Lymphocyte transformation in vitro : V. Thymidine incorporation into unstimulated lymphocytes

In vitro conditions and kinetics of ¹⁴C-thymidine incorporation into unstimulated lymphocytes were studied. Lymphocytes cultured during 3 to 9 days displayed a progressive increase of thymidine uptake with time. The addition of varying numbers of autologous mitomycin-treated lymphocytes to cultures containing a fixed number of untreated lymphocytes markedly enhanced thymidine uptake per non-mitomycin-treated lymphocytes. In the latter cultures a sharp rise of thymidine uptake between the 7th day of culture was seen. Supernatants of non-stimulated lymphocytes, whether mitomycin-treated or not, showed no stimulating effect on autologous normal cells in these experiments. Although the mechanism of the enhancing effect of mitomycin-treated autologous lymphocytes remains unclear, it obviously may interfere in culture experiments in which the antigen specific stimulatory effect of mitomycin-treated cells is measured, which can only be detected by including the appropriate controls.     

Bi-functional action of transforming growth factor-beta on DNA synthesis in early passage human fetal fibroblasts

We investigated the influence of transforming growth factor-beta (TGF-beta) on DNA synthesis in human fetal fibroblasts, as measured by the incorporation of [3H]thymidine and cell replication. In serum-free medium, without additional peptide growth factors, TGF-beta had no action on thymidine incorporation. However, in the presence of 0.1% v/v fetal calf serum, TGF-beta exhibited a bi-functional action on the cells. A dose-dependent stimulation of [3H]thymidine incorporation, and an increase in cell number, occurred with fibroblasts established from fetuses under 50 g body weight, with a maximum stimulation seen at 1.25 ng/ml. For fibroblasts from fetuses of 100 g or greater body weight, TGF-beta caused a dose-related decrease in thymidine uptake with a maximal inhibition at 2.5 ng/ml, and a small decrease in cell number. When DNA synthesis was stimulated by the addition of somatomedin-C/insulin-like growth factor I, epidermal growth factor, or platelet-derived growth factor, their actions were potentiated by the presence of TGF-beta on cells derived from fetuses under 50 g body weight, but inhibited on cells obtained from the larger fetuses weighing more than 100 g. Similar results were found for changes in cell number in response to TGF-beta when stimulated by SM-C/IGF I. The ability of TGF-beta to modulate [3H] thymidine incorporation did not involve a change in the time required for growth-restricted cells to enter the S phase of the replication cycle. These data suggest that TGF-beta may exert either a growth-promoting or growth-inhibiting action on human fetal connective tissues in the presence of other peptide growth factors, which is dependent on fetal age and development.     

Coordination of growth and differentiation in the fetal lung

The male fetal lung begins to synthesize surfactant later in gestation than the female. This delay appears to be caused by androgens. We hypothesized that male fetal lung differentiation is delayed as a consequence of an extended phase of growth which is elicited by androgens. We observed that in vivo fetal lung protein synthesis relative to DNA synthesis peaked earlier in gestation in the female fetal lung and that this event was synchronous with the onset of differentiation. Pregnant rats were treated with dihydrotestosterone (DHT) during pregnancy, and fetal lung growth parameters were measured. Lung wet weight, dry weight, and DNA and protein concentrations were significantly elevated by DHT treatment. Type II cells and fibroblasts were isolated from lungs of DHT-treated fetuses. The number of total cells recovered was increased by 30%; the number of type II cells recovered was increased by 87%; and the number of fibroblasts recovered was increased by 42%. The type II cells which were recovered exhibited increased incorporation of [3H]thymidine into DNA and a reduced ratio of radiolabeled protein to radiolabeled DNA compared to that of cells from control lungs. Further studies were done in vitro with fibroblasts and type II cells isolated from untreated fetal rat lungs. Treatment of the fibroblasts with DHT during culture caused an increase in thymidine incorporation into DNA. This effect was not blocked by simultaneous treatment with cortisol, which normally causes reduced DNA synthesis and induces fibroblast differentiation. Treatment of the type II cells with DHT in culture caused a dose-dependent increase in cell number but a decrease in synthesis of disaturated phosphatidylcholine. These studies provide more direct evidence of the interrelationships between the control of growth and the control of differentiation in the fetal lung. DHT, a signal which delays the onset of expression of differentiation, also induces growth. We conclude that the controls of growth and of differentiation of the fetal lung are reciprocally linked.     

Gonadotropin induction of a regulatory mechanism of steroidogenesis in fetal Leydig cell cultures

Studies were conducted to define further the development of the gonadotropin induced, E2 mediated steroidogenic lesion (17-alpha-hydroxylase/17,20-desmolase) in fetal Leydig cell cultures. Analysis of dispersed fetal testes purified by centrifugal elutriation demonstrated a group of cells with sedimentation velocity 12 less than to less than 16.8 mm/h.g containing a small population of adult like "transitional" Leydig cells and homogeneous "fetal" Leydig cell population collected at greater than 19.3 mm/h.g. After cells were cultured for 3 days with addition of 1 microgram oLH at 3 day intervals, the transitional cells showed testosterone accumulation comparable to the fetal cells. In contrast, transitional cells had 10-fold higher basal and hCG-stimulated aromatase activity than fetal cells, and a lack of testosterone response to acute (3 h) hCG stimulation. At day 6, transitional cells steroidogenic ability declined markedly. The fetal population maintained in culture with LH additions every 3 days, showed typical immature Leydig cell response, with enhancement of acute testosterone response to hCG at 3 day (1-fold) and at 6 day of culture (5-fold). Higher doses of LH (5 micrograms/day) or daily treatment of 1 microgram to fetal cultures, induced a lesion of 17 alpha-hydroxylase/17,20-desmolase with reduction of enzymatic activities (P less than 0.01) and impaired testosterone production (P less than 0.01) in response to acute hCG stimulation. Also aromatase was stimulated by hCG + 140% and 50% and E2 receptors were increased by 100 and 180% at 3 days and 6 days of cultures with daily or high dose LH addition, findings consistent with the observation of the E2-mediated lesion during LH action. In conclusion, the cultured fetal Leydig cell provides a useful model to elucidate molecular mechanisms involved in the development of gonadotropin-induced estradiol-mediated desensitization. Treatment of fetal Leydig cell cultures with multiple or frequent doses of LH elevate aromatase activity to necessary levels for the induction of desensitization. We have isolated small population of transitional Leydig cells with morphological characteristics of cells found in 15 day post-natal testis but functional capabilities of adult cells. We have also demonstrated the emergence of a functional adult-like population from the fetal Leydig cell.     

Cultured fetal rat myoblasts release peptide growth factors which are immunologically and biologically similar to somatomedin

The production of immunologically and biologically active somatomedin activity from isolated myoblasts and fibroblasts from fetal rats of 21 days gestational age was investigated. Myoblast-rich cell populations were derived from primary cultures of dispersed muscle cells by the tendency of myoblasts to become detached from the culture dish in the presence of cytochalasin B. Fibroblasts were obtained from fetal muscle. Culture medium conditioned by exposure to myoblasts for 48 hours produced an increased incorporation of both [35S]sulphate and [3H]thymidine by explants of fetal rat costal cartilage in vitro compared to fresh medium. Myoblast-conditioned medium also contained somatomedin-C-like immunoreactivity which diluted in parallel with partially purified human somatomedin-C (3,271 +/- 446 mU/mg cell protein; mean +/- SEM, seven experiments). Medium conditioned by exposure to fetal rat fibroblasts did not promote isotope uptake by fetal rat cartilage above control values, and contained only low levels of somatomedin-C-like immunoreactivity (343 +/- 89 mU/mg cell protein, three experiments). The release of both somatomedin bioactivity and immunoreactivity into conditioned medium was significantly reduced by the incubation of myoblasts in the presence of rat growth hormone (100 ng/ml and 500 ng/ml). We conclude that fetal rat myoblasts released growth factor activity during culture which exhibited biological and immunologic characteristics of somatomedin. Since the bioactivity was demonstrated on skeletal tissues from rat fetuses of the same gestational age as those that yielded myoblasts such growth factor release may be physiological.     

Effects of retinoic acid on cell proliferation and cell differentiation in a rat tracheal epithelial cell line

Abstract. The effects of 3.3 times 10-⁷ M to 3.3 times 10^-5 M all- trans -retinoic acid (vitamin-A acid) on the total cell population dynamics of 165 S, a keratinizing epithelial cell line from carcinogen-exposed rat trachea, were studied. During the first 6 days of culture, cells accumulated on the dish in the presence of the vitamin to twice the density of controls. [³H]thymidine incorporation into DNA and percentage of [³H]thymidine-labelled cells in autoradiographs were stimulated in a dose-dependent fashion to a maximum of 25- and 34-fold, respectively. Exfoliation of cells from the cultures was also enhanced 2–3-fold, resulting in nearly twice the total number of cells (attached plus exfoliated) in the presence of the vitamin.
During 19 days of culture, retinoic acid maintained a higher level of [³H]thymidine incorporation and cell exfoliation in 165 S cells so that by day 19, total cell production was more than three times that seen in controls. At this time, vitamin-treated cultures showed a reduced cell saturation density compared to controls. The higher final cell density in the control cultures was a result of multilayering and papillary formation which did not occur in the presence of retinoic acid. The papillae in control cultures stained specifically with Rhodamine B or with the eosin and orange G components of the Papanicolaou method. A count of the number of eosin and orange G positive cells in the attached and exfoliated cell compartments showed an 8-fold reduction of keratinization in retinoic acid-exposed cultures. Our results show that retinoic acid is a growth stimulant in these cell cultures, causing increased cell proliferation and exfoliation accompanied by inhibition of keratinization.     

Spontaneous proliferation in unfractionated spleen cell cultures: autologous mixed-lymphocyte reactions (AMLR) which can be differentially regulated by prostaglandins and lymphokines

The present studies were undertaken to define the contribution of the autologous or syngeneic mixed-leukocyte reactions (AMLR/SMLR) to the cellular proliferation observed in unfractionated spleen cell cultures. Proliferation was studied in whole, untreated 6-day murine spleen cell cultures supplemented with syngeneic serum. These cultures exhibited relatively low but significant levels of cellular proliferation as measured by uptake of radioactive thymidine ([3H]TdR). Treatment of spleen cells with monoclonal anti-Thy 1.2 antibody and complement before culture, the addition of specific anti-I-A monoclonal antibodies to the cultures or removal of Ia+ adherent cells before initiation of culture all inhibited the proliferative response significantly. Thus, the autologous proliferation of untreated and unfractionated spleen cells manifests the main characteristics of the AMLR/SMLR, namely, its dependence on T (responder) and Ia+ (stimulator) cells and specific inhibition by anti-I-A antibodies. A marked augmentation in cellular proliferation was observed in unfractionated spleen cell cultures treated for the initial 24 hr of culture with 5 X 10(-6) M indomethacin, an inhibitor of prostaglandin synthesis. Conversely, the addition of 7 X 10(-9) M prostaglandin E1 (PGE1) to these cultures depressed cellular proliferation. This suppression of autologous splenic cell proliferation induced by PGE1 could be partially reversed by the addition of concanavalin A-induced lymphokine (LK) preparations early in the culture. These findings indicate that (a) the proliferation of unfractionated spleen cell cultures occurring in the absence of exogenous stimulatory signals is due largely to an ongoing AMLR, and (b) biologically active mediators with opposing influences, namely, prostaglandins and immunostimulatory LK, participate in the regulation of the AMLR.     

Juvenile sandhoff disease: Complementation tests with Sandhoff and Tay-Sachs disease using polyethylene glycol-induced cell fusion

Summary Juvenile Sandhoff, Sandhoff, and Tay-Sachs fibroblasts were mixed in paired combinations and treated with polyethylene glycol (PEG) to promote cell fusion. The hexosaminidase (hex) isozymes of PEG-treated mixed-cell cultures were determined and compared with those of untreated control cultures. Fusions involving juvenile Sandhoff and Sandhoff fibroblasts did not show an increase in either total hexosaminidase or heat-stable hex B. Fusions of juvenile Sandhoff (or Sandhoff) and Tay-Sachs fibroblasts showed an increase of heat-labile hex A. Thus, juvenile Sandhoff cells show complementation with Tay-Sachs cells but not Sandhoff cells. Consequently, the genetic defect in juvenile Sandhoff disease probably represents an allelic mutation of the gene that is defective in Sandhoff disease.     

Mechanical stretch promotes alveolar epithelial type II cell differentiation.

Functional maturation of pulmonary alveolar epithelial cells is crucial for extrauterine survival. Mechanical distension and mesenchymal-epithelial interactions play important roles in this process. We hypothesized that mechanical stretch simulating fetal breathing movements is an important regulator of pulmonary epithelial cell differentiation. Using a Flexercell Strain Unit, we analyzed effects of stretch on primary cultures of type II cells and cocultures of epithelial and mesenchymal cells isolated from fetal rat lungs during late development. Cyclic stretch of isolated type II cells increased surfactant protein (SP) C mRNA expression by 150 +/- 30% over controls (P < 0.02) on gestational day 18 and by 130 +/- 30% on day 19 (P < 0.03). Stretch of cocultures with fibroblasts increased SP-C expression on days 18 and 19 by 170 +/- 40 and 270 +/- 40%, respectively, compared with unstretched cocultures. On day 19, stretch of isolated type II cells increased SP-B mRNA expression by 50% (P < 0.003). Unlike SP-C, addition of fibroblasts did not produce significant additional effects on SP-B mRNA levels. Under these conditions, we observed only modest increases in cellular immunoreactive SP-B, but secreted saturated phosphatidylcholine rose by 40% (P < 0.002). These results indicate that cyclic stretch promotes developmentally timed differentiation of fetal type II cells, as a direct effect on epithelial cell function and via mesenchymal-epithelial interactions. Expression of the SP-C gene appears to be highly responsive to mechanical stimulation.     

Mitogenic effect of fibroblast growth factor on early passage cultures of human and murine fibroblasts

Fibroblast growth factor (FGF), a polypeptide that has been shown to stimulate division in 3T3 cells, was tested for mitogenic effects on diploid, early-passage cells from human and murine sources. The quantitative assay of [3H]thymidine incorporation into acid-insoluble material showed that FGF at low concentrations (10 minus 9 M) was more effective than additional serum for provoking the initiation of DNA synthesis in human foreskin fibroblasts or mouse fibroblasts maintained in 5 or 10% serum, respectively. The growth of the human fibroblasts was twice as fast in the presence of FGF plus 10% calf serum as it was in the presence of 10% calf serum or 20% fetal calf serum alone. The addition of FGF to primary cultures of mouse fibroblasts in 0.4% serum resulted in a twofold increase in cell number compared to controls. In contrast to results obtained with 3T3 cells, neither insulin nor a glucocorticoid potentiated the effects of FGF on either human or mouse cells.     

Inosine 5'-monophosphate vs inosine and hypoxanthine as substrates for purine salvage in human lymphoid cells

The ability of inosine 5'-monophosphate vs inosine or hypoxanthine to supply the total purine requirements of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells was evaluated. Mitogen-stimulated human peripheral blood T cells were treated with aminopterin to inhibit purine synthesis de novo and make the cells dependent upon an exogenous purine source. Thymidine was added as a source of pyrimidines. Under these conditions, 25 microM inosine 5'-monophosphate, inosine, and hypoxanthine showed comparable abilities to support [3H]thymidine incorporation into DNA at rates equal to that of untreated control cultures. In parallel experiments with the rapidly dividing human B lymphoblastoid cell line, WI-L2, treatment with aminopterin (plus thymidine) inhibited the growth rate by greater than 95%. The normal growth rate was restored by the addition of 30 microM inosine 5'-monophosphate, inosine, or hypoxanthine to the medium. However, in similar experiments with cell line No. 1254, a derivative of WI-L2 which lacks detectable ecto-5'-nucleotidase activity, only inosine and hypoxanthine (plus thymidine), but not inosine 5'-monophosphate (and thymidine) were able to restore the growth inhibition due to aminopterin. These results show that the catalytic activity of ecto-5'-nucleotidase is sufficient to meet the total purine requirements of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells and suggest that this enzyme may have functional significance when rates of purine synthesis de novo are limited and/or an extracellular source of purine nucleotides is available.     

The effect of prostaglandin E2 (PGE2) on amino acid uptake and protein formation by lung fibroblasts

The effect of prostaglandin E2 (PGE2) on the utilization of extracellular amino acids by fetal lung fibroblasts was examined. PGE2 decreased the uptake of proline and aminoisobutyric acid (AIB) by quiescent fibroblasts in culture. The uptake of AIB by serum-activated cultures was also dramatically decreased by PGE2. The PGE2-induced decrease in the uptake of AIB was first observed at 4 h following the addition of the effector molecule to the cultures. PGE2 did not affect the uptake of leucine. The addition of cycloheximide also resulted in a decrease in the uptake of proline, similar to that induced by PGE2 at 5 X 10(-8) M. The combination of cycloheximide and PGE2 resulted in a further decrease in proline uptake. Kinetic analysis of AIB uptake following a 24-h PGE2 treatment showed an increase in the apparent Km as compared with untreated cultures. The prostaglandin remained active for at least 72 h after the addition of the molecule. Removal of the PGE2 was followed by an influx of proline into the cells. The decrease in proline uptake was associated with a decrease in the amount of intracellular free proline and an overall decrease in the amount of cell-associated protein. While PGE2 is known to increase intracellular protein degradation, the effect of PGE2 on amino acid uptake was not the result of an increase in the intracellular concentration of amino acids (transinhibition).     

The effects of ethanol on cellular calcium content in primary myocardial cell cultures from offspring of sedentary and swim-trained pregnant rats

Primary myocardial cell cultures obtained from offspring of swim-trained (T) and sedentary (S) Sprague-Dawley rat mothers (dams) were used to evaluate the influence of exercise training and ethanol on cellular calcium (45Ca) content. The pregnant rats swam in water maintained at 37 degrees C 6 days/week during gestation. The dams swam continuously for 30 min on the first day of gestation. The swimming time was increased by 5 min until the rats swam continuously for 1.5 hr. After the cultures had been in incubation for 4 days, the cells were treated with 600, 800, and 1000 mg% ethanol for 30 min and 1 hr. 45Ca content (nmol/mg protein) of the untreated controls and all but one of the ethanol treated groups from the T cultures (1000 mg%) were significantly elevated over the comparable groups from the S cultures for both 30 min and 1 hr of incubation (p less than or equal to 0.05). The data suggest that exercise during pregnancy induces adaptations in myocardial cell cultures from the offspring such that 45Ca content levels are elevated which may provide protection against ethanol toxicity.     

Repair of benzo[a]pyrene-initiated DNA damage in human cells requires activation of DNA polymerase alpha

Normal human fibroblasts treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) yielded DNA polymerase alpha with elevated levels of activity, incorporated [3H]thymidine as a function of unscheduled DNA synthesis, and exhibited restoration of normal DNA-strand length as a function of unscheduled DNA synthesis. Lipoprotein-deficient fibroblasts treated with BPDE did not show elevated levels of DNA polymerase alpha activity, exhibited minimal [3H]thymidine incorporation, and had fragmented DNA after 24 h of repair in the absence of lipoprotein or phosphatidylinositol supplementation. When DNA polymerase beta activity was inhibited, cells with normal lipoprotein uptake exhibited [3H]thymidine incorporation into BPDE-damaged DNA but did not show an increase in DNA-strand length. DNA polymerase alpha activity and [3H]thymidine incorporation in lipoprotein-deficient fibroblasts increased to normal levels when the cells were permeabilized and low-density lipoproteins or phosphatidylinositol were introduced into the cells. DNA polymerase alpha isolated from normal human fibroblasts, but not from lipoprotein-deficient fibroblasts, showed increased specific activity after the cells were treated with BPDE. When BPDE-treated lipoprotein-deficient fibroblasts were permeabilized and 32P-ATP was introduced into the cells along with lipoproteins, 32P-labeled DNA polymerase alpha with significantly increased specific activity was isolated from the cells. These data suggest that treatment of human fibroblasts with BPDE initiates unscheduled DNA synthesis, as a function of DNA excision repair, which is correlated with increased activity of DNA polymerase alpha, and that increased DNA polymerase alpha activity may be correlated with phosphorylation of the enzyme in a reaction that is stimulated by low-density lipoprotein or by the lipoprotein component, phosphatidylinositol.     

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.     

Thymidine as a synchronizing agent. I. Nucleic acid and protein formation in synchronous HeLa cultures treated with excess thymidine

Synchronous cultures of HeLa cells obtained by selective detachment of mitoses were treated with high concentrations of thymidine. The inhibitor was added soon after completion of cell division and rates of cell enlargement and accumulation of DNA, RNA and protein were compared for untreated and thymidine-treated cultures at various points of the cell cycle. It was found that concentrations of thymidine which in randomly growing cultures inhibit the rate of cell division by more than 90% allowed a considerable degree of DNA synthesis and did not affect the rate of accumulation of RNA and protein, when applied to cells in the G1 phase of synchronous culture. Treated and untreated cells enlarged at the same rate throughout their life cycle. The results show that concentrations of thymidine commonly employed to produce cell synchrony do not arrest the cells at the G1-S boundary, but allow slow progress through S in respect to DNA synthesis, and near-normal progress towards G2 as regards RNA and protein accumulation and cell enlargement.     

Inhibitory effect of Boerhaavia diffusa on experimental metastasis by B16F10 melanoma in C57BL/6 mice

Administration of the aqueous methanol (3:7) extract of B.diffusa was found to be effective in reducing the metastases formation by B16F10 melanoma cells. Prophylactic administration of the extract (0.5 mg/dose) inhibited the metastases formation by about 95% as compared to untreated control animals. There was 87% of inhibition in the lung metastases formation in syngenic C57BL/6 mice, when the extract was administered simultaneously with tumour challenge. Biochemical parameters such as lung collagen hydroxyproline, hexosamines and uronic acid levels were also reduced significantly (P < 0.001) in the treated animals. Levels of serum sialic acids and gamma-glutamyltranspeptidase that are markers of neoplastic proliferation were also reduced in the tumour plus extract treated animals. More over treatment with the extract enhanced the survival of the animals more than double that of untreated control animals. When a non-toxic concentration of the extract was treated directly to the B16F10 cells in vitro, it inhibited the cell proliferation as estimated by the 3H - thymidine uptake assay. From the Zymogram analysis using culture supernatant from the extract treated cells it became evident that the components of the extract inhibited the expression or activity of gelatinases A and B (MMP-2 and MMP-9). Since the MMPs are intimately associated with cell invasion and angiogenesis, inhibition of these functions along with the anti-proliferative activity (cytostatic) may be contributing to the antimetastatic property shown by B. diffusa.     

The interactions of benzo(a)pyrene with cell membranes: uptake into Chinese hamster ovary (CHO) cells and fluorescence studies with isolated membranes.

The interactions of benzo(a)pyrene (B(a)P) with the cell surface membrane were studied by measuring B(a)P uptake into intact mammalian cells and by determining B(a)P fluorescence in the presence of isolated cell surface membranes. It was found that 0.19 mu-g B(a)P were taken up by 10-6 Chinese hamster ovary (CHO) cells after 30 min exposure to a solution containing 0.59 mu-g/ml. Culture conditions were found to markedly alter B(a)P uptake. Low cell culture densities resulted in a four-fold increase in rate of B(a)P uptake per cell relative to confluent monolayer cultures. The uptake rate of B(a)P was reduced in the presence of bovine serum (BS) and, under some conditions, perylene. This information should be considered in the design of experiments on the biological effects of B(a)P. Another aspect of B(a)P membrane interaction was that the binding of B(a)P to cell surface membranes could be measured by fluorescence. The additional B(a)P fluorescence, found in the presence of cell surface membranes, was sufficiently large that the methods of data treatment used in the study of fluorescent probe-membrane interactions could be applied to get quantitative information on B(a)P-membrane interactions. It was found that 0.6 x 10-8 moles B(a)P were bound per mg membrane protein and that the apparent statistical dissociation constant for the complex was 3.8 x 10-7 M. The data suggest that the mechanism of uptake of B(a)P is probably passive diffusion.     

Primary cultures of hepatocytes on human fibroblasts

Summary Parenchymal hepatocytes isolated from adult rats were cultured on three types of collagen-containing substrata: collagen-coated plates, collagen membranes and confluent diploid human fibroblasts. Hepatocytes on the latter two substrata maintained characteristic morphology for at least 10 days in culture, whereas degenerative changes (cell death and formation of multinucleated hepatocytes) and growth of nonparenchymal elements were seen after 5 days in cultures on collagen-coated plates. Parallel findings were seen on basal and induced levels of cytochrome P-450 and NADPH-cytochrome C reductase. The basal levels of cytochrome P-450 were not measurable after day 3 in hepatocytes cultured on collagen-coated plates, whereas measurable levels were maintained in the hepatocytes cultured on the other two substrata. Addition of phenobarbital or methylcholanthrene at day 5 in culture caused an increase in cytochromes P-450 and P-448, respectively, only in hepatocytes cultured on collagen membranes and confluent fibroblasts. Analogous results were seen for the enzyme NADPH-cytochrome C reductase. The similarities in performance between hepatocytes on collagen membranes and on human fibroblasts show that a continuous collagen-containing substratum is important for optimal performance of hepatocytes in primary culture. The possible importance of cultures of hepatocytes on human fibroblasts for carcinogenesis studies is discussed.