DNA adduct formation from tobacco-specific N-nitrosamines

Tobacco-specific N-nitrosamines are a group of carcinogens derived from the tobacco alkaloids. They are likely causative factors for cancers of the lung, esophagus, pancreas, and oral cavity in people who use tobacco products. The most carcinogenic tobacco-specific nitrosamines in laboratory animals are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and N'-nitrosonornicotine (NNN). DNA adduct formation from NNK and NNN has been studied extensively and is reviewed here. NNK is metabolically activated by cytochromes P450 to intermediates which methylate and pyridyloxobutylate DNA. The resulting adducts have been detected in cells and tissues susceptible to NNK carcinogenesis in rodents. The methylation and pyridyloxobutylation pathways are both important in carcinogenesis by NNK. NNK also induces single strand breaks and increases levels of 8-oxodeoxyguanosine in DNA of treated animals. NNAL, which like NNK is a potent pulmonary carcinogen, is also metabolically activated to methylating and pyridyloxobutylating intermediates. NNN pyridyloxobutylates DNA in its rat target tissues, esophagus and nasal mucosa. Methyl and pyridyloxobutyl DNA adducts are detected in human tissues. The methyl adducts most likely result in part from exposure of smokers to NNK, but these adducts are also detected in non-smokers. Some of the methyl adducts detected in non-smokers may be due to environmental tobacco smoke exposure. There are also potential dietary and endogenous sources of these adducts. Pyridyloxobutyl DNA adducts in human tissues result mainly from exposure to tobacco-specific N-nitrosamines. In laboratory animals, DNA adduct formation and carcinogenicity of tobacco-specific N-nitrosamines are closely correlated in many instances, and it is likely that similar relationships will hold in humans.     

Effects of carcinogenic halogenated aliphatic hydrocarbons on [3H]thymidine incorporation into various organs of the mouse. A comparison between 1,2-dibromoethane and 1,2-dichloroethane

The effects of 1,2-dibromoethane (DBE) and 1,2-dichloroethane (DCE) on the incorporation of [3H]thymidine into DNA were evaluated in various tissues of mice. The compounds were given intraperitoneally 24 h before sacrifice in an equimolar dose (293 mumoles/kg body weight). 2 h before the animals were killed, 0.5 mu Ci [3H]thymidine/g body weight was injected intraperitoneally. Both agents inhibited the [3H]thymidine incorporation in the forestomach, a site for their carcinogenic action. Whereas DBE also suppressed the [3H]thymidine incorporation in the nasal mucosa, the thymus, and the "glandular stomach", DCE was inhibitory only in the kidney. The observed difference in the effect of DBE and DCE on the thymus had its counterpart in a DBE-induced decrease of acid-insoluble radioactivity, demonstrated with whole-body autoradiography. The results indicate that in vivo screening of [3H]thymidine incorporation into various organs of an intact experimental animal is a sensitive technique for comparing cyto- and/or genotoxic effects of chemicals with a similar chemical structure.     

Formation of genotoxic products from N-nitrosoheptamethyleneimine (NHMI), 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone (NNK) and N′-Nitrosonornicotine (NNN) by isolated rabbit lung cells

The genotoxic potentials of N-nitrosoheptamethyleneimine (NHMI), 4-(methylnitrosamino)-1-(3 pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) were studied in fresh preparations of Clara cells and type II cells isolated by centrifugal elutriation and density gradient centrifugation, and macrophages from rabbit lung. The activation of the compounds to bacterial mutagens was assayed in the Salmonella mutagenicity test using strains of TA 100 and TA 1530 preincubated with test chemicals and cells placed in chambers with nucleopore membranes to separate cells and bacteria. Unscheduled DNA synthesis was measured by incorporation of [³H]-thymidine in the cells after exposure to the compounds. NHMI, NNK and NNN were not activated to bacterial mutagens by Clara cells, type II cells or macrophages, presumably because the reactive metabolites generated were not released into the incubation medium. However, NHMI and NNK increased unscheduled DNA synthesis in Clara cells, and the highest repair activity was found after incubation with NNK. The effect of NNN was only marginal. This indicates that NHHI and NNK are genotoxic in the rabbit lung and that the Clara cells are involved in the metabolic activation of these compounds.Abbreviations NHMI N-nitrosoheptamethyleneimine - NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone - NNN N-nitrosonornicotine Supported by a grant and a fellowship (R.B.) from the Royal Norwegian Council for Scientific and Industrial Research.     

High-performance liquid chromatographic analysis of metabolites of the nicotine-derived nitrosamines, N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

An improved high-performance liquid chromatographic system was developed for separation of 11 metabolites of the nicotine-derived nitrosamines N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The new system employed a 5-microns octadecylsilane bonded column eluted with aqueous sodium acetate-methanol gradients of varying pH. Analysis times were typically 30 min for NNN metabolites and 50 min for NNK metabolites, compared to 80 and 90 min, respectively, when 10-microns columns were used. The E and Z isomers of all nitrosamine-containing metabolites of NNK were separated. The chromatographic behavior of the 11 metabolites as well as NNN and NNK was studied between pH 4.0 and 7.5. The retention times of several metabolites were altered significantly as a function of pH. The results of the pH study provide valuable additional criteria for metabolite identification as well as optimized conditions for their separation. Applications of the system to the metabolism of [2'-14C]NNN in cultured rat esophagus and [carbonyl-14C]NNK in rat liver slices are presented.     

Long-term effects of deendothelialization of rabbit aorta: in vitro synthesis of DNA, protein, and lipid

To study the long-term local effects of a single balloon catheter deendothelialization of the aorta in the rabbit, the incorporation of [3H]leucine and [3H]thymidine into protein and DNA, respectively, and [14C]acetate and [14C]mevalonate into sterols was measured in incubations of intima-media sections prepared from vessels taken 1 year following the procedure. The uptake of [3H]thymidine by the tissue was essentially the same as in the nonballooned controls, but the incorporation of [3H]leucine and [14C]acetate into tissue residue (proteins and glycoproteins) was approximately nine times and four times control values, respectively. At the same time, sections from the ballooned animals incorporated over six times the amount of radioactive acetate into nonsaponifiable lipids and cholesterol than did controls. In animals ballooned 3 months before sacrifice, when about half of the aortic luminal surface was covered with endothelium, intima-media tissue from both covered and uncovered areas showed increased uptake of labeled precursors into protein, nonsaponifiables, and cholesterol but there was no significant difference in incorporation between reendothelialized and nonendothelialized areas. The persistence of increased metabolic activity in the vessel following the loss of endothelium could be a contributing factor in the atherogenic process.     

Studies on lung tumours. IV. Correlation between [3H]thymidine labelling of lung and liver cells and tumour formation in GRS/A and C3Hf/A male mice following administration of dimethylnitrosamine.

Male mice of the inbred strain GRS/A are highly susceptible to lung tumour but refractory to liver tumour formation, whereas the opposite relation holds for C3Hf/A male mice. Liver and lung cells of these 2 mouse strains were studied autoradiographically after intraperitoneal injection of [3H]dimethylnitrosamine (DMN) and of [3H]thymidine at days 1--14 after administration of unlabelled DMN. Corresponding cell types in the lungs or livers of these 2 mouse strains bound similar amount of [3H]DMN. Among the various types of lung cells only the alveolar Type II cells, from which the lung adenomas derive, showed a strain-specific difference in [3H]thymidine labelling indices, much more cells becoming labelled in the case of the GRS/A than of the C3Hf/A strain at days 3--7 after carcinogen administration. Opposite thymidine labelling indices were exhibited by the parenchymal liver cells of the 2 strains, with C3Hf/A now showing a greater response than did GRS/A males. Thus thymidine-labelling and tumourigenic responses of target lung and liver cells to carcinogen in the 2 strains coincided. Sulphur dioxide and carbon tetrachloride mimicked the effects of DMN on the thymidine labelling indices of, respectively, the lung alveolar Type II and the thymidine labelling indices of, respectively, the lung alveolar Type II and the liver parenchymal cells of the 2 strains. The nature of the differential effect of carcinogen on the [3H]thymidine labelling of the cells and the correlation of these patterns with susceptibility to tumour formation, are briefly discussed.     

Evaluation of radiation dose resulting from the ingestion of [3H]- and [14C]thymidine in the rat

Average doses to rat tissues from the ingestion of 2-[14C]thymidine were compared with those from methyl-[3H]thymidine or 6-[3H]thymidine. Among the three precursors, [14C]thymidine gave the highest dose to spleen and small intestine. The doses to other tissues from [14C]thymidine were almost the same or lower as compared with those from [3H]thymidine, irrespective of the 9 times higher beta-ray energy of 14C than that of 3H. In the case of [14C]thymidine, most of the dose was given by radioactivity incorporated into the organic tissue constituents (non-volatile radioactivity). In the case of [3H]thymidine, however, the dose contributions by non-volatile radioactivity were very small and the major contributions were rather from volatile radioactivity (3HHO), formed by degradation of [3H]thymidine. No significant difference in their total doses was found between the two [3H]precursors, but the dose from non-volatile radioactivity alone was 2-3 times higher with methyl-[3H]thymidine than with 6-[3H]thymidine. Estimates of the dose to cell nuclei in various tissues after the ingestion of [3H]thymidine were also made in order to predict more precisely possible radiation hazards.     

Direct evidence that the insulin receptor mediates a mitogenic response in cultured human fibroblasts

To define the role of the insulin receptor in mediating a mitogenic response in cultured human fibroblasts, the effects of specific monoclonal antibodies against the insulin and the type I IGF receptor on insulin-stimulated [3H]thymidine incorporation were investigated. Insulin stimulated [3H]thymidine incorporation in a biphasic fashion. In the first phase, a half-maximal effect was observed at 20 ng/ml, and a seemingly maximal effect was obtained at 100-1000 ng/ml. With 10 micrograms/ml insulin, a secondary increase in [3H]thymidine incorporation was seen which was similar to the maximal effect of IGF-I. These [3H]thymidine incorporation results were corroborated with cell replication studies. MC-51, a highly specific monoclonal antibody for the insulin receptor, inhibited the stimulation of [3H]thymidine incorporation by 25 ng/ml of insulin. AlphaIR-3, a monoclonal antibody specifically directed against the type I IGF receptor, had no significant effect on insulin-stimulated [3H]thymidine incorporation at low (10-1000 ng/ml) concentrations of insulin. However, alpha IR-3 interfered with the incremental increase in [3H]thymidine incorporation observed at 10-100 micrograms/ml insulin. These data demonstrate that insulin, at low concentrations, is capable of stimulating DNA synthesis and replication of human fibroblasts through interaction with its own receptor, while at supraphysiological concentrations, much of insulin's mitogenic effect is mediated through the type I IGF receptor.     

3H]thymidine incorporation into whole liver as an alternative to [3H]thymidine incorporation into DNA as a parameter of cell proliferation in regenerating liver tissue in rats

OBJECTIVE: To monitor liver regeneration following partial hepatectomy, liver cell proliferation can be measured by assaying in vivo [3H]thymidine incorporation into liver cell DNA. We hypothesized that [3H]thymidine incorporation into whole liver tissue parallels [3H]thymidine incorporation into liver cell DNA, both in high proliferating and low proliferating liver. STUDY DESIGN: Liver cell proliferation in rats after partial hepatectomy or a sham operation was studied by measuring incorporation of [3H]thymidine into various fractions of liver tissue on days 1, 2, 3, 4 and 10 after surgery. RESULTS: [3H]thymidine incorporation into whole liver tissue and in the protein fraction correlated well with DNA-specific [3H]thymidine incorporation into regenerating (r > .80, P < .0001) and nonregenerating liver (r > .69, P < .005). [3H]thymidine incorporation into DNA was < 5% of the total amount of administered [3H]thymidine in both sham-operated and hepatectomized rats. Significant differences in [3H]thymidine incorporation into partially hepatectomized livers as compared to sham-operated rat livers were found on days 1 and 2 (whole liver tissue and protein fraction) or day 1 (DNA) after surgery. CONCLUSION: [3H]thymidine incorporation into whole liver tissue is a simple technique that can be used for the study of liver cell proliferation after partial hepatectomy in rats.     

Synergistic actions of cytokines and growth factors in enhancing porcine granulosa cell growth.

In our studies of the growth-promoting effect of a cytokine, interleukin-1 (IL-1), on cultured porcine granulosa cells, we found that the potency of IL-1 action correlated with the serum concentration in the culture medium and that IL-1 acted synergistically with insulin to increase the number of cells in the presence of low serum concentrations (0.1-1%). With granulosa cells maintained in a quiescent state under serum-free conditions, we therefore examined the effects of combined treatment with IL-1 and peptide growth factors, including insulin, on [3H]thymidine incorporation by these cells. IL-1 by itself enhanced [3H]thymidine incorporation in a concentration-dependent manner. Moreover, IL-1 acted synergistically with insulin, epidermal growth factor (EGF), or fibroblast growth factor (FGF) to enhance [3H]thymidine incorporation. Combinations of maximally effective concentrations of insulin (1 micrograms/ml), EGF (1 ng/ml), or FGF (50 ng/ml) with the maximally effective concentration of IL-1 (10 ng/ml) increased the levels of [3H]thymidine incorporation to 10-, 22-, and 20-fold, respectively, over the control values. Whereas IL-2 (0.1-100 ng/ml) did not affect [3H]thymidine incorporation, tumor necrosis factor alpha (TNF alpha) stimulated [3H]thymidine incorporation by itself and reproduced the actions of IL-1 to act synergistically with insulin, EGF, or FGF. When IL-1 and TNF alpha were added together in relatively low concentrations (1 ng/ml each), the combination had synergistic effects in enhancing [3H]thymidine incorporation. The present study demonstrates that cytokines and peptide growth factors act synergistically to markedly enhance porcine granulosa cell growth in vitro.     

Glucose-stimulated DNA synthesis through mammalian target of rapamycin (mTOR) is regulated by KATP channels: effects on cell cycle progression in rodent islets

The aim of this study was to define metabolic signaling pathways that mediate DNA synthesis and cell cycle progression in adult rodent islets to devise strategies to enhance survival, growth, and proliferation. Since previous studies indicated that glucose-stimulated activation of mammalian target of rapamycin (mTOR) leads to [3H]thymidine incorporation and that mTOR activation is mediated, in part, through the K(ATP) channel and changes in cytosolic Ca2+, we determined whether glyburide, an inhibitor of K(ATP) channels that stimulates Ca2+ influx, modulates [3H]thymidine incorporation. Glyburide (10-100 nm) at basal glucose stimulated [3H]thymidine incorporation to the same magnitude as elevated glucose and further enhanced the ability of elevated glucose to increase [3H]thymidine incorporation. Diazoxide (250 microm), an activator of KATP channels, paradoxically potentiated glucose-stimulated [3H]thymidine incorporation 2-4-fold above elevated glucose alone. Cell cycle analysis demonstrated that chronic exposure of islets to basal glucose resulted in a typical cell cycle progression pattern that is consistent with a low level of proliferation. In contrast, chronic exposure to elevated glucose or glyburide resulted in progression from G0/G1 to an accumulation in S phase and a reduction in G2/M phase. Rapamycin (100 nm) resulted in an approximately 62% reduction of S phase accumulation. The enhanced [3H]thymidine incorporation with chronic elevated glucose or glyburide therefore appears to be associated with S phase accumulation. Since diazoxide significantly enhanced [3H]thymidine incorporation without altering S phase accumulation under chronic elevated glucose, this increase in DNA synthesis also appears to be primarily related to an arrest in S phase and not cell proliferation.     

Tumor necrosis factor stimulates DNA synthesis in the liver of intact rats

TNF is cytotoxic to tumor cell lines but enhances growth of some nontransformed cells. Because animals administered TNF have an increase in liver size, we studied the [3H]thymidine incorporation into DNA in the liver of intact rats. A significant increase in [3H]thymidine incorporation is seen 20 hours following TNF administration and peaks at 24 hours. The lowest dose of TNF that increases DNA synthesis is 10 micrograms/200 g rat with a maximal increase occurring with 25 micrograms/200 g, considerably less than the dose required for maximally increasing plasma triglycerides. The increase in [3H]thymidine incorporation was shown to be due to an increase in DNA polymerase alpha activity (associated with the replication of DNA) rather than DNA polymerases beta (associated with DNA repair) plus gamma activity. These results indicate that TNF administration stimulates DNA replication in the liver of intact animals.     

Whole body and hepatic cholesterol synthesis rates in the guinea-pig: effect of dietary fat quality

The effects of polyunsaturated, monounsaturated and saturated dietary fat on total and hepatic cholesterol synthesis were studied in the guinea-pig. Male Hartley guinea-pigs were fed semi-synthetic diets containing 7.5% (w/w) of either corn oil (CO), olive oil (OL) or lard for a period of 5 weeks and rates of endogenous cholesterol synthesis were determined from the incorporation of [3H]water into digitonin-precipitable sterols (DPS) and by measurement of sterol balance. In addition, total and expressed 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activities were determined in hepatic microsomes. Rates of whole body cholesterol synthesis determined by incorporation of [3H]water into DPS were significantly lower for guinea-pigs on the CO diet with values of 18.7 +/- 1.8 mumol/h (n = 4) vs. 26.7 +/- 4.8 and 24.6 +/- 1.8 mumol/h for animals on the OL (n = 4) and lard (n = 3) diets (P less than 0.001), respectively. Hepatic cholesterol synthesis rates were significantly decreased in animals on the OL diet, whether determined from incorporation of [3H]water into DPS or by analysis of HMG-CoA reductase activity. Hepatic total and free cholesterol levels were not different for animals on the three dietary fats; however, cholesteryl ester levels were 35% lower in guinea-pigs fed the lard diet (P less than 0.02). Sterol balance measurements indicated that whole body cholesterol synthesis rates were not affected by dietary fat quality (51.9 +/- 12.2, 42.8 +/- 7.6 and 51.2 +/- 20.2 mg/kg per day for animals on the CO, OL and lard diets, respectively). This is in striking contrast to the observed reduction in cholesterol synthesis rates for animals on the polyunsaturated CO diet as determined by incorporation of [3H]water into DPS. One possible explanation for the discrepancy between the sterol balance and [3H]water incorporation data is a polyunsaturated fat-mediated effect on energy utilization, which affects the equilibration of NADPH with the body water pool such that the [3H]NADPH has a lower specific activity than body [3H]water.     

Immunohistochemical identification of proliferating cells in organ culture using bromodeoxyuridine and a monoclonal antibody

The ability to measure cell proliferation is important in the study of cancer biology. The usual technique for quantitating proliferating cells in tissue explant and organ culture by detection of [3H]-thymidine incorporation into DNA by autoradiography is tedious and time-consuming. We have developed a technique for identification and quantitation of bromodeoxyuridine (an analogue of thymidine) in cultured tissue explants. Fetal mouse colon explants were exposed in vitro to bromodeoxyuridine (BUdR) or [3H]-thymidine for 3 to 72 hr and then for various periods to unlabeled thymidine. The tissues were stained with a monoclonal anti-bromodeoxyuridine antibody and in parallel [3H]-thymidine incorporation was detected by autoradiography. Incorporation of BUdR was measured by quantitating the amount of pigment deposited over nuclei after immunohistochemical staining, using an optical data digitizer. It was found that both techniques identified proliferating cells. Dividing cells were present both in crypts and in the surrounding stroma in Day 14 fetal mouse colon cultures. The immunohistochemical technique was more rapid and less cumbersome than autoradiography.     

Growth stimulatory effect of pancreatic spasmolytic polypeptide on cultured colon and breast tumor cells

The effects of a novel polypeptide, pancreatic spasmolytic polypeptide (PSP) on a colon carcinoma cell line (HCT 116) were examined. PSP stimulated the incorporation of [3H]thymidine into HCT 116 cells as well as cell proliferation in a dose-dependent manner. Maximal increase in [3H]thymidine incorporation of 50-60% occurred at 3-300 microM PSP. The VIP-mediated-increase in cAMP levels was reduced by PSP at greater than 1 microM concentrations. PSP is highly homologous to the estrogen-induced pS2 protein in MCF-7 breast cancer cells. We find that PSP also enhanced [3H]thymidine incorporation in MCF-7 cells. These findings indicate for the first time that PSP has growth stimulatory properties.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of (3H] uridine incorporation into RNA and [3H] leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10-21 M). Insulin stimulated the rate of [3H] thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100-1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [3H] thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of 3H- uridine, [3H] thymidine and [3H] leucine into their respective precursor pools is not responsible for the apparent stimulation of RNA, DNA and protein synthesis.     

Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide [3H]thymidine incorporation into macrophages

Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.     

Age related changes in total DNA and RNA and incorporation of uridine and thymidine in rat brain

1. Total brain DNA and total brain RNA and the incorporation of thymidine[14C] and uridine[3H] were measured in young and aged rats. 2. From 20 days to the time of sexual maturation, both DNA and RNA levels increase. Total RNA exceeds total DNA at all ages. Comparatively, the ratio of total DNA/RNA is higher in young than in aged animals. 3. The incorporation of thymidine[14C]/g of DNA and of uridine[3H]/g of RNA decreases with age. This decrease is rapid in young animals. After 350 days of age, the incorporation becomes very low. The significance of data is discussed.     

Incorporation of [3H]thymidine by isolated fetal myoblasts and fibroblasts in response to human placental lactogen (HPL): possible mediation of HPL action by release of immunoreactive SM-C

We investigated the actions of human placental lactogen (HPL) and human growth hormone (HGH) on [3H]thymidine incorporation and the release of immunoassayable somatomedin-C (SM-C) by isolated myoblasts, dermal fibroblasts, and costal cartilage explants taken from human fetuses at 11-21 weeks of gestation. The incorporation of [3H]thymidine by myoblasts and fibroblasts was significantly increased after incubation for 20 hr or 44 hr, and cell number after incubation for 7 days, in the presence of 50-250 ng/ml HPL. Incubation with HPL did not increase [3H]thymidine incorporation into cartilage explants, whereas incubation with HGH failed to enhance the uptake of this isotope by any of the tissues. Following extraction with acid-ethanol, culture medium conditioned by exposure to myoblasts or fibroblasts for 44 hr, and to cartilage explants for 7 days, contained radioimmunoassayable SM-C. Myoblast-conditioned medium contained significantly more SM-C [1,609 +/- 953 mU/mg cell protein (mean +/- SD); n = 10] than did that conditioned by fibroblasts (637 +/- 323; n = 5; P less than 0.02). In 1 week of culture, cartilage explants released 4.1 +/- 1.1 mU/mg wet weight (n = 7). The release of immunoassayable SM-C from cultured cells was significantly increased in the presence of 250 ng/ml HPL in five of eight experiments with myoblasts and two of four experiments with fibroblasts. Neither fibroblasts or myoblasts showed increased SM-C release following exposure to HGH. The results suggest that HPL, but not HGH, is growth-promoting for some human fetal tissues in vitro and that this action is mediated, at least in part, by an increased release of somatomedins.     

Effect of 5-Fluoro-2′-Deoxyuridine on [3H]Thymidine Incorporation by Bacterioplankton in the Waters of Southwest Florida

The effect of 5-fluoro-2′-deoxyuridine (FdUrd) on [methyl-³H] thymidine incorporation by bacterioplankton populations in subtropical freshwater, estuarine, and oceanic environments was examined. In estuarine waters, intracellular isotope dilution was inhibited by FdUrd, which enabled us to estimate both intracellular and extracellular isotope dilution. In 2 of 10 cases, extracellular isotope dilution was significant. At low concentrations of [methyl-³H]thymidine or [6-³H]thymidine, FdUrd completely inhibited incorporation of radioactivity into protein and RNA. At high concentrations of [³H]thymidine, however, FdUrd had little effect on labeling patterns. The dihydrofolate reductase inhibitors amethopterin and trimethoprim had no effect on macromolecular labeling patterns. These results suggest that thymidylate synthase is not involved in nonspecific labeling and that FdUrd inhibits nonspecific labeling by blocking some other enzyme involved in thymidine catabolism. In oligotrophic oceanic and freshwater samples, FdUrd did not inhibit intracellular isotope dilution or [³H]thymidine labeling of protein and RNA, but caused some inhibition of [³H]thymidine incorporation into DNA. The ability of FdUrd to inhibit nonspecific macromolecular labeling during [³H]thymidine incorporation was significantly correlated (r = 0.84) with total thymidine incorporation (in picomoles per liter per hour). The results are discussed in terms of applications of FdUrd to routine bacterial production measurements and the general assumptions of [³H]thymidine incorporation.