Effects of four aromatic organic pollutants on microbial glucose metabolism and thymidine incorporation in marine sediments

The metabolism of D-[U-14C]glucose and the incorporation of [methyl-3H]thymidine by aerobic and anaerobic marine sediment microbes exposed to 1 to 1,000 ppm anthracene, naphthalene, p,p'-dichlorodiphenyltrichloroethane, and pentachlorophenol were examined. Cell-specific rates of [14C]glucose metabolism averaged 1.7 X 10(-21) and 0.5 X 10(-21) mol/min per cell for aerobic and anaerobic sediment slurries, respectively; [3H]thymidine incorporation rates averaged 43 X 10(-24) and 9 X 10(-24) mol/min per cell for aerobic and anaerobic slurries, respectively. Aerobic sediments exposed to three of the organic pollutants for 2 to 7 days showed recovery of both activities. Anaerobic sediments showed little recovery after 2 days of pre-exposure to the pollutants. We conclude that (i) anaerobic sediments are more sensitive than aerobic sediments to pollutant additions; (ii) [3H]thymidine incorporation is more sensitive to pollutant additions than is [14C]glucose metabolism; and (iii) the toxicity of the pollutants increased in the following order: anthracene, p,p'-dichlorodiphenyltrichloroethane, naphthalene, and pentachlorophenol.     

Bacterial [methyl-3H]thymidine incorporation in substrate-amended estuarine sediment slurries

Abstract Five different bacterial communities were enriched in substrate-amended slurries of sediment from the Tay Estuary, Scotland. During incubation of the slurries, concentrations of volatile fatty acids, sulphate, sulphide and methane were monitored to clearly define the activity of the stimulated populations. An aerobic population, a ‘microaerophilic’ population and three anaerobic populations (fermentative heterotrophs, sulphate-reducing bacteria and methanogens plus acetogens) were established to reflect community growth and metabolism both in surface oxic and deeper anoxic layers. Similar numbers of cells involved in division were observed in all five slurries, demonstrating the potential for bacterial production. Thymidine incorporation rates in glucose-stimulated slurries under both aerobic and fully anaerobic conditions were similar, confirming the ability of fermentative anaerobic heterotrophs to incorporate [ methyl -³H]thymidine into DNA during growth. Although anaerobic communities of sulphate-reducing, acetogenic plus methanogenic bacteria were stimulated and actively growing, they did not incorporate [ methyl -³H]thymidine into DNA. Since the thymidine technique does not measure the growth of these important groups, calculated productivity values based upon thymidine incorporation within anoxic sediment systems will be substantially underestimated, even if growth substrates are not limiting.     

Effects of co-occurring aromatic hydrocarbons on degradation of individual polycyclic aromatic hydrocarbons in marine sediment slurries.

Rates of polycyclic aromatic hydrocarbon (PAH) degradation and mineralization were influenced by preexposure to alternate PAHs and a monoaromatic hydrocarbon at relatively high (100 ppm) concentrations in organic-rich aerobic marine sediments. Prior exposure to three PAHs and benzene resulted in enhanced [14C]naphthalene mineralization, while [14C]anthracene mineralization was stimulated only by benzene and anthracene preexposure. Preexposure of sediment slurries to phenanthrene stimulated the initial degradation of anthracene. Prior exposure to naphthalene stimulated the initial degradation of phenanthrene but had no effect on either the initial degradation or mineralization of anthracene. For those compounds which stimulated [14C]anthracene or [14C]naphthalene mineralization, longer preexposures (2 weeks) to alternative aromatic hydrocarbons resulted in an even greater stimulation response. Enrichment with individual PAHs followed by subsequent incubation with one or two PAHs showed no alteration in degradation patterns due to the simultaneous presence of PAHs. The evidence suggests that exposure of marine sediments to a particular PAH or benzene results in the enhanced ability of these sediments to subsequently degrade that PAH as well as certain other PAHs. The enhanced degradation of a particular PAH after sediments have been exposed to it may result from the selection and proliferation of specific microbial populations capable of degrading it. The enhanced degradation of other PAHs after exposure to a single PAH suggests that the populations selected have either broad specificity for PAHs, common pathways of PAH degradation, or both.     

Effects of co-occurring aromatic hydrocarbons on degradation of individual polycyclic aromatic hydrocarbons in marine sediment slurries

Rates of polycyclic aromatic hydrocarbon (PAH) degradation and mineralization were influenced by preexposure to alternate PAHs and a monoaromatic hydrocarbon at relatively high (100 ppm) concentrations in organic-rich aerobic marine sediments. Prior exposure to three PAHs and benzene resulted in enhanced [14C]naphthalene mineralization, while [14C]anthracene mineralization was stimulated only by benzene and anthracene preexposure. Preexposure of sediment slurries to phenanthrene stimulated the initial degradation of anthracene. Prior exposure to naphthalene stimulated the initial degradation of phenanthrene but had no effect on either the initial degradation or mineralization of anthracene. For those compounds which stimulated [14C]anthracene or [14C]naphthalene mineralization, longer preexposures (2 weeks) to alternative aromatic hydrocarbons resulted in an even greater stimulation response. Enrichment with individual PAHs followed by subsequent incubation with one or two PAHs showed no alteration in degradation patterns due to the simultaneous presence of PAHs. The evidence suggests that exposure of marine sediments to a particular PAH or benzene results in the enhanced ability of these sediments to subsequently degrade that PAH as well as certain other PAHs. The enhanced degradation of a particular PAH after sediments have been exposed to it may result from the selection and proliferation of specific microbial populations capable of degrading it. The enhanced degradation of other PAHs after exposure to a single PAH suggests that the populations selected have either broad specificity for PAHs, common pathways of PAH degradation, or both.     

Aerobic glycolysis and lymphocyte transformation

1. The role of enhanced aerobic glycolysis in the transformation of rat thymocytes by concanavalin A has been investigated. Concanavalin A addition doubled [U-(14)C]glucose uptake by rat thymocytes over 3h and caused an equivalent increased incorporation into protein, lipids and RNA. A disproportionately large percentage of the extra glucose taken up was converted into lactate, but concanavalin A also caused a specific increase in pyruvate oxidation, leading to an increase in the percentage contribution of glucose to the respiratory fuel. 2. Acetoacetate metabolism, which was not affected by concanavalin A, strongly suppressed pyruvate oxidation in the presence of [U-(14)C]glucose, but did not prevent the concanavalin A-induced stimulation of this process. Glucose uptake was not affected by acetoacetate in the presence or absence of concanavalin A, but in each case acetoacetate increased the percentage of glucose uptake accounted for by lactate production. 3. [(3)H]Thymidine incorporation into DNA in concanavalin A-treated thymocyte cultures was sensitive to the glucose concentration in the medium in a biphasic manner. Very low concentrations of glucose (25mum) stimulated DNA synthesis half-maximally, but maximum [(3)H]thymidine incorporation was observed only when the glucose concentration was raised to 1mm. Lactate addition did not alter the sensitivity of [(3)H]-thymidine uptake to glucose, but inosine blocked the effect of added glucose and strongly inhibited DNA synthesis. 4. It is suggested that the major function of enhanced aerobic glycolysis in transforming lymphocytes is to maintain higher steady-state amounts of glycolytic intermediates to act as precursors for macromolecule synthesis.     

Depth Distribution of Bacterial Production in a Stratified Lake with an Anoxic Hypolimnion

The purpose of this study was to determine the depth distribution of bacterial biomass and production in a stratified lake and to test techniques to measure bacterial production in anaerobic waters. Bacterial abundance and incorporation of both [³H]thymidine and [³H]leucine into protein were highest in the metalimnion, at the depth at which oxygen first became unmeasurable. In contrast, [³H]thymidine incorporation into DNA was highest in the epilimnion. The ratios of incorporation into DNA/protein averaged 2.2, 0.49, and 0.95 for the epilimnion, metalimnion, and hypolimnion, respectively. Low incorporation into DNA was not due to artifacts associated with the DNA isolation procedure. Recovery of added [³H]DNA was about 90% in waters in which the portion of [³H]thymidine incorporation into DNA was about 40%. At least some obligate anaerobic bacteria were capable of assimilating thymidine since aeration of anaerobic hypolimnion waters substantially inhibited thymidine incorporation. The depth profile of bacterial production estimated from total thymidine and leucine incorporation and the frequency of dividing cells were all similar, with maximal rates in the metalimnion. However, estimates of bacterial production based on frequency of dividing cells and leucine incorporation were usually significantly higher than estimates based on thymidine incorporation (using conversion factors from the literature), especially in anaerobic hypolimnion waters. These data indicate that the thymidine approach must be examined carefully if it is to be applied to aquatic systems with low oxygen concentrations. Our results also indicate that the interface between the aerobic epilimnion and anaerobic hypolimnion is the site of intense bacterial mineralization and biomass production which deserves further study.     

Anaerobic degradation of polycyclic aromatic hydrocarbons and alkanes in petroleum-contaminated marine harbor sediments.

Although polycyclic aromatic hydrocarbons (PAHs) have usually been found to persist under strict anaerobic conditions, in a previous study an unusual site was found in San Diego Bay in which two PAHs, naphthalene and phenanthrene, were oxidized to carbon dioxide under sulfate-reducing conditions. Further investigations with these sediments revealed that methylnaphthalene, fluorene, and fluoranthene were also anaerobically oxidized to carbon dioxide in these sediments, while pyrene and benzo[a]pyrene were not. Studies with naphthalene indicated that PAH oxidation was sulfate dependent. Incubating the sediments with additional naphthalene for 1 month resulted in a significant increase in the oxidation of [14C]naphthalene. In sediments from a less heavily contaminated site in San diego Bay where PAHs were not readily degraded, naphthalene degradation could be stimulated through inoculation with active PAH-degrading sediments from the most heavily contaminated site. Sediments from the less heavily contaminated site that had been adapted for rapid anaerobic degradation of high concentrations of benzene did not oxidize naphthalene, suggesting that the benzene- and naphthalene-degrading populations were different. When fuels containing complex mixtures of alkanes were added to sediments from the two sites, there was significant degradation in the alkanes. [14C]hexadecane was also anaerobically oxidized to 14CO2 in these sediments. Molybdate, a specific inhibitor of sulfate reduction, inhibited hexadecane oxidation. These results demonstrate that a wide variety of hydrocarbon contaminants can be degraded under sulfate-reducing conditions in hydrocarbon-contaminated sediments, and they suggest that it may be possible to use sulfate reduction rather than aerobic respiration as a treatment strategy for hydrocarbon-contaminated dredged sediments.     

Incorporation of [methyl-3H]thymidine by obligate and facultative anaerobic bacteria when grown under defined culture conditions

Abstract Incorporation of [ methyl -³H]thymidine into bacterial DNA was determined for a range of axenic anaerobic bacterial cultures: fermentative heterotrophs, sulphate-reducing bacteria, purple sulphur bacteria, acetogens and methanogens. Anaerobically growing Bacillus sp. and the obligate aerobe Thiobacillus ferrooxidans were also investigated. Actively growing cultures of sulphate-reducing bacteria belonging to the genera Desulfovibrio, Desulfotomaculum, Desulfobacter, Desulfobotulus and Desulfobulbus , purple sulphur bacteria ( Chromatium vinosum OP2 and Thiocapsa roseopersicina OP1), methanogens ( Methanococcus GS16 and Methanosarcina barkeri ) and an acetogen ( Acetobacterium woodii ) did not incorporate [ methyl -³H]thymidine into DNA. The only obligate anaerobes in which thymidine incorporation into DNA could be unequivocally demonstrated were members of the genus Clostridium . Anaerobically growing Bacillus sp. also incorporated thymidine. These data demonstrate that pure culture representatives of major groups of anaerobic bacteria involved in the terminal oxidation of organic carbon and anoxygenic phototrophs within sediments are unable to incorporate [ methyl -³H]thymidine into DNA, although some obligate and facultative anaerobes can. Variability in thymidine incorporation amongst pure culture isolates indicates that unless existing techniques can be calibrated to take this into consideration then productivity estimates in both aerobic and anaerobic environments may be greatly underestimated using the [ methyl -³H]thymidine technique.     

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.     

H]thymidine incorporation to estimate growth rates of anaerobic bacterial strains

The incorporation of [H]thymidine by axenic cultures of anaerobic bacteria was investigated as a means to measure growth. The three fermentative strains and one of the methanogenic strains tested incorporated [H]thymidine, whereas the sulfate-reducing bacterium and two of the methanogenic bacteria were unable to incorporate [H]thymidine during growth. It is concluded that the [H]thymidine incorporation method underestimates bacterial growth in anaerobic environments.     

Microbial carbon monoxide consumption in salt marsh sediments

We have examined sediments from a fringing salt marsh in Maine to further understand marine CO metabolism, about which relatively little is known. Intact cores from the marsh emitted CO during dark oxic incubations, but emission rates were significantly higher during anoxic incubations, which provided evidence for simultaneous production and aerobic consumption in surface sediments. CO emission rates were also elevated when cores were exposed to light, which indicated that photochemical reactions play a role in CO production. A kinetic analysis of marsh surface sediments yielded an apparent K(m) of about 82 ppm, which exceeded values reported for well-aerated soils that consume atmospheric CO (65nM). Surface (0-0.2 cm depth interval) sediment slurries incubated under oxic conditions rapidly consumed CO, and methyl fluoride did not inhibit uptake, which indicated that neither ammonia nor methane oxidizers contributed to the observed activity. In contrast, aerobic CO uptake was inhibited by additions of readily available organic substrates (pyruvate, glucose and glycine), but not by cellulose. CO was also consumed by surface and sub-surface sediment slurries incubated under anaerobic conditions, but rates were less than during aerobic incubations. Molybdate and nitrate or nitrite, but not 2-bromoethanesulfonic acid, partially inhibited anaerobic uptake. These results suggest that sulfidogens and acetogens, but not dissimilatory nitrate reducers or methanogens, actively consume CO. Sediment-free plant roots also oxidized CO aerobically; rates for Spartina patens and Limonium carolinianum roots were significantly higher than rates for Spartina alterniflora roots. Thus plants may also impact CO cycling in estuarine environments.     

Stimulation of protein synthesis in cultured heart muscle cells by glucose.

Glucose stimulated the rate of incorporation of [3H]leucine into HCLO4-insoluble fraction of cultured rat heart muscle cells under both aerobic and anaerobic conditions. In the aerobic system the incorporation proceeded at a constant rate during 3h of incubation with and without glucose whereas in the anaeorbic system the incorporation ceased after approx. 60 min and could be renewed only by the addition of glucose. No correlation was found to exist between the above effect of glucose on protein synthesis and glucose-dependent changes in the intracellular ATP concentration. The extent of the stimulation of protein synthesis was related to the concentration of glucose. The effect of glucose was suppressed by cycloheximide but was not affected by actinomycin D. Glucose had no effect on the rate of transport of alpha-aminoisobutyric acid. Mannose also stimulated [3H]leucine incorporation. Substances that did not produce lactate were ineffective. Iodoacetate inhibited the stimulatory effect of glucose, but pyruvate, which by itself had no apprecialbe stimulatory action, relieved the inhibition induced by iodoacetate. There was no concomitant change in the concentration of ATP when iodoacetate inhibition was reversed by pyruvate. L-Lactate or other intermediates of energy metabolism could not relieve the inhibitory effect of iodoacetate.     

[3H]Thymidine Incorporation To Estimate Growth Rates of Anaerobic Bacterial Strains

The incorporation of [³H]thymidine by axenic cultures of anaerobic bacteria was investigated as a means to measure growth. The three fermentative strains and one of the methanogenic strains tested incorporated [³H]thymidine, whereas the sulfate-reducing bacterium and two of the methanogenic bacteria were unable to incorporate [³H]thymidine during growth. It is concluded that the [³H]thymidine incorporation method underestimates bacterial growth in anaerobic environments.     

3,7-Dichloroquinolinecarboxylic Acid Inhibits Cell-Wall Biosynthesis in Maize Roots

The mode of action of the herbicide 3,7-dichloroquinolinecar-boxylic acid (quinclorac) was examined by measuring incorporation of [14C]glucose, [14C]acetate, [3H]thymidine, and [3H]uridine into maize (Zea mays) root cell walls, fatty acids, DNA, and RNA, respectively. Among the precursors examined, 10 [mu]M quinclorac inhibited [14C]glucose incorporation into the cell wall within 3 h. Fatty acid and DNA biosynthesis were subsequently inhibited, whereas RNA biosynthesis was unaffected. In contrast to the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile, quinclorac strongly inhibited cellulose and a hemicellulose fraction presumed to be glucuronoarabinoxylan. However, the synthesis of (1->3),(1->4)-[beta]-D-glucans was only slightly inhibited. The degree of inhibition was time- and dose-dependent. By 4 h after treatment, the concentration that inhibited [14C]glucose incorporation into the cell wall, cellulose, and the sensitive hemicellulose fraction by 50% was about 15, 5, and 20 [mu]M, respectively. Concomitant with an inhibition of [14C]glucose incorporation into the cell wall, quinclorac treatment led to a marked accumulation of radioactivity in the cytosol. The increased radioactivity was found mostly in glucose and fructose. However, total levels of glucose, fructose, and uridine diphosphate-glucose were not changed greatly by quinclorac. These data suggest that quinclorac acts primarily as a cell-wall biosynthesis inhibitor in a susceptible grass by a mechanism that is different from that of 2,6-dichlorobenzonitrile.     

Microbial mineralization of VC and DCE under different terminal electron accepting conditions

Production of 14CO2 from [1,2-14C] dichloroethene (DCE) or [1,2-14C] vinyl chloride (VC) was quantified in aquifer and stream-bed sediment microcosms to evaluate the potential for microbial mineralization as a pathway for DCE and VC biodegradation under aerobic, Fe(III)-reducing, SO4-reducing, and methanogenic conditions. Mineralization of [1,2-14C] DCE and [1,2-14C] VC to 14CO2 decreased under increasingly reducing conditions, but significant mineralization was observed for both sediments even under anaerobic conditions. VC mineralization decreased in the order of aerobic > Fe(III)-reducing > SO4-reducing > methanogenic conditions. For both sediments, VC mineralization was greater than DCE mineralization under all electron-accepting conditions examined. For both sediments, DCE mineralization was at least two times greater under aerobic conditions than under anaerobic conditions. Although significant microbial mineralization of DCE was observed under anaerobic conditions, recovery of 14CO2 did not differ substantially between anaerobic treatments.     

Microbial activities in deep subsurface environments

Activities of microorganisms residing in terrestrial deep subsurface sediments were examined in 46 sediment samples from three boreholes. Radiolabeled time course experiments assessing in situ microbial activities were initiated within 30 min of core recovery. [1‐C⁴] Acetate incorporation into lipids, [ methyl‐³H] thymidine incorporation into DNA, [2‐¹⁴C]acetate, and [U‐¹⁴C]glucose mineralization in addition to microbial enrichment and enumeration studies were examined in surface and subsurface sediments. Surface soils contained the greatest biomass and activities, followed by the shallow aquifer zones. Water‐saturated subsurface sands exhibited three to four orders of magnitude greater activity and culturable microorganisms than the dense clay zones, which had low permeability. Regardless of depth, sediments that contained more than 20% clays exhibited the lowest activities and culturable microorganisms.     

Microbial biomass and utilization of dissolved organic matter in the okefenokee swamp ecosystem

The Okefenokee Swamp exhibited levels of microbial biomass and aerobic glucose uptake comparable to those of other organically rich, detritus-based aquatic ecosystems. In contrast to other peat-accumulating systems, this acidic (pH 3.7), low-nutrient environment does not show diminished water column or surface sediment microbial biomass or heterotrophic activity. The total particular ATP varied between 0.03 and 6.6 mug liter (mean, 1.6 mug liter) in water and between 1 and 28 mug g (dry weight) (mean, 10.0 mug g [dry weight] in sediments. The turnover times for dissolved d-glucose were 1.26 to 701.25 h (mean, 110.25 h) in aerobic waters and 2.4 to 72 min (mean, 10.2 min) in aerobic surface sediments. Water column bacterial secondary production, measured as the incorporation of [H]thymidine into cold-trichloroacetic acid-insoluble material, ranged from 0.06 to 1.67 nmol liter day (mean, 0.45 nmol liter day). The kinetics of d-glucose uptake by water column microflora are multiphasic and suggest the presence of a diverse microbial population capable of using labile substrates at nanomolar concentrations and at substantial rates. The presence of a large and active aerobic microbial community in the Okefenokee Swamp is indicative of an important role for microbes in swamp geochemistry and strongly suggests the existence of a detritus-based food web.     

High glucose increase cell cycle regulatory proteins level of mouse embryonic stem cells via PI3-K/Akt and MAPKs signal pathways

This study examined the effects of high glucose on cell proliferation and its related signal pathways using mouse embryonic stem (ES) cells. Here, we showed that high glucose level significantly increased [3H]thymidine incorporation, BrdU incorporation, the number of cells, [3H]leucine, and [3H]proline incorporation in a time-( >3 hr) and dose-(> 25 mM) dependent manner. Moreover, high glucose level increased the cellular reactive oxygen species (ROS), Akt, and mitogen-activated protein kinases (MAPKs) phosphorylation. Subsequently, these signaling molecules involved in high glucose-induced increase of [3H]thymidine incorporation. High glucose level also increased cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4 protein levels, which is cell cycle regulatory proteins acting in G1-S phase of cell cycle. Inhibition of phosphatidylinositol 3-kinase (PI3-K) (LY 294002: PI3-kinase inhibitor, 10(-6) M), Akt (Akt inhibitor, 10(-5) M), and p44/42 MAPKs (PD 98059: MEK inhibitor, 10(-5) M) decreased these proteins. High glucose level phosphorylated the RB protein, which was decreased by inhibition of PI3-K and Akt. In conclusion, high glucose level stimulates mouse ES cell proliferation via the PI3-K/Akt and MAPKs pathways.     

Synthesis and phosphorylation of histone H1 and high mobility group proteins in the regenerating rat liver after X irradiation

A rat was irradiated to the upper abdomen including the liver and then partially hepatectomized. The subsequent synthesis and phosphorylation of histone H1 and nonhistone chromosomal high mobility group (HMG) proteins were investigated. Incorporation of [3H]lysine into histone H1 was increased and reached its peak at 27 h after hepatectomy, and 14 Gy of X rays inhibited the increase. Increase in the incorporation of [3H]lysine into HMG (1 + 2), 14, and 17 which occurred around 27 h after hepatectomy was not inhibited by 14 Gy irradiation. Phosphorylation of histone H1, measured with 32Pi incorporation in vivo, was maximal between 21 and 24 h, and it was inhibited by 4.8 Gy of X rays and delayed with 1.9 Gy. Phosphorylation of HMG 14, which was the only HMG protein phosphorylated under present conditions, was not affected by X irradiation. The [3H]thymidine incorporation into nuclear DNA started increasing at 21 h and reached its maximum at 27 h after hepatectomy. X irradiation with 4.8 Gy inhibited the incorporation, and 1.9 Gy lowered it.     

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.