Thymidine Incorporation In DNA Research Articles

Antioxidant and nephroprotective potential of butylated hydroxyanisole against ferric nitrilotriacetate-induced oxidative stress and early tumor events

The present study was aimed to study protective effect of butylated hydroxyanisole (BHA), a phenolic antioxidant used in foods on ferric nitrilotriacetate (Fe-NTA)-induced nephrotoxicity. Male albino rats of Wistar strain (4-6 weeks old) weighing 125-150 g were used in this study. Animals were given a single dose of Fe-NTA (9 mg kg(-1) body weight) after treatment with BHA (1 and 2 mg animal(-1) day(-1)). Fe-NTA treatment enhanced ornithine decarboxylase (ODC) activity to 5.3-fold, and [(3)H]-thymidine incorporation in DNA to 2.5-fold in kidney compared with the corresponding saline-treated control, whereas glutathione (GSH) levels and the activities of antioxidant enzymes decreased to a range of 2- to 2.5-fold in kidney. These changes were reversed significantly in animals receiving a pretreatment of BHA. The enhanced ODC activity and DNA synthesis showed a reduction to 2.12-fold and 1.15-fold, respectively, at a higher dose of 2 mg BHA day(-1) animal(-1), compared with the Fe-NTA-treated groups. Pretreatment with BHA prior to Fe-NTA treatment increased GSH and the activities of antioxidant enzymes to a range of 1.5- to 2-fold in kidney. The results indicate that BHA suppresses Fe-NTA-induced nephrotoxicity in male Wistar rats.

Ascorbic acid inhibits ferric nitrilotriacetate induction of ornithine decarboxylase, DNA synthesis, oxidative stress, and hepatotoxicity in rats

Ascorbic acid (AA) is a naturally occurring phenolic compound with antioxidant properties used in food, cosmetics, and pharmaceutical products. In this study, the effect of AA on ferric nitrilotriacetate (Fe-NTA)-induced hepatotoxicity in rats has been examined. Fe-NTA alone enhances ornithine decarboxylase activity to 4.5-fold and tritiated thymidine incorporation in DNA to 3.6-fold in livers compared with the corresponding saline-treated controls. The enhanced ornithine decarboxylase activity and DNA synthesis showed a reduction to 3.02- and 1.88-fold, respectively, at a higher dose of 2 mg AA per day per animal, compared with the Fe-NTA-treated groups. Fe-NTA treatment also enhanced the hepatic microsomal lipid peroxidation to 1.7-fold compared to saline-treated controls. These changes were reversed significantly in animals receiving pretreatment of AA. The present data shows that AA can reciprocate the toxic effects of Fe-NTA and can serve as a potent chemopreventive agent to suppress oxidant-induced tissue injury and hepatotoxicity in rats.

Antioxidant effect of butylated hydroxytoluene on ferric nitrilotriacetate induced renal hyper proliferation and injury in rats

This study was designed to investigate the effect of butylated hydroxy toluene (BHT), a phenolic antioxidant used in foods, cosmetics and pharmaceutical products, on Fe-NTA-induced nephrotoxicity in rats. Fe-NTA (alone) treatment enhances ornithine decarboxylase activity to 5.3-fold, and [(3)H] thymidine incorporation in DNA to 3.5-fold compared with the corresponding saline treated control. The enhanced ornithine decarboxylase activity and DNA synthesis showed a reduction to 2.12-2.15-fold respectively at a higher dose of 2 mg BHT/day/animal, compared with the Fe-NTA treated group. Fe-NTA treatment also enhanced the renal microsomal lipid peroxidation to 2.0-fold and decreased the activities of glutathione and antioxidant enzymes to a range of 2.2-2.5-fold in kidney. These changes were reversed significantly in animals receiving a pretreatment of BHT. Present data suggests that BHT can prevent the toxic effects of Fe-NTA and can serve as a potent chemopreventive agent to suppress oxidant-induced tissue injury and nephrotoxicity in rats.

Emblica officinalis reverses thioacetamide-induced oxidative stress and early promotional events of primary hepatocarcinogenesis

Emblica officinalis is widely used in Indian medicine for the treatment of various diseases. In the present study, it was found that fruits of E. officinalis inhibit thioacetamide-induced oxidative stress and hyper-proliferation in rat liver. The administration of a single necrotic dose of thioacetamide(6.6 mM kg(-1)) resulted in a significant (P < 0.001) increase in serum glutamic oxaloacetic transaminase(SGOT), serum glutamic pyruvic transaminase (SGPT) and gamma-glutamyl transpeptidase (GGT) levels compared with saline-treated control values. Thioacetamide caused hepatic glutathione (GSH) depletion and a concomitant increase in malanodialdehyde (MDA) content. It also resulted in an increase(P < 0.001) in the activity of glutathione-S-transferase (GST), glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PD) and a decrease in glutathione peroxidase (GPx) activity (P < 0.001). Hepatic ornithine decarboxylase activity and thymidine incorporation in DNA were increased bythioacetamide administration. Prophylactic treatment with E. officinalis for 7 consecutive days before thioacetamide administration inhibited SGOT, SGPT and GGT release in serum compared with treated control values. It also modulated the hepatic GSH content and MDA formation. The plant extract caused a marked reduction in levels of GSH content and simultaneous inhibition of MDA formation. E. officinalis also caused a reduction in the activity of GST, GR and G6PD. GPx activity was increased after treatment with the plant extract at doses of 100 mg kg(-1) and 200 mg kg(-1). Prophylactic treatment with the plant caused a significant down-regulation of ornithine decarboxylase activity (P < 0.001) and profound inhibition in the rate of DNA synthesis (P < 0.001). In conclusion, the acute effects of thioacetamide in rat liver can be prevented by pre-treatment with E. officinalis extract.

Effect of Luteolin on Nickel Chloride–Induced Renal Hyperproliferation and Biotransformation Parameters in Wistar Rats

Nickel, a major environmental pollutant, is a known potent nephrotoxic agent. In this communication, we report the chemopreventive effect of luteolin on nickel chloride (NiCl2)-induced renal oxidative stress, toxicity, and cell proliferation response in male Wistar rats. NiCl2 (250 µmol/kg b.wt./2 mL) enhances reduced renal glutathione content (GSH), glutathione S.-transferase (GST), glutathione reductase (GR), lipid peroxidation (LPO), H2O2 generation, blood urea nitrogen (BUN), and serum creatinine with a concomitant decrease in the activity of glutathione peroxidase (GPx) (p < 0.001). NiCl2 administration also dose-dependently induced the renal ornithine decarboxylase (ODC) activity several-fold compared with the activity in saline-treated rats. Similarly, renal DNA synthesis, which is measured as [3H]thymidine incorporation in DNA, is also increased after NiCl2 treatment. Prophylactic treatment of rats with luteolin (10 and 20 µmol/kg b.wt.) daily for 1 week resulted in the diminution of NiCl2-mediated damage as evident from the downregulation of glutathione content, GST, GR, LPO, H2O2 generation, BUN, serum creatinine, DNA synthesis (p < 0.001), and ODC activity (p < 0.01) with concomitant restoration of GPx activity. Thus, the current investigation suggests that luteolin can be used as a chemopreventive agent for cancer prevention as evident from the study where it blocks or suppresses the events associated with chemical carcinogenesis.

Acorus calamus Extracts and Nickel Chloride: Prevention of Oxidative Damage and Hyperproliferation Response in Rat Kidney

Nickel, a major environmental pollutant, is known for its clastogenic, toxic, and carcinogenic potential. In this article, we report the effect of Acorus calamus on nickel chloride (NiCl2)-induced renal oxidative stress, toxicity, and cell proliferation response in male Wistar rats. NiCl2 (250 micromol/kg body weight/mL) enhanced reduced renal glutathione content (GSH), glutathione- S-transferase (GST), glutathione reductase (GR), lipid peroxidation (LPO), H2O2 generation, blood urea nitrogen (BUN), and serum creatinine with a concomitant decrease in the activity of glutathione peroxidase (GPx) (p < 0.001). NiCl2 administration also dose-dependently induced the renal ornithine decarboxylase (ODC) activity several-fold as compared to salinetreated control rats. Similarly, renal DNA synthesis, which is measured in terms of [3H] thymidine incorporation in DNA, was elevated following NiCl2 treatment. Prophylactic treatment of rats with A. calamus (100 and 200 mg/kg body weight po) daily for 1 wk resulted in the diminution of NiCl2- mediated damage, as evident from the downregulation of glutathione content, GST, GR, LPO, H2O2 generation, BUN, serum creatinine, DNA synthesis (p < 0.001), and ODC activity (p < 0.01) with concomitant restoration of GPx activity. These results clearly demonstrate the role of oxidative stress and its relation to renal disfunctioning and suggest a protective effect of A. calamus on NiCl2-induced nephrotoxicity in a rat experimental model.

Inhibitory effect of 18β-glycyrrhetinic acid on 12-O-tetradecanoyl phorbol-13-acetate-induced cutaneous oxidative stress and tumor promotion in mice

Glycyrrhetinic acid is an aglycone of glycyrrhizic acid, another major active component of licorice roots. Licorice root extract has been used for a long time as a medicine and a natural sweetening additive. In the present study, we found that glycyrrhetinic acid inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA) mediated oxidative stress and tumor promotion in murine skin. Topical application of TPA alone in mouse skin enhances ornithine decarboxylase activity and also increases [3H]-thymidine incorporation in DNA. Topical application of TPA also resulted in the depletion of glutathione, activities of glutathione metabolizing and antioxidant enzymes. Application of glycyrrhetinic acid prior to TPA treatment reduces this enhanced ODC activity, [3H]-thymidine incorporation in DNA and oxidative stress. Glycyrrhetinic acid was also found to inhibit DMBA/TPA-induced skin tumor formation at doses of 1.25 and 2.5 mg by reducing the number of tumors per mouse by 24% (P < 0.05) and 62% (P < 0.05), respectively. These results suggest that glycyrrhetinic acid, an antioxidant, is a potential chemopreventive agent that can inhibit DMBA/TPA-induced cutaneous oxidative stress and tumor promotion.

Effect of Hibiscus rosa sinensis Extract on Hyperproliferation and Oxidative Damage Caused by Benzoyl Peroxide and Ultraviolet Radiations in Mouse Skin

The present study was conducted to investigate the ameliorative potential of Hibiscus rosa sinensis extract in mice skin. Combination of a single topical application of benzoyl peroxide (20 mg/0.2 ml/animal) followed by ultraviolet radiations (0.420 J/m2/s) was used to induce hyperproliferation and oxidative stress. Single benzoyl peroxide application prior to ultraviolet B radiations exposure caused significant depletion in the detoxification and antioxidant enzymes, while malondialdehyde formation, hydrogen peroxide content, ornithine decarboxylase activity and DNA synthesis were raised significantly. However, pretreatment of H. rosa sinensis extract (3.5 mg and 7 mg/ kg b.wt.) partly restored the levels of cellular protective enzymes (P<0.05). Besides, malondialdehyde formation and hydrogen peroxide content (P<0.05) were statistically significantly reduced at both doses. The ornithine decarboxylase activity and thymidine incorporation in DNA were also reduced dose dependently (P<0.05) by the plant extract. Therefore, we propose that H. rosa sinensis extract exerts a protective effect against the tumour promotion stage of cancer development.

EVALUATION OF THE EFFECT OT IMMUNOSUPPRESSIVE AGENTS ON HEPATITIS C

O35 Aims: Hepatitis C virus (HCV) infection is the leading indication for liver transplantation. HCV recurrence is universal and can lead to graft loss. Clinical studies suggest that the type and intensity of immunosuppression can significantly influence the evolution of this infection. Despite the risk of increased rejection, the current trend in the management of these patients is to lower immunosuppression in order to reduce viral replication. We hypothesized that the different immunosuppressive drugs didn’t have the same effect on HCV replication. The aim of our study was to evaluate HCV replication in the HUH-7 cell line carrying the subgenomic HCV replicon with or without exposure to non toxic concentrations of the different immunosuppressive agents currently used in liver transplantation. Methods: HCV replication was evaluated by measuring genomic RNA by real time PCR and viral protein expression by western blotting. Cell proliferation was evaluated by measuring [3H]-thymidine incorporation in DNA. Results: Thymidine incorporation was similar in cells exposed to tacrolimus, hydrocortisone, interferon and control, and slightly reduced in cells exposed to cyclosporin and rapamycin (respectively 87% and 75% of control, p<0.05). Mycophenolate mofetyl (MMF) dramatically reduced thymidine incorporation in these cells (2% of control, p<0.01). As expected, interferon-α (500UI/mL) significantly reduced the number of positive strand RNA by 1.76 log after 48 hours of exposure (p<0.03), the level of expression of the non-structural protein NS5a (39% of control, p<0.03) and the amount of negative strand HCV RNA (43% of control, p<0.03). Rapamycin (10 nM), tacrolimus (250 nM) and hydrocortisone (250 μM) did not significantly modify HCV replication. On the other hand, MMF (10 μg/ml) reduced the amount of positive strand by 1.09 log (p<0.01) and the level of NS5a expression (46% of control, p<0.01). Cyclosporin (2.5μg/ml) also reduced the amount of positive strand by 1.85 log (p<0.01), the level of NS5a expression (46% of control, p<0.05) and the amount of HCV RNA negative strand (66% of control, p<0.05). Verapamil and quinine, which are P-glycoprotein blockers such as cyclosporin also had the capacity to reduce HCV replication. Conclusions: These results suggest that immunosuppressive agents can significantly modify HCV replication in the replicon model and that the response differs depending on the type of agents. Since HCV replication is affected by cell proliferation in the replicon model and that MMF significantly affected this parameter, cyclosporin is the only agent that actually decreases HCV replication in our study.

Study on prevention of two-stage skin carcinogenesis by Hibiscus rosa sinensis extract and the role of its chemical constituent, gentisic acid, in the inhibition of tumour promotion response and oxidative stress in mice.

We designed the present study to investigate the role of gentisic acid in the chemopreventive activity of Hibiscus rosa sinensis extract on 7,12-dimethyl benz(a)anthracene (DMBA)/croton oil-mediated carcinogenesis in mouse skin via 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced tumour promotion response and oxidative stress. Single topical application of DMBA followed by twice weekly applications of croton oil after one week for 20 weeks resulted in 100% incidence of tumours in animals in 15 weeks. However, application of H. rosa sinensis extract 30 minutes prior to the application of croton oil twice weekly for 20 weeks caused significant reduction in the number of tumours per mouse and the percentage of tumour-bearing mice. Also, the latency period for the appearance of the first tumour was delayed on H. rosa sinensis pretreatment. A single topical application of TPA caused significant depletion in reduced glutathione (GSH) content, activities of its metabolizing and antioxidant enzymes, while malondialdehyde (MDA) formation, H2O2 content, ornithine decarboxylase (ODC) activity and DNA synthesis were significantly increased. Interestingly, pretreatment of H. rosa sinensis extract (3.5 mg and 7 mg/kg body weight) and gentisic acid (2.0 microg and 4.0 microg/0.2 ml acetone per animal) restored the levels of GSH, and its metabolizing and antioxidant enzymes (P<0.05). There was also a statistically significant reduction in MDA formation and H2O2 content (P<0.05) at both doses. Although inhibition of ODC activity by gentisic acid was not dose-dependent, thymidine incorporation in DNA (P<0.05) was dose-dependently recovered by the plant extract and its chemical constituent. We therefore propose that gentisic acid has a role in the modulatory activity of H. rosa sinensis extract.

Potassium bromate (KBrO 3) induces renal proliferative response and damage by elaborating oxidative stress

Many chemical compounds which induce oxidative stress in the tissue produce carcinogenesis either alone or act as a tumor promoter in carcinogen-initiated animals after prolonged exposure. Here, we report that potassium bromate (KBrO 3) induces renal proliferative response and damage by elaborating oxidative stress. KBrO 3 administration dose dependently induced renal ornithine decarboxylase (ODC) activity several fold compared to its activity in saline-treated rats. Similarly renal DNA synthesis which has been measured as [ 3H]thymidine incorporation in DNA also increases. KBrO 3 administration also depleted the level of renal glutathione and glutathione reductase activity in a time dependent manner. The maximum depletion in the levels of renal glutathione and glutathione reductase activity was observed 3 h after KBrO 3 treatment which was 60 and 40%, respectively, of saline-treated controls. Parallel to these changes, a sharp increase in the blood urea nitrogen and serum creatinine levels was observed which is indicative of the concurrent renal damage. These results suggest that oxidant generating KBrO 3 acts as a potent proliferator of kidney and acts by producing oxidative damage.

Alpha-Tocopherol (vitamin-E) ameliorates ferric nitrilotriacetate (Fe-NTA)-dependent renal proliferative response and toxicity: diminution of oxidative stress.

Ferric nitrilotriacetate (Fe-NTA) is a potent nephrotoxic agent. In this communication, we show the modulatory effect of DL-alpha-tocopherol (Vitamin-E) on ferric nitrilotriacetate (Fe-NTA)-induced renal oxidative stress, toxicity and hyperproliferative response in rats. Fe-NTA-treatment enhances the susceptibility of renal microsomal membrane for iron-ascorbate-induced lipid peroxidation and hydrogen peroxide generation which are accompanied by a decrease in the activities of renal antioxidant enzymes, catalase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase and depletion in the level of renal glutathione. Parallel to these changes, a sharp increase in blood urea nitrogen and serum creatinine has been observed. In addition, Fe-NTA-treatment also enhances renal ornithine decarboxylase activity (ODC) and increases [3H]thymidine incorporation in renal DNA. Prophylactic treatment of animals with Vit.E daily for 1 week prior to the administration of Fe-NTA resulted in the diminution of Fe-NTA-mediated damage. Enhanced susceptibility of renal microsomal membrane for lipid peroxidation induced by iron-ascorbate and hydrogen peroxide generation were significantly reduced (P < 0.05). In addition, the depleted level of glutathione and inhibited activities of antioxidant enzymes recovered to significant levels (P < 0.05). Similarly, the enhanced blood urea nitrogen and serum creatinine levels which are indicative of renal injury showed a reduction of about 50% at a higher dose of Vit.E. The pretreatment of rats with Vit.E reduced the Fe-NTA-mediated induction in ODC activity and enhancement in [3H]thymidine incorporation in DNA. The protective effect of Vit.E was dose dependent. In summary, our data suggest that Vit.E is an effective chemopreventive agent in kidney and may suppress Fe-NTA-induced renal toxicity.

Ferric Nitrilotriacetate (Fe-NTA) Is a Potent Hepatic Tumor Promoter and Acts Through the Generation of Oxidative Stress

Fe-NTA is a known renal carcinogen. However, little is known about its carcinogenic potential in liver. In this study we for the first time show that Fe-NTA is a potent hepatic tumor promoter. Fe-NTA administration induced dose dependently the hepatic ornithine decarboxylase (ODC) activity several folds as compared to its activity in the saline-treated rats. Similarly, hepatic DNA synthesis which is measured as [3H]thymidine incorporation in DNA is also increased following Fe-NTA treatment. The effects of Fe-NTA were similar to other tumor promoters not only with respect to inducing ODC activity and [3H]thymidine incorporation in DNA but also in depleting antioxidant armory of the tissue. Fe-NTA depleted levels of glutathione to about 35% of the saline-treated control and activities of antioxidant enzymes catalase, glutathione peroxidase, glutathione reductase and glucose 6-phosphate dehydrogenase decreased significantly (45-55% of saline-treated control). Concomitant with the depletion in antioxidant armory, Fe-NTA augmented hepatic microsomal lipid peroxidation more than three folds. The pretreatment of rats with antioxidants BHA or BHT diminished the observed effects of Fe-NTA. Our data indicate that Fe-NTA is a potent hepatic tumor promoter and acts through a mechanism involving oxidative stress.

Specific stimulation of human endothelial cells by Triticum vulgare extract and its biologically active fraction

AbstractThe effects of an active fraction, prepared by bioassay‐guided isolation from the aqueous extract derived from Triticum vulgare, the common wheat plant, on cell stimulation and DNA‐synthesis of different cell lines was investigated. This active fraction contained one or more non‐proteinous small molecules, which stimulate the growth of human endothelial cells and/or interfere with the incorporation of thymidine in DNA without affecting growth stimulation. They also enhanced the growth and DNA synthesis of fetal bovine aortic endothelial and baby hamster kidney cells clone 21, but not that of human retinal pigment epithelial cells and human fibroblasts.

Radiolabeling of DNA can induce its fragmentation in HL-60 human promyelocytic leukemic cells

Incorporation of radiolabeled thymidine is commonly used to investigate DNA damage. Using a filter-binding assay, we observed that the addition of various doses of [ methyl- 3H]thymidine (0.2 and 2 μCi/ml) or [2- 14C]thymidine (0.02 and 0.2 μCi/ml) in the culture medium for 2 days, a standard method for cell-labeling, induces DNA fragmentation in HL-60 human promyelocytic cells. This effect was dose- and time-dependent and the DNA fragments were not protein-linked since the levels of DNA fragmentation were identical in the presence and in the absence of proteinase K (0.5 mg/ml). Radiolabeled thymidine-induced DNA fragmentation was associated with an inhibition of cell growth, but cells remained able to exclude trypan blue, suggesting that plasma membrane integrity was conserved, except at very high doses of [ methyl- 3H]thymidine (2 μCi/ml). By agarose-gel electrophoresis, the DNA-fragmentation was demonstrated to be internucleosomal with a typical ladder pattern. Addition of unlabeled thymidine to the culture medium prevented DNA fragmentation in a dose-dependent manner, indicating that radiolabeled thymidine incorporation in DNA was directly responsible for DNA fragmentation. We conclude that radiolabeling of DNA using thymidine incorporation can induce DNA fragmentation in some cell lines such as HL-60. This observation must be taken into account in methods using radiolabeling to study DNA damage in these cells.

Proliferative effect of phospholipase A 2 in rat chondrocyte via its specific binding sites

We studied the presence of specific binding sites for pancreatic-type group I phospholipase A 2 (PLA 2-I), EC 3.1.1.4, and a PLA 2-I action on the DNA synthesis of rat chondrocytes. Rat chondrocytes, derived from the xiphisternum of adult rats, had a single class of PLA 2-I binding site with an equilibrium binding constant value of 0.9 nM and a maximum binding capacity of 53.9 fmol/10 6 cells. PLA 2-I alone did not show any proliferative effect, however, PLA 2-I dose-dependently stimulated thymidine incorporation in DNA in the presence of basic fibroblast growth factor (bFGF). The mammalian mature type of PLA 2s-I specifically recognized the binding sites in these cells and had a synergistic effect on DNA synthesis with bFGF, whereas its inactive zymogen and group II PLA 2 showed much lesser activities. The type-specific action of PLA 2s implicated the involvement of PLA 2-I specific binding sites in this activation process.

Mitotic responsiveness of cultured adult human hepatocytes to epidermal growth factor, transforming growth factor α, and human serum

The present study was undertaken to evaluate the ability of human hepatocytes to respond in culture to various mitotic agents including epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), or serum from patients with fulminant hepatitis. Human hepatocytes were maintained in culture on collagen-coated plates in a chemically and hormonally defined serum-free medium at low cell density. Twelve hours after plating, cultures were treated with increasing amounts of EGF (1–100 ng/mL), TGF-α (1–100 ng/mL), or human serum (1%–10%) for 0–96 hours. Proliferative response was assessed by determining against time the rate of DNA synthesis by [3H]thymidine incorporation in DNA, the labeling index, the expression of cyclin A, the amount of DNA, and the number of cells. The rate of DNA synthesis reached a maximum after 48 hours of treatment with 20 ng/mL EGF, 40 ng/mL TGF-α, or 5%–10% of human serum (fulminant hepatitis); the average increase with respect to untreated cells was 4.35 times with EGF, 5.4 times with TGF-α, and 4–6 times with serum from patients with fulminant hepatitis. The maximum expression of cyclin A coincided with the maximum of DNA synthesis. After 72 hours of treatment with EGF or human serum (fulminant hepatitis), the amount of DNA increased by 75%–100% (P < 0.001) and the number of cells by 50% (P < 0.001). These results show that adult human hepatocytes respond to mitogens, as expected from previous studies on animal hepatocytes, and provide experimental basis for future investigations in the field of human liver regeneration.

Mitotic responsiveness of cultured adult human hepatocytes to epidermal growth factor, transforming growth factor alpha, and human serum

The present study was undertaken to evaluate the ability of human hepatocytes to respond in culture to various mitotic agents including epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), or serum from patients with fulminant hepatitis. Human hepatocytes were maintained in culture on collagen-coated plates in a chemically and hormonally defined serum-free medium at low cell density. Twelve hours after plating, cultures were treated with increasing amounts of EGF (1-100 ng/mL), TGF-alpha (1-100 ng/mL), or human serum (1%-10%) for 0-96 hours. Proliferative response was assessed by determining against time the rate of DNA synthesis by [3H]thymidine incorporation in DNA, the labeling index, the expression of cyclin A, the amount of DNA, and the number of cells. The rate of DNA synthesis reached a maximum after 48 hours of treatment with 20 ng/mL EGF, 40 ng/mL TGF-alpha, or 5%-10% of human serum (fulminant hepatitis); the average increase with respect to untreated cells was 4.35 times with EGF, 5.4 times with TGF-alpha, and 4-6 times with serum from patients with fulminant hepatitis. The maximum expression of cyclin A coincided with the maximum of DNA synthesis. After 72 hours of treatment with EGF or human serum (fulminant hepatitis), the amount of DNA increased by 75%-100% (P less than 0.001) and the number of cells by 50% (P less than 0.001). These results show that adult human hepatocytes respond to mitogens, as expected from previous studies on animal hepatocytes, and provide experimental basis for future investigations in the field of human liver regeneration. (Gastroenterology 1992 Apr;102(4 Pt 1):1340-50)

Novel proliferative effect of phospholipase A2 in Swiss 3T3 cells via specific binding site.

Phospholipase A2 (PLA2), EC 3.1.1.4, which catalyzes the release of free fatty acids from the sn-2 position of glycerophospholipids, has been extensively studied from the viewpoint of eicosanoid production (Arita, H., Nakano, T., and Hanasaki, K. (1989) Prog. Lipid Res. 28, 273-301). Several lines of evidence suggest that extracellular PLA2 is pathophysiologically related to some disorders, including inflammation and hypersensitivity. Despite this, little is known of the precise mechanism of the pathological processes as well as their intrinsic correlation with dysfunction. Here, we report a novel PLA2 action on the proliferation of Swiss 3T3 fibroblasts via specific binding sites of approximately Mr 200,000. Pancreatic type PLA2 in the active form specifically recognized the sites and stimulated thymidine incorporation in DNA. Its inactive zymogen and other PLA2s from platelets, snake, and bee venoms showed much lesser activities. Although the physiological significance remains to be identified, our finding is the first to offer a new viewpoint on the effect of mammalian extracellular PLA2 on cellular function.

The effects of cortisol, 11-epicortisol, and lysine vasopressin on DNA and RNA synthesis in isolated human adrenocorticotropic hormone-secreting pituitary tumor cells

The effects of cortisol, its steric analog 11-epicortisol, and lysine vasopressin (LVP) on DNA and RNA synthesis in isolated adrenocorticotropic hormone-secreting human pituitary tumor cells obtained by transsphenoidal surgery were studied using [ 3H]thymidine incorporation in DNA and [ 3H]uridine in RNA. Cortisol suppressed RNA and, to a greater extent, DNA synthesis in these cells. This could explain the slow growth of pituitary tumors in patients with Gushing's disease and the rapid growth of Nelson's pituitary tumors after bilateral adrenalectomy. 11-Epicortisol also suppressed RNA synthesis, but it had a stimulatory effect on DNA synthesis, which indicates a high specificity of glucocorticoid receptors. When applied together with cortisol, 11-epicortisol antagonized the suppressive effects of cortisol on DNA synthesis. Although LVP stimulated RNA synthesis, it suppressed DNA synthesis in most of the tumor cells.