Release of EPA and DHA from salmon oil - a comparison of in vitro digestion with human and porcine gastrointestinal enzymes.

In the present study, we hypothesised whether in vitro digestion of salmon oil would release different amounts of PUFA depending on the origin of the lipolytic enzymes used. For this purpose, in vitro digestion of salmon oil (SO) was performed using human duodenal juice (HDJ) or a commercial enzyme preparation consisting of porcine pancreatin and bile (PB). The lipolytic effect was determined by measuring the release of fatty acids (FA) using solid-phase extraction and GC-flame ionisation detection, withdrawing samples every 20 min during digestion. The amount of FA released indicated that a plateau was reached after 80 min with approximately similar amounts of FA detected using both HDJ and PB (379 (sd 18) and 352 (sd 23) mg/g SO, respectively). However, the release of 18 : 2, EPA (20 : 5) and DHA (22 : 6) was significantly different during in vitro digestion. At 80 min, HDJ and PB released 43 and 33% of 18 : 2, 14 and 9% of EPA and 11 and 9% of DHA, respectively. Both enzyme preparations released approximately the same amounts of the other FA analysed. The effect of the addition of bile salts (BS) was significantly different in the two enzyme systems, where porcine pancreatin highly responded to the increase in BS concentration, in contrast to HDJ.

Static in vitro digestion model adapted to the general older adult population: an INFOGEST international consensus.

Understanding the mechanisms of food digestion is of paramount importance to determine the effect foods have on human health. Significant knowledge on the fate of food during digestion has been generated in healthy adults due to the development of physiologically-relevant in vitro digestion models. However, it appears that the performance of the oro-gastrointestinal tract is affected by ageing and that a model simulating the digestive conditions found in a younger adult (<65 years) is not relevant for an older adult (>65 years). The objectives of the present paper were: (1) to conduct an exhaustive literature search to find data on the physiological parameters of the older adult oro-gastrointestinal tract, (2) to define the parameters of an in vitro digestion model adapted to the older adult. International experts have discussed all the parameters during a dedicated workshop organized within the INFOGEST network. Data on food bolus properties collected in the older adult were gathered, including food particle size found in older adult boluses. In the stomach and small intestine, data suggest that significant physiological changes are observed between younger and older adults. In the latter, the rate of gastric emptying is slowed down, the pH of the stomach content is higher, the amount of secretions and thus the hydrolytic activities of gastric and intestinal digestive enzymes are reduced and the concentration of bile salts lower. The consensus in vitro digestion model of the older adult proposed here will allow significant progress to be made in understanding the fate of food in this specific population, facilitating the development of foods adapted to their nutritional needs. Nevertheless, better foundational data when available and further refinement of the parameters will be needed to implement the proposed model in the future.

Direct contact ultrasound assisted freezing of chicken breast samples.

Nowadays, rapid freezing is sought to favor the formation of small ice crystals. Several studies have shown that the application of ultrasounds (US) accelerates the processes of energy and mass transfer when they are applied through immersion systems. However, there are hardly any studies on its application in direct systems without the use of a liquid medium for its transmission. Therefore, the objective of this work was to evaluate the potential of the application of US for improving the freezing process of chicken breast samples. First, the application of intermittent US treatments at different net sonication times of 7, 17, 37, 50 and 67% during the freezing of distilled water samples in a conventional freezer was evaluated. It was observed that net sonication times of 37, 50 and 67% reduced the phase change period by 30.0, 21.4, 27.0%, respectively. The effective freezing time was also reduced by 12.4 and 12.8% by applying net sonication times of 37 and 50%. Considering these results, an intermittent US treatment with a net sonication time of 37% was chosen for chicken breast freezing in an air-forced cooling tunnel at ambient temperatures from -13 to -22 °C. The length of all the freezing phases was reduced upon application of US, leading to an overall process time reduction of approx. 11%. On the other hand, no significant differences were found either in the Water Holding Capacity (WHC) or Cooking Loss (CL) values between control and US assisted frozen chicken breast samples. Furthermore, in vitro experiments showed that US-assisted freezing did not influence protein digestibility of chicken meat samples. This study demonstrates the potential of the application of US by direct contact to favor energy transfer processes during freezing of water and chicken breasts samples. However, its effect on the quality of the frozen products should be further studied.

Plant sterols from rapeseed and tall oils: effects on lipids, fat-soluble vitamins and plant sterol concentrations.

BACKGROUND AND AIMS: Data comparing the impact of different sources of plant sterols on CVD risk factors and antioxidant levels is scarce. We evaluated the effects of plant sterols from rapeseed and tall oils on serum lipids, lipoproteins, fat-soluble vitamins and plant sterol concentrations. METHODS AND RESULTS: This was a double-blinded, randomized, crossover trial in which 59 hypercholesterolemic subjects consumed 25 g/day of margarine for 4 weeks separated by 1 week washout periods. The two experimental margarines provided 2g/day of plant sterols from rapeseed or tall oil. The control margarine had no added plant sterols. The control margarine reduced LDL cholesterol by 4.5% (95% CI 1.4, 7.6%). The tall and rapeseed sterol margarines additionally reduced LDL cholesterol by 9.0% (95% CI 5.5, 12.4%) and 8.2% (95% CI 5.2, 11.4%) and apolipoprotein B by 5.3% (95% CI 1.0, 9.6%) and 6.9% (95% CI 3.6, 10.2%), respectively. Lipid-adjusted beta-carotene concentrations were reduced by both sterol margarines (P<0.017). alpha-Tocopherol concentrations were reduced by the tall sterol compared to the rapeseed sterol margarine (P=0.001). Campesterol concentrations increased more markedly with the rapeseed sterol versus tall sterol margarine (P<0.001). The rapeseed sterol margarine increased while the tall sterol margarine decreased brassicasterol concentrations (P<0.001). CONCLUSIONS: Plant sterols from tall and rapeseed oils reduce atherogenic lipids and lipoproteins similarly. The rapeseed sterol margarine may have more favorable effects on serum alpha-tocopherol concentrations.

Cell cycle progression kinetics of regenerating mouse epidermal cells: an in vivo study combining DNA flow cytometry, cell sorting, and [3H]dThd autoradiography.

Cantharidin application to mouse skin induces cell injury followed by a regenerative wave of cells entering S phase in partial synchrony about 16 h after application. After pulse labeling with [3H]dThd the synchronized cohort of cells was traced through subsequent cell cycles during regeneration. This was accomplished by DNA flow cytometry of isolated basal cells combined with sorting from G1, S, and G2 phases followed by autoradiography at intervals after pulse labeling. Successive peaks of labeled cells in S phase at about 12-h intervals, followed by subsequent peaks in G2 and G1 phases were seen. This shows that the peaks of S-phase cells seen at 16 and 28 h after cantharidin application represent mother and daughter cells, respectively, the latter still cycling in partial synchrony. These 2 peaks of S-phase cells, therefore, are not keratinocyte subpopulations with different time lags between the stimulus to regeneration and the subsequent response. It is further shown that the mean cell cycle time is reduced from about 55 h in normal epidermis to 12 h during early regeneration. This is mainly due to a considerably reduced G1 phase duration, but the S and G2 phase durations are also reduced, although still within the range of circadian variations seen in normal animals. It is reasonable to assume a causal relationship between the considerably reduced G1 duration and loss of growth restriction. Cells with a slow progression rate through G2 phase (70% of all G2 cells) in normal mouse epidermis seem to maintain a slow progression rate during regeneration. Normal growth homeostasis seems to be gradually reestablished during the second day of regeneration.

DNA synthesis in mouse epidermis: S phase cells that remain unlabeled after pulse labeling with DNA precursors progress slowly through S.

Epidermal basal cells from hairless mice were isolated after pulse labeling with tritiated DNA precursors and subjected to DNA flow cytometry combined with cell sorting. Cells were sorted from a window in the middle of the S phase, collected on glass slides, and subjected to autoradiography. Unlabeled cells in the middle of the S phase were found in normal mouse epidermis after optimal pulse labeling with tritiated thymidine [( 3H]dThd), in accordance with previous results. The proportion of unlabeled S phase cells was considerably increased among basal cells from mice treated with growth-inhibitory epidermal extracts. Reanalysis and re-sorting of cells previously sorted from mid S showed that unlabeled cells could not be accounted for by G1 contamination. Furthermore, labeling with precursors incorporated into DNA by "de novo" metabolic pathway [( 3H]Urd) did not reduce the proportion of unlabeled S phase cells, either when given alone or when given in combination with the precursor for DNA incorporated by the "salvage" pathway [( 3H]dThd). This strongly indicates that the unlabeled S phase cells do not synthesize DNA continuously, or are synthesizing DNA at a rate below the level of detection. A reduced proportion of unlabeled S phase cells was found in regenerating epidermis. This may be explained by a dilution effect caused by the 3-fold increase in the total number of cells within S phase at this condition. The observation that essentially all cells in mid S phase were labeled during 4 days of continuous labeling with [3H]dThd, indicates that cells in S phase that remain unlabeled after optimal pulse labeling are cycling, albeit slowly. Two-parameter sorting based on DNA and light scatter indicated that slowly cycling cells are larger than the average. These cells may represent a subpopulation of basal cells going through their last division cycle before differentiation.

Evidence of mouse epidermal subpopulations with different cell cycle times.

In order to obtain information on the distribution of total cell cycle times in hairless mouse epidermis, basal cells were isolated and prepared for DNA flow cytometry at intervals after a pulse labeling with 50 microCi of thymidine. The DNA distributions were recorded, and cells were sorted from windows in the S, G2, and G1 phases of the cell cycle, collected on glass slides, and subjected to autoradiography. The proportions of labeled cells were scored in each fraction, and the percentage of labeled mitoses was determined in histologic sections from the same animals. Grain count distributions were recorded at selected time points over labeled cells in sorted fractions and over labeled mitoses. The movement of the labeled S-phase cohort was thus followed through all cell cycle phases. Peaks in labeled cells were observed at about 36 h in S phase, G2 phase, and mitosis, and high levels of labeled G2 cells and mitoses were seen at about 80 h. These results indicate the existence of one rapidly cycling subpopulation of keratinocytes with a cell cycle time slightly less than 30 h, in addition to keratinocytes with considerably longer cell cycle times. The first peak of labeled G2 cells reached only about 30%. This is consistent with earlier findings of about 30% G2 cells with a rapid traverse, and 70% with a considerably delayed traverse through G2 phase. The proportion of labeled G1 cells reached a value corresponding to twice the initial labeling index at 8 h after pulse labeling. This is consistent with previously obtained phase durations, indicating an unperturbed cell cycle traverse of labeled cells from S phase through G2 and mitosis.

Heterogeneity in the mouse epidermal cell cycle analysed by computer simulations.

Different sets of cell kinetic data obtained over many years from hairless mouse epidermis have been simulated by a mathematical model including circadian variations. Simulating several independent sets of data with the same mathematical model strengthens the validity of the results obtained. The data simulated in this investigation were all obtained with the experimental system in a state of natural synchrony. The data include cell cycle phase distributions measured by DNA flow cytometry of isolated epidermal basal cells, fractions of tritiated thymidine ([3H]TdR) labelled cells within the cell cycle phases measured by cell sorting at intervals after [3H]TdR pulse labelling, bivariate bromodeoxyuridine (BrdUrd)/DNA data from epidermal basal cells isolated at intervals after pulse labelling with BrdUrd, mitotic rate and per cent labelled mitosis (PLM) data from histologic sections. The following main new findings were made from the simulations: the second PLM peak observed at about 35 h after pulse labelling is hardly influenced by circadian variations; the peak is mainly determined by persisting synchrony of a rapidly cycling population with a G1-duration (TG1) of 20 h to 30 h; and there is a highly significant population of slowly cycling G1-cells (G1 sigma). However, no significant circadian variations were found in the number of these cells.

Characterization of bladder tumours by multiparameter flow cytometry with special reference to grade II tumours.

Sixty-three human transitional cell carcinomas of the urinary bladder were studied by multiparameter flow cytometry (FCM). The cellular DNA content, the cellular protein content, the fraction of cells in S phase, and the nuclear size were registered and correlated to histological grade (WHO) and histologically determined infiltration through the basement membrane. Aneuploidy was found in the great majority of grade III tumours, but in only 24% of grade II tumours. A new, combined variable, viz. the cellular DNA to protein ratio, indicated a possibility for further subdivision of the tumours. Grade II tumours, which constitute a rather heterogeneous group with regard to prognosis, could be classified in two subgroups: One group of diploid tumours with the FCM characteristics of grade I tumours, and another group of diploid and aneuploid tumours with the characteristics of grade III tumours. Infiltration was most frequently seen in the latter subgroup. The putative prognostic relevance of such a subdivision will be the subject of a future study. Compared to FCM measurement of DNA alone, multiparameter FCM, including measurement of the total cellular protein content, has given additional information that may be of prognostic value.

The epidermal cell kinetic response to ultraviolet B irradiation combines regenerative proliferation and carcinogen associated cell cycle delay.

The cell cycle traverse of epidermal basal cells 24 h after in vivo exposure of ultraviolet B (UVB) irradiation was studied by immunochemical staining of incorporated bromodeoxyuridine (BrdU) and bivariate BrdU/DNA flow cytometric analysis. The results were compared with the cell kinetic patterns following topical application of the skin carcinogen methylnitrosourea (MNU) as well as the skin irritant cantharidin. Hairless mice were injected intraperitoneally with BrdU 24 h after treatment of their back skin with either a minimal erythema dose of UVB, or a single application of MNU or cantharidin dissolved in acetone. The cell cycle traverse of the BrdU-labelled cohorts of epidermal basal cells were then followed for the subsequent 12 h. At 6 h after BrdU-injection, when all labelled cells in the control group as well as in the cantharidin group had left the S phase, the bivariate distributions of the UVB-exposed and the MNU group showed that BrdU-positive cells were still present in S phase. Hence, UVB irradiation, similar to the carcinogen MNU, prolonged the S phase duration in some of the basal cells. At 12 h after pulse labelling, however, BrdU-positive cells from UVB-exposed mice were re-entering S phase from G1 phase, indicating that UVB irradiation induced a shortening of the cell cycle time as well, similar to the response observed after cantharidin. The present data can not tell whether these cells also were delayed in S phase. Thus, the cell cycle traverse in hairless mouse epidermis 24 h after in vivo exposure to UVB seemed to be a combination of the cell kinetic effects following chemical skin carcinogens and skin irritants. UVB irradiation induced both a delay in transit time through S phase, probably due to DNA damage and subsequent repair, as well as a reduction in the total cell cycle time consistent with rapid regenerative proliferation.

Serum cholesterol predictive equations with special emphasis on trans and saturated fatty acids. an analysis from designed controlled studies.

The effects of dietary trans fatty acids on serum total and low density lipoprotein (LDL) cholesterol have been evaluated by incorporating trans fatty acids into predictive equations and comparing their effects with the effects of the individual saturated fatty acids 12:0, 14:0, and 16:0. Trans fatty acids from partially hydrogenated soybean oil (TRANS V) and fish oil (TRANS F) were included in previously published equations by constrained regression analysis, allowing slight adjustments of existing coefficients. Prior knowledge about the signs and ordering of the regression coefficients was explicitly incorporated into the regression modeling by adding lower and upper bounds to the coefficients. The amounts of oleic acid (18:1) and polyunsaturated fatty acids (18:2, 18:3) were not sufficiently varied in the studies, and the respective regression coefficients were therefore set equal to those found by Yu et al. [Yu, S., Derr, J., Etherton, T.D., and Kris-Etherton, P.M. (1995) Plasma Cholesterol-Predictive Equations Demonstrate That Stearic Acid Is Neutral and Monounsaturated Fatty Acids Are Hypocholesterolemic, Am. J. Clin. Nutr. 61, 1129-1139]. Stearic acid (18:0), considered to be neutral, was not included in the equations. The regression analyses were based on results from four controlled dietary studies with a total of 95 participants and including 10 diets differing in fatty acid composition and with 30-38% of energy (E%) as fat. The analyses resulted in the following equations, where the change in cholesterol is expressed in mmol/L and the change in intake of fatty acids is expressed in E%: delta Total cholesterol = 0.01 delta(12:0) + 0.12 delta(14:0) + 0.057 delta(16:0) + 0.039 delta(TRANS F) + 0.031 delta(TRANS V) - 0.0044 delta(18:1) - 0.017 delta(18:2, 18:3) and deltaLDL cholesterol = 0.01 delta(12:0) + 0.071 delta(14:0) + 0.047 delta(16:0) + 0.043 delta(TRANS F) + 0.025 delta(TRANS V) - 0.0044 delta(18:1) - 0.017 delta(18:2, 18:3). The regression analyses confirm previous findings that 14:0 is the most hypercholesterolemic fatty acid and indicate that trans fatty acids are less hypercholesterolemic than the saturated fatty acids 14:0 and 16:0. TRANS F may be slightly more hypercholesterolemic than TRANS V or there may be other hypercholesterolemic fatty acids in partially hydrogenated fish oil than those included in the equations. The test set used for validation consisted of 22 data points from seven recently published dietary studies. The equation for total cholesterol showed good prediction ability with a correlation coefficient of 0.981 between observed and predicted values. The equation has been used by the Norwegian food industry in reformulating margarines into more healthful products with reduced content of cholesterol-raising fatty acids.

Replacement of partially hydrogenated soybean oil by palm oil in margarine without unfavorable effects on serum lipoproteins.

We have compared the effects of three different margarines, one based on palm oil (PALM-margarine), one based on partially hydrogenated soybean oil (TRANS-margarine) and one with a high content of polyunsaturated fatty acids (PUFA-margarine), on serum lipids in 27 young women. The main purpose of the study was to test if replacement of trans fatty acids in margarine by palmitic acid results in unfavorable effects on serum lipids. The sum of saturated fatty acids (12:0, 14:0, 16:0) was 36.3% of total fatty acids in the PALM-diet, the same as the sum of saturated (12:0, 14:0, 16:0) (12.5%) and trans (23.1%) fatty acids in the TRANS-diet. This sum was 20.7% in the PUFA-diet. The content of oleic acid was 37.9, 35.2, and 38.6%, respectively, in the three diets, whereas linoleic acid amounted to 16, 13.5, and 27.3%, respectively. Total fat provided 30-31% and the test margarines 26% of total energy in all three diets. The subjects consumed each of the diets for 17 d in a Latin-square crossover design. There were no significant differences in total cholesterol, low density lipoprotein (LDL)-cholesterol and apolipoprotein B (apoB) between the TRANS- and the PALM-diets. High density lipoprotein (HDL)-cholesterol and apoA-1 were significantly higher on the PALM-diet compared to the TRANS-diet whereas the ratio of LDL-cholesterol to HDL-cholesterol was lower, although not significantly (P = 0.077) on the PALM-diet. Total cholesterol, LDL-cholesterol, and apoB were significantly lower on the PUFA-diet compared to the two other diets. HDL-cholesterol was not different on the PALM- and the PUFA-diets but it was significantly lower on the TRANS-diet compared to the PUFA diet. Compared to the PUFA-diet the ratio of LDL- to HDL-cholesterol was higher on both the PALM- and the TRANS-diets whereas apoA-1 was not different. Triglycerides and lipoprotein (a) were not significantly different among the three diets. We concluded that nutritionally, palmitic acid from palm oil may be a reasonable alternative to trans fatty acids from partially hydrogenated soybean oil in margarine if the aim is to avoid trans fatty acids. A palm oil-based margarine is, however, less favorable than one based on a more polyunsaturated vegetable oil.

Serum cholesterol predictive equations in product development.

The aim of the study was to incorporate trans fatty acids into predictive equations for serum cholesterol and compare their effects with the effects of the individual saturated fatty acids 12:0, 14:0 and 16:0. We have introduced trans fatty acids from partially hydrogenated soybean oil (TransV) and fish oil (TransF) into previously published equations by constrained regression analysis. Prior knowledge about the signs and ordering of existing regression coefficients were incorporated into the regression modelling by adding lower and upper bounds to the coefficients. Oleic acid (18:1) and polyunsaturated fatty acids (18:2, 18:3) were not sufficiently varied in the studies and the respective regression coefficients therefore set equal to those found by Yu et al. (Am J Clin Nutr 1995;61:1129-39). Stearic acid (18:0) considered to be neutral was not included in the equations. The regression analyses were based on results from four controlled dietary studies with a total of 95 participants and including 10 diets differing in fatty acid composition. The analyses resulted in the following equations where the change in cholesterol is expressed in mmol/L and the change in intake of fatty acids is expressed in E%: Delta Total cholesterol = 0.01 delta(12:0) + 0.12 Delta(14:0) + 0.057 delta(16:0) + 0.039 delta(TransF) + 0.031 delta(TransV)- 0.0044 delta(18:1) - 0.017 delta(18:2, 18:3) and deltaLDL cholesterol = 0.01 delta(12:0) + 0.071 delta(14:0) + 0.047 delta(16:0) + 0.043 delta(TransF) + 0.025 delta(TransV) - 0.0044 delta(18:1) - 0.017 delta(18:2, 18:3). The test set used for validation consisted of 22 data points from seven recently published dietary studies. The equation for total cholesterol showed good prediction ability with a correlation coefficient of 0.981 between observed and predicted values. The equation has been used to reformulate margarines into "trans free" products all with more favourable effects on serum cholesterol than previous products. Also a cholesterol reducing margarine has been produced. When tested against butter in an open clinical trial among subjects with mild hypercholesterolemia the observed cholesterol-lowering effect of this margarine corresponded reasonably well with the predicted (0.77 vs. 0.64 mmol/L). We conclude that the equation has practical applicability and can be used to formulate and nutritionally optimise fat products as well as to evaluate already existing products on the market.

Effect of broccoli phytochemical extract on release of fatty acids from salmon muscle and salmon oil during in vitro digestion.

The aim of the present work was to study the effect of a broccoli phytochemical extract (Br-ex) on the release of fatty acids (FA) from salmon muscle (SM) and salmon oil (SO) during in vitro digestion. The hypothesis of the study was that Br-ex contains polyphenols which might act as pancreatic lipase inhibitors. The effect on the release of specific FA, in particular the long-chain n-3 polyunsaturated fatty acids (PUFAs), EPA (C20:5 n-3) and DHA (C22:6 n-3), was recorded, and the impact of the SM matrix was studied by comparing the release of FA from SM and SO. In vitro digestion was performed and lipolytic activity, measured as the release of fatty acids (FFA) by solid phase extraction and GC-FID, was recorded at 20, 40, 80 and 140 minutes in the intestinal phase. The results showed, unexpectedly, that Br-ex stimulated the release of FA during digestion of SO and SM, showing the highest increases in FFA, 67% and 64%, respectively, at 20 min. No difference in the release of FA from SO compared to SM was observed, suggesting that the SM matrix had minor influence on the lipolytic activity. The results also demonstrated that the increase in lipolytic activity caused by Br-ex was not affected by the SM matrix. However, addition of Br-ex resulted in a lower percentage of EPA and DHA in the FFA fraction, suggesting that the lipase sn-position preference was altered. Whether this affects the bioaccessibility of EPA and DHA needs further investigation.

A standardised static in vitro digestion method suitable for food - an international consensus.

Simulated gastro-intestinal digestion is widely employed in many fields of food and nutritional sciences, as conducting human trials are often costly, resource intensive, and ethically disputable. As a consequence, in vitro alternatives that determine endpoints such as the bioaccessibility of nutrients and non-nutrients or the digestibility of macronutrients (e.g. lipids, proteins and carbohydrates) are used for screening and building new hypotheses. Various digestion models have been proposed, often impeding the possibility to compare results across research teams. For example, a large variety of enzymes from different sources such as of porcine, rabbit or human origin have been used, differing in their activity and characterization. Differences in pH, mineral type, ionic strength and digestion time, which alter enzyme activity and other phenomena, may also considerably alter results. Other parameters such as the presence of phospholipids, individual enzymes such as gastric lipase and digestive emulsifiers vs. their mixtures (e.g. pancreatin and bile salts), and the ratio of food bolus to digestive fluids, have also been discussed at length. In the present consensus paper, within the COST Infogest network, we propose a general standardised and practical static digestion method based on physiologically relevant conditions that can be applied for various endpoints, which may be amended to accommodate further specific requirements. A frameset of parameters including the oral, gastric and small intestinal digestion are outlined and their relevance discussed in relation to available in vivo data and enzymes. This consensus paper will give a detailed protocol and a line-by-line, guidance, recommendations and justifications but also limitation of the proposed model. This harmonised static, in vitro digestion method for food should aid the production of more comparable data in the future.

Circadian variations in cell cycle phase progression of mouse epidermal cells measured directly by bivariate BrdUrd/DNA flow cytometry.

Circadian stage-dependent variations in cell cycle traverse of mouse epidermal cells in vivo were investigated. The fate of cohorts of basal cells pulse-labelled with bromodeoxyuridine (BrdUrd) at different times of the day were studied by bivariate BrdUrd/DNA flow cytometry of isolated epidermal basal cells. Basal cells were tracked through the cell cycle up to 96 h after intraperitoneal injection of BrdUrd at 0800 and 2000, or followed for 6 h after BrdUrd injection at 0400, 1200, 1600 and 2400. The results confirmed our previous assumption that the cell cycle progression through S phase and G2 phase is considerably delayed at night, i.e. from 1600 to 0400, compared with daytime. The results indicate variations in G1 phase as well. The data strongly support the hypothesis that the main parameters responsible for circadian fluctuations in mitotic activity are variations in the S and G2 phase durations. The data are also consistent with the notion of proliferative heterogeneity among basal cells as described by a hierarchical proliferation model.

Persisting long-term effects of a single carcinogenic dose of methylnitrosourea on epidermal growth in mice.

The cell proliferation in hairless mouse epidermis was studied before tumor development following a single application of a carcinogenic dose (2 mg) of N-methyl-N-nitrosourea (MNU). The number of basal and suprabasal cells, the mitotic index (MI) and the mitotic rate (MR) were scored in histological sections. The [3H]thymidine labeling index (LI) and the mean grain count (MGC) were scored in histological sections or in smears of basal cells. Flow cytometric two-parameter analyses of cellular DNA and protein content were performed on isolated epidermal basal cells. An increased MR and a slight but consistent epidermal hyperplasia were found. A 24-h study performed 25 weeks after MNU application showed that the MR and MI were altered in a circadian stage-dependent manner with considerably increased values around noon when the circadian rhythms had their peaks, followed by normal values around midnight. The LI was generally increased, but showed a normal circadian rhythm with high values at night and low values during day. The MGC was reduced at night and in the morning when the LI values were high. The results show that a single carcinogenic dose of MNU caused alterations in the epidermal growth kinetics that persisted until tumor development. The altered growth parameters, however, had circadian rhythms that were in phase with control rhythms. Assuming a constant size of the proliferative compartment, the increased mitotic activity indicated a considerable shortening of the mean cell cycle time.

Cell kinetics in mouse epidermis studied by bivariate DNA/bromodeoxyuridine and DNA/keratin flow cytometry.

Hairless mice were injected intraperitoneally with bromodeoxyuridine (Brd-Urd). Basal cells were isolated from epidermis, fixed in 70% ethanol, and prepared for bivariate BrdUrd/DNA flow cytometric (FCM) analysis. Optimum detection of incorporated BrdUrd in DNA was obtained by combining pepsin digestion and acid denaturation. The cell loss was reduced to a minimum by using phosphate-buffered saline containing Ca2+ and Mg2+ to neutralize the acid. The percentage of cells in S phase and the average uptake of BrdUrd per labelled cell in eight consecutive windows throughout the S phase were measured after pulse labelling at intervals during a 24 h period. Furthermore, the cell cycle progression of a pulse-labelled cohort of cells was followed up to 96 h after BrdUrd injection. In general the results from both experiments were in good agreement with previous data from 3H-thymidine labelling studies. The percentage of cells in S phase was highest at night and lowest in the afternoon, whereas the average uptake of BrdUrd per labelled cell showed only minor circadian variations. There were no indications that BrdUrd significantly perturbed normal epidermal growth kinetics. A cell cycle time of about 36 h was observed for the labelled cohort. Indications of heterogeneity in traverse through G1 phase were found, and the existence of slowly cycling or temporarily resting cells in G2 phase was confirmed. There was, however, no evidence of a significant population of temporarily resting cells in the S phase. Bivariate DNA/keratin FCM analysis revealed a high purity of basal cells in the suspensions and indicated that the synthesis of the differentiation-keratin K10 was turned on only in G1 phase and after the last division.

Effects of pulse labelling with tritiated thymidine on the circadian rhythmicity in epidermal cell-cycle distribution in mice.

The influence of pulse labelling with 50 microCi tritiated thymidine ( [3H]TdR) (2 microCi/g) on epidermal cell-cycle distribution in mice was investigated. Animals were injected intraperitoneally with the radioactive tracer or with saline at 08.00 hours, and groups of animals were sacrificed at intervals during the following 32 hr. Epidermal basal cells were isolated from the back skin of the animals and prepared for DNA flow cytometry, and the proportions of cells in the S and G2 phases of the cell cycle were estimated from the obtained DNA frequency distributions. The proportions of mitoses among basal cells were determined in histological sections from the same animals, as were the numbers of [3H]TdR-labelled cells per microscopic field by means of autoradiography. The results showed that the [3H]TdR activity did not affect the pattern of circadian rhythms in the proportions of cells in S, G2 and M phase during the first 32 hr after the injection. The number of labelled cells per vision field was approximately doubled between 8 and 12 hr after tracer injection, indicating an unperturbed cell-cycle progression of the labelled cohort. In agreement with previous reports, an increase in the mitotic index was seen during the first 2 hr. These data are in agreement with the assumption that 50 microCi [3H]TdR given as a pulse does not perturb cell-cycle progression in mouse epidermis in a way that invalidates percentage labelled mitosis (PLM) and double-labelling experiments.

A comparison between the epidermal regenerative responses provoked by a skin irritant and a tumor promoter using anti-BrdUrd/DNA flow cytometry.

The proliferative responses induced in hairless mouse epidermis after application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the skin irritant cantharidin were investigated. Doses known to give the same degree of hyperplasia were chosen. Mice were pulse-labeled with bromodeoxyuridine (BrdUrd) 30 min prior to or 24 h after a single application of either cantharidin or TPA, and thereafter killed at various time intervals. The basal cells were isolated from epidermis, fixed in 70% ethanol and prepared for bivariate BrdUrd/DNA flow cytometric analysis. Cells pulse-labeled in S phase 30 min prior to treatment with cantharidin or TPA were slightly delayed in their progression through S phase and temporarily blocked in G2 phase. However, they were still able to re-enter S phase 18 h later, indicating a shortening of the G1 phase. Cells pulse-labeled 24 h after treatment had a considerably reduced cell cycle time, i.e. reduced G1 transit time. Hence, the wave of cells entering S phase from 16 h after injury could be explained by an immediate reduction in G1 transit time, without assuming recruitment of temporarily resting G0/G1 cells. Although cantharidin caused the longest initial delay in cell cycle progression, the subsequent proliferative response was less pronounced than that provoked by TPA. Rapid proliferation may allow for clonal expansion of initiated cells. The higher ability of TPA to induce rapid proliferation, apparently without causing any severe initial cell damage, may thus be related to its higher tumor promoting ability.