjugated putrescine levels increased in ripe fruit, they were nonetheless substantially lower than the free-form levels. The rise in putrescine levels in ripening cherimoya fruit was correlated with greatly enhanced ADC activity. No ODC activity was detected under any of the experimental conditions. It is suggested that the ripening-related changes in putrescine levels are regulated via ADC-activity-mediated synthesis and that no conjugation is involved in the process.

Ornithine Decarboxylase Induction during Liver Regeneration in IRS-1-Deficient Mice

We investigated the induction of ornithine decarboxylase during liver regeneration after partial hepatectomy in IRS-1-deficient mice. There were no significant differences in ODC activity or the time course of changes in ODC activity between IRS-1-deficient mice and wild-type mice. PI 3′-kinase activity showed similar increases in both groups of mice. Furthermore, ODC induction in IRS-1 transfected CHO cells was studied after stimulation by addition of FCS. The maximal ODC activity was 2.5-fold greater in IRS-1-transfected CHO cells than in control CHO cells. Our results suggest that the IRS-1 pathway may be involved in ODC induction. The absence of a difference in ODC and PI 3′-kinase activity in the regenerating liver between IRS-1-deficient mice and wild-type mice may have been related to the compensatory effects of IRS-2/pp190 [Araki et al, Nature (1994) 372, 186-190; Tobe et al. J.Biol.Chem. (1995) 270, 5698-5701].

Transcription-dependent and -independent regulation of hepatic ornithine decarboxylase activity by CNS beta-endorphin in rat pups.

We have previously shown that intracerebroventricular administration of relatively low doses of beta-endorphin suppresses basal levels of hepatic ODC activity as well as tissue ODC responsiveness to administered insulin in developing rats. Using Northern blotting analysis, the current studies examine whether these effects of CNS beta-endorphin may be mediated by changes in ODC gene expression. Subcutaneous administration of insulin (20 IU/kg body weight) rapidly and profoundly increased liver ODC activity. The time course of the response was characterized by proportionally increased levels of ODC mRNA, suggesting that insulin-induced stimulation of ODC activity is due to an increased transcription of ODC mRNA. Pretreatment with actinomycin D (2 mg/kg body weight, intraperitoneally) completely prevented the insulin-induced increase in ODC activity, confirming the requirement for the de novo synthesis of ODC mRNA for the effect. More importantly, intracerebroventricular but not subcutaneous injection of beta-endorphin (1 microgram) markedly diminished the stimulatory effect of insulin on hepatic ODC mRNA accumulation. The time course and magnitude of the inhibition of mRNA accumulation essentially mirrored that of the peptide on ODC activity. On the other hand, contrary to the inhibitory effect of beta-endorphin on basal ODC activity, the peptide did not lower basal ODC mRNA levels when given alone. Taken together, the results from these studies provide evidence for the existence of at least two separate mechanisms through which CNS beta-endorphin might downregulate ODC activity in peripheral organs of rat pups. The peptide can suppress insulin-induced ODC activity in the liver tissue by decreasing the rate of transcription of the ODC gene, whereas the inhibition of basal ODC activity appears to involve posttranscriptional mechanisms.

Synthesis of isoornithines and methylputrescines. An evaluation of their inhibitory effects on ornithine decarboxylase.

2-(Aminomethyl)-4-aminobutyric acid (isoornithine), 3-methylisoornithine, and 2,3-dimethylisoornithine were not decarboxylated by liver ornithine decarboxylase (ODC, EC 4.1.1.17) of thioacetamide-treated rats but were good competitive inhibitors of the enzyme (Ki ranged from 0.72 to 1.79 mM). When assayed in vivo in the treated rats, the above mentioned isoornithines were also found to inhibit liver ODC when administered 1 h before sacrifice. When the methylputrescines formally derived from the decarboxylation of several isoornithines were assayed on rat liver ODC, it was found that only 2,3-dimethylputrescine decreased the enzymatic activity. When assayed in vivo, it was found to decrease ODC activity by 60%, when the latter was measured 1 h after administration. The effect was reverted 4 h after administration of the drug. Isoornithines were not taken up by H-35 hepatoma cells; hence they did not affect their ODC activity. 2,3-Dimethylputrescine however, was transported into the cells and significantly decreased its ODC activity.

L-glutamine and L-asparagine stimulate ODC activity and proliferation in a porcine jejunal enterocyte line.

We studied the effect of L-glutamine (Gln), the principal intestinal fuel, on proliferation of a porcine jejunal cell line, IPEC-J2. In cells synchronized by serum deprivation for 4 h, Gln stimulated ornithine decarboxylase (ODC; EC 4.1.1.17) in a dose- and time-dependent manner, with maximal effects at 10 mM in 3 h (P < 0.01). Similar effects were seen for the structurally related amino acid L-asparagine and serum. The Gln effect on ODC was specific, as isosmolar mannitol, glucose, methyl-beta-D-glucoside, L-phenylalanine, ammonia, and aminoisobutyric acid were ineffective. The alanine aminotransferase inhibitor aminooxyacetate (AO) inhibited the ODC stimulation by Gln in a dose-dependent manner (half-maximal inhibitory concentration = 0.5 mM). AO was not toxic to cells, as determined by propidium iodide uptake into nuclei. In addition, Gln stimulated a twofold increase of cellular 24-h [3H]thymidine incorporation above rates of control cells bathed in standard media (P < 0.01); this effect was also blocked by AO. Gln and phorbol 12-myristate 13-acetate stimulated ODC in a synergistic manner. The Na+/H+ exchange inhibitor methylisobutyl amiloride blocked the enhancement of ODC by Gln. Gln also induced the mRNA of the immediate-early gene c-jun. Gln stimulates proliferation in a porcine jejunal cell line through a mechanism requiring transamination and intact Na+/H+ exchange. This stimulation of enterocyte proliferation by Gln suggests that therapeutic Gln administration could facilitate epithelial recovery in the injured small intestine.

The inhibition of ornithine decarboxylase activity in developing rat tissues by central nervous system β-endorphin is mediated by μ-opioid receptors, but not by δ- or ϵ-opioid receptors

Our laboratory has previously shown that intracerebroventricular (i.c.v.) administration of β-endorphin suppresses brain and liver ornithine decarboxylase activity (ODC; a growth regulatory enzyme) in preweanling rats. This investigation examined, in 6-day-old rats, the relative participation of brain μ-, δ- and ϵ-opioid receptors in β-endorphin's ODC effects, by comparing tissue ODC responses to β-endorphin given alone i.c.v. and in the presence of d-Phe-Cys-Tyr- d-Trp-Orn-Thr-Pen-Thr-NH 2 (CTOP; μ-opioid receptor antagonist), N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI-174,864; δ-opioid receptor antagonist) or β-endorphin-(1–27) (ϵ-opioid receptor antagonist). Administration of 0.5 μg of β-endorphin alone significantly decreased brain and liver ODC activity 4 h after injection, and the effect was completely blocked by coinjection of CTOP (0.075 μg) but not by ICI-174,864 (0.75 or 3.75 μg) or β-endorphin-(1–27) (3.75 or 7.5 μg). The blockade of endogenous opioid:opioid receptor interactions by either CTOP (at doses > 0.075 μg) or ICI-174,864 alone was accompanied by increased levels of basal ODC activity. The results obtained demonstrate that i.c.v. β-endorphin downregulates ODC expression in central as well as in peripheral tissues by interacting with brain μ-opioid receptors, but not with δ- or ϵ-opioid receptors or μ/δ-opioid receptor complexes. Also, they indicate that endogenous opioid systems have a tonic inhibitory influence on ODC activity which is mediated, at least in part, by μ- and δ-opioid receptors.

Identification and evaluation of the role of endogenous tyrosine kinases in azoxymethane induction of proliferative processes in the colonic mucosa of rats

Although tyrosine kinases (Tyr-k) are known to play a role in regulating proliferation of normal, preneoplastic and neoplastic cells, little is known about the identity of different species of Tyr-k involved in this process. Utilizing a non-denaturing polyacrylamide gel electrophoresis system, in which the separated proteins from tissue extracts are assayed directly for Tyr-k, we attempted to identify the species of Tyr-k that may be involved in azoxymethane (AOM) induction of colonic mucosal ornithine decar☐ylase (ODC) activity, an enzyme whose activity is known to rise in rapidly proliferating cells. We have observed that 5 days after a single injection of the colonic carcinogen AOM (20 mg/kg body wt.) to 3–4-month old rats, a significant 230% rise in colonic mucosal proliferative activity (as evidenced by 5-bromo-2′-deoxyuridine (BrdU) immunoreactivity) was also accompanied by a 550% increase in ODC activity. This was also associated with a marked rise (140–240%) in the relative activity of Tyr-k of three mucosal proteins with M r of 165, 145 and 125 kDa. Since the molecular mass of one of the Tyr-k (165 kDa) corresponded to that of EGF-receptor (EGF-R), this led us to examine the role of EGF-R Tyr-k in AOM induction of colonic mucosal ODC. We observed that a 320% increase in mucosal ODC activity, 5 days after AOM injection, was accompanied by over 200% rise in Tyr-k activity of EGF-R. Daily injection of tyrphostin (300 μg/kg body wt.), a Tyr-k inhibitor with a higher specificity for EGF-R Tyr-k, significantly attenuated AOM-induced stimulation of both ODC and Tyr-k activity of EGF-R. Administration of AOM also stimulated the rate of synthesis and secretion of TGF-α in isolated colonocytes. In addition, the levels of TGF-α and its mRNA in the colonic mucosa were also found to be 100% and 250% higher, respectively, in AOM-treated rats when compared with the controls. We suggest that (a) activation of intrinsic Tyr-k of EGF-R is an important event in AOM induction of colonic mucosal proliferative processes, and (b) this activation is thought to be mediated by TGF-α through an autocrine mechanism.

L-519, a phenolic compound, inhibits metabolism of benzo(a)pyrene and mutagenesis induced by benzo(a)pyrene

L-519 is a phenolic compound. In this study, L-519 was found to inhibit the mutagenicity of benzo(a)pyrene [B(a)P] in Salmonella Typhimurium TA 97 and TA 100 and inhibit the cytochrome P-450 mediated metabolism of B(a)P. It also inhibited the epidermal ODC activity induced by croton oil. L-519 also decreased the lipid peroxidation induced by FeSO4 and Cysteine in rat liver microsome system. Our results demonstrated that L-519 exhibited anti-mutagenicity, anti-initiation as well as anti-promotion activities.