Unilateral Renal Ischemia as a Model of Acute Kidney Injury and Renal Fibrosis in Cats.

The objectives of this study were to define the acute and chronic effects of 1-hour unilateral in vivo renal ischemia on renal function and histology in cats. Twenty-one adult purpose-bred research cats were anesthetized, and 1 kidney underwent renal artery and vein occlusion for 1 hour. Serum creatinine and urea concentrations, urine protein:creatinine ratio, urine-specific gravity, glomerular filtration rate, hematocrit, platelet concentration and function, and white blood cell count were measured at baseline and variable time points after ischemia. Renal histopathology was evaluated on days 3, 6, 12, 21, 42, and 70 postischemia; changes in smooth muscle actin and interstitial collagen were examined. Following ischemia, whole animal glomerular filtration rate was significantly reduced (57% of baseline on day 6; P < .05). At the early time points, the ischemic kidneys exhibited severe acute epithelial necrosis accompanied by evidence of regeneration of tubules predominantly within the corticomedullary junction. At later periods, postischemic kidneys had evidence of tubular atrophy and interstitial inflammation with significantly more smooth muscle actin and interstitial collagen staining and interstitial fibrosis when compared with the contralateral control kidneys. This study characterizes the course of ischemic acute kidney injury in cats and demonstrates that ischemic acute kidney injury triggers chronic fibrosis, interstitial inflammation, and tubular atrophy in feline kidneys. These late changes are typical of those observed in cats with naturally occurring chronic kidney disease.

The effect of isoproterenol and hydroxyurea on the presence of ubiquitin and protein A24 in the rat salivary gland.

The in vivo administration of hydroxyurea for 12 h counteracts DNA synthesis and cell cycling stimulated by 72 h of isoproterenol treatment in rat salivary gland, as determined by fluorescence-activated flow cytometry. Hydroxyurea has little effect on [3H]leucine incorporation (protein synthesis) of the nuclear proteins soluble in 0.35 M NaCl, when examined by polyacrylamide gel chromatography and autoradiography from electrostatically sorted nuclei of (G0 + G1) and (G2 + M) phases of the in vivo cell cycle. Differential incorporation of [3H]leucine into nuclear proteins was observed during various phases of the cell cycle. Proteins 'X' and 'Z', observed in stained gel chromatographs of the 0.35 M NaCl-soluble nuclear proteins, were identified by biochemical analyses as ubiquitin and protein A24, respectively. Ubiquitin appeared transiently while A24 increased in gel chromatograms concomitant with progressive quiescence of the salivary gland induced by hydroxyurea.

The modulating effect of isoproterenol on DNA replication and protein synthesis. Synthesis patterns of the HMG proteins from electrostatically sorted salivary gland nuclei during the in vivo cell cycle.

Within 96 h after initial isoproterenol administration, DNA replication and cell cycling were activated, as reflected in the bimodal distribution of nuclear fluorescence determined by flow-microfluorometric techniques. A group of proteins, the cetyltrimethylammonium bromide extractable nuclear proteins (CTAB-proteins), isolated form electrostatically sorted nuclei of rat salivary glands, was shown by staining and autoradiography after two-dimensional electrophoresis to undergo differential synthesis during various phases of the in vivo cell cycle after isoproterenol administration. Stained chromatographs revealed quantitative differences in protein synthesis. Gel autoradiography was a more sensitive technique than staining for detecting nuclear protein synthesis during cell cycling. As observed in the autoradiographs of the CTAB-proteins, isoproterenol initiated two distinct periods of protein synthesis in the salivary gland cell cycle: one during the 2C population G0/G1), and one during the 4C population (G2/M). Protein synthesis after isoproterenol administration was much more dramatic in the 2C (isoproterenol) population, where five new spots were seen. There was less radioactive incorporation in the 4C (isoproterenol) population. Two spots 'a' and 'b' that demonstrate differential protein synthesis in stained gel chromatographs and gel autoradiographs were shown to have electrophoretic mobilities, molecular weights and amino acid compositions highly similar to those of HMG1 and HMG2, respectively. A positive correlation could also be drawn between quantitative levels of 'a' and 'b' and their levels of incorporation during cellular activity with HMG (high mobility group) proteins. For example protein 'b' (HMG2) was consistently more abundant in proliferating cell populations than in the quiescent ones. Autoradiographic patterns of the CTAB-proteins indicated that proteins 'a' and 'b' were synthesized during the G0/G1 phase of the cell cycle, as were the majority of CTAB-proteins.

Cell cycle-specific effects of sodium arsenite and hyperthermic exposure on incorporation of radioactive leucine and phosphate by stress proteins from mouse lymphoma cell nuclei.

Cultured mouse lymphoma cells incorporated [3H]leucine and [32P]phosphate into nuclear stress proteins within 3 h after exposure to either elevated temperature (45 degrees C) or sodium arsenite. Radiolabeled proteins were detected by autoradiography after two-dimensional polyacrylamide gel electrophoresis. To determine the cell cycle stage specificity of labeling, nuclei were isolated and sorted into two cell cycle phases using a fluorescent activated cell sorter. After either heat shock or sodium arsenite treatment, the majority of [3H]leucine incorporation into stress proteins occurred during the G0 + G1 phase with minimal labeling in the G2 phase. On the other hand, 32P labeling of stress proteins occurred in both the G0 + G1 and G2 phases after exposure to sodium arsenite, while incorporation of 32P was limited after heat stress. Following sodium arsenite treatment, a distinct set of four stress proteins (80-84 kDa) was detected with [3H]leucine only in G0 + G1 phase, but with [32P]phosphate these stress proteins were labeled in both G0 + G1 and G2. There was differential [32P]phosphate labeling between proteins of the 80-84 kDa set during cell cycling. Individual proteins of this set were isolated from gel plugs after sodium arsenite or heat-shock treatment. Coelectrophoresis of proteins from the two treatment groups showed that they had similar electrophoretic mobilities. All four proteins of the 80-84 kDa set (sodium arsenite induced) possessed similar polypeptide maps after digestion with V8 protease. Cytofluorometric analysis demonstrated a reduction in the number of nuclei in both S and G2 phases of the cell cycle two h after heat shock, but not following sodium arsenite treatment. However, there was a significant depression in the number of nuclei in S and G2 4 h after exposure to sodium arsenite and very modest labeling with 32P of stress proteins was observed at this time.

The relationship of p53 and stress proteins in response to bleomycin and retinoic acid in the p53 heterozygous mouse.

A single, i.p. dose of bleomycin was administered simultaneously with [35S]methionine to 4-month-old p53 wild type (+/+) and p53 heterozygous (+/-) C57BL/6 mice. Following a period of 3.5 h from dosing, the bone marrow nuclei were examined by two-dimensional PAGE and fluorography for induction of stress proteins (sps). Eight sps ranging from 22000 to 100000 Mr were synthesized in p53+/- and p53+/+ mice following elicitation by bleomycin. No quantitative or qualitative differences were observed in sp expression in these two groups of animals. In a second experiment, three doses of retinoic acid were given i.p. to p53+/- and p53+/+ mice over a 36 h period. The p53 isoforms in bone marrow nuclei from these mice were analyzed by PAGE for incorporation of [35S]methionine following retinoic acid injections. Quantitative and qualitative alterations in p53 isotypes were substantially increased in p53+/+ as compared with p53+/- mice. The increased complexity in the synthesis patterns in both groups of dosed mice consisted of additional isoforms possessing more acidic isoelectric values. In an in vitro binding assay, individual p53 isoforms demonstrated varying degrees of association with sps 25a, 70i, 72c and 90 which was consistently greater in p53+/+ mice. Both the synthesis and binding of isoforms were greater in G1 than in S+G2 phase, in both groups of animals, reflecting a cell cycle regulated mechanism for these events. Collectively, these data implied that the synthesis and the binding characteristics of p53 isoforms with sps were enhanced in the p53+/+ mice relative to the p53+/- mouse; however, sp labeling was not affected by p53 genotype.

Evaluation of magnetic resonance imaging for quantification of intraabdominal fat in human beings by spin-echo and inversion-recovery protocols.

We evaluated two magnetic resonance imaging (MRI) methods, spin echo and inversion recovery (IR), for quantification of intraabdominal fat in a subgroup of participants from the Atherosclerosis Risk in Communities (ARIC) Study. Both methods were used previously to quantify visceral fat, and the IR but not the spin echo method has been validated by comparison with computed tomography in human beings. In the present study, the reliability of both methods was excellent: reliability coefficients comparing two readers on the same scan were 0.9574 for IR (n = 158) and 0.9254 for spin echo (n = 47) when random effects models with log-transformed data were used. A comparison of visceral fat areas in 47 subjects with both IR and spin echo indicated that IR gave a slightly higher mean area than did spin echo: 134.9 compared with 129.8 cm2. However, a mixed-model analysis of variance (ANOVA) of the log-transformed data showed no statistical difference between either method or readers in the comparison of IR and spin echo. These data suggest that the IR and spin echo protocols evaluated in this communication are comparable with one another and reliable for estimation of intraabdominal fat.

Relationship of intraabdominal fat as measured by magnetic resonance imaging to postprandial lipemia in middle-aged subjects.

Adipose distribution has been associated with coronary artery disease and its risk factors. We previously described an association between postprandial triglyceride response (pptg response) to a standard high-fat meal and extracranial carotid atherosclerosis. This study was designed to evaluate the association between intraabdominal fat and pptg response. Twenty-nine subjects were recalled for determination of intraabdominal fat by magnetic resonance imaging (MRI). Fat was quantified according to an inversion recovery protocol previously validated and the cross-sectional area of intraabdominal fat at the umbilicus was analyzed as an independent variable. We observed a strong independent correlation between intraabdominal fat and pptg response to a fatty meal (r = 0.521, P < 0.05). Baseline triglyceride was also independently correlated with postprandial triglycerides (r = 0.631, P < 0.05). In univariate analysis, intraabdominal fat was correlated with age, sex, body mass index (BMI), waist-to-hip ratio (WHR), and dietary saturated fatty acids. The association with age and BMI persisted in multivariate analyses.

Cell cycle analysis in bone marrow and kidney tissues of dietary restricted rats.

The effect of dietary restriction (DR) on the proportion of cells in various phases of the cell cycle as determined by flow cytometry was investigated in the bone marrow and kidney of young and old Fischer 344 rats. Control rats were fed a standard occurrence of numerous age-associated diseases, including cancer, renal diseases and by the control rats starting at 16 weeks of age until killed at 5 or 20 months old. The relative proportion of cells in the various phases of the cell cycle was independent of tissue type, treatment condition and age, consistently showing an order of G1- greater than S- greater than G2M-phase. In old rats DR did not affect cell cycling in bone marrow of either sex, however, it did cause an increase in the percentage of G1-phase cells in the kidney of male rats. Additionally, DR caused a mathematically significant change in the percentage of cells in all phases of the cell cycle in the bone marrow of young male rats but had no effect in young females. The percentage of S-phase cells in both tissues of both sexes decreased in old rats when compared to young rats regardless of treatment conditions, indicating a parallel decline in cell proliferating activity with aging. To summarize, DR produces a greater cell cycle effect in the young male than the old male rats. Proliferative capacity is enhanced when the young male rats are dietary restricted. This may aid in DNA repair mechanisms and/or immune system response.

Cell proliferation by cell cycle analysis in young and old dietary restricted mice.

The effect of dietary restriction (DR) on cell proliferation determined by cell cycle analysis in tissues of young and old mice was investigated. Using the percentage of S-phase cells as an index of cell proliferation, we found that DR inhibited cell proliferation in spleen and thymus in young mice. No significant changes were found in bone marrow and kidney in the ad libitum (AL) or DR mice regardless of age. In old mice, the DR effect was observed in spleen only. When age increased, a parallel decline in cell proliferation was evidenced by a reduced % of S-phase cells. DR produces a greater cell cycle effect in the young mice than in the old mice. The present data suggests that inhibition of cell proliferation by DR may be affected by type of tissue, age, length of DR, and capacity or rate of cell proliferation.

Temporal and substrate-dependent patterns of stress protein expression in the hypothalamus of caloric restricted rats.

Stress proteins (sps) 27, 34, 70 and 90 (Mr x 10(3)) were induced in the hypothalamus of caloric restricted (CR) rats by feeding stress. A definite time pattern for sps synthesis was observed when their induction was examined at several time points after the rats were fed, and the level of sps expression was found to vary significantly at different times of the day. The same group of proteins was induced in ad libitum fed rats when they were subjected to food deprivation for 48 h. Stress protein 34 expression in the hypothalamus of old caloric restricted rats was found to be dependent on blood glucose levels, and was substantially reduced when insulin was added to the glucose infusion. The expression of sps 27, 70 and 90, however, was little changed with glucose and/or insulin infusion.

Chronic caloric restriction induces stress proteins in the hypothalamus of rats.

The induction of stress proteins (sps) in the hypothalamus of female Fischer 344 rats in response to caloric restriction (CR) and to heat stress was investigated. Caloric restriction was found to elicit sps 27, 34, 70, and 90 in the hypothalamus of both young and old rats while none was found in the hypothalamus of ad libitum (AL) fed controls. Heat stress initiated heat shock proteins (hsps/sps) 27, 70, and 90 in the hypothalamus of the young (AL) fed animals, the same proteins evoked by feeding stress. The same sps were induced in the old (AL) rats although the expression showed substantial decline with age. This reduction was less marked, however, with the old CR rats. Stress protein 34, an infrequently reported protein, was related to feeding and was not induced by heat shock. Recent reports point to the important role sps play in the cellular reaction to stress, as well as their involvement in the higher functions. The findings reported here suggest that sps are involved in the regulatory mechanisms allowing CR animals to tolerate stress related to metabolic substrate deprivation.

P53 synthesis and phosphorylation in the aging diet-restricted rat following retinoic acid administration.

Multiple doses of retinoic acid (RA) were administered intraperitoneally to three groups of male Fischer 344 rats over a 36 h period. The p53 isoforms from bone marrow nuclei in these three groups of rats were analyzed over time by two-dimensional polyacrylamide gel electrophoresis (PAGE) and fluorography for the incorporation of [35S]methionine (p53-synthesis) and [32P]phosphate (p53-phosphorylation). Two groups of rats, young (3.5 months) ad libitum (Y/AL) and old (28 months) ad libitum (O/AL), had free access to Purina rat chow; a third group of old (28 months) diet-restricted rats (O/DR) were maintained on a restricted caloric intake (60% of the AL diet) from 3 months of age. After 36 h of RA dosing, the PAGE patterns of p53 synthesis and phosphorylation in Y/AL and O/DR rats were very similar. In both groups, an increase in complexity was observed with labeling of additional isotypes possessing more acidic isoelectric values. In contrast, the O/AL animals showed a pattern of p53 isoform synthesis and phosphorylation that was considerably less complex and lacked the pronounced shift to more acidic forms following RA dosing. The p53 isoforms of O/AL rats as recognized by wild type (wt) Pab 246 antibody, were also much less dramatic in their increase to more acidic forms. Two-dimensional phospho-tryptic maps of Y/AL and O/DR rats were also very similar, both exhibiting two additional minor 32P-labeled fragments after RA dosing. The maps of O/AL rats did not show the two additional fragments following RA administration. After RA dosing, cyclin protein inhibitors (p16, p21, p27) revealed robust labeling with their respective antibodies in Y/AL and O/DR rats as analyzed by Western blotting. The O/AL animals showed marginally detectable antibody recognition of the cyclin inhibitors after RA dosing. Taken together, these data suggest that the biosynthesis and phosphorylation of p53 isoforms and the expression of cyclin dependent kinase inhibitor proteins is not significantly different between Y/AL and O/DR rats. Further, these results confirm and extend our previous observations that chronic diet-restriction attenuates the age related decline in the metabolic activity of nuclear protein products.

Use of a paramagnetic substance, colloidal manganese sulfide, as an NMR contrast material in rats.

Paramagnetic pharmaceuticals ( magnetopharmaceuticals ) that are suitably distributed into specific organ systems or diseased sites might be clinically useful for tissue contrast enhancement in nuclear magnetic resonance images. To determine whether an insoluble magnetopharmaceutical might be useful in such service, we investigated the effect of a colloidal preparation of manganese sulfide ( MnSC ) upon liver and lung spin-lattice relaxation times (T1) in rats following intravenous administration. NMR tissue sample measurements were made at 24 MHz, ahd showed that after MnSC treatment, liver T1 values--and to a lesser extent lung T1 values--were depressed below control values. Liver manganese content (as determined by flame atomic absorption spectrophotometry) increased in proportion to the dose of MnSC , and the reciprocal of the liver T1 values also increased in proportion to the dose of MnSC .

Hepatic nuclear ploidy distribution of dietary-restricted mice.

Hepatic parenchymal cells in most adult mammals are polyploid, with most of the cells in the quiescent or low-proliferation state. Polyploidization has been related to carcinogenesis and aging, and both end points are significantly affected by dietary restriction (DR). Direct measures of hepatic nuclear polyploidization in DR B6C3F1 mice have not been examined. We examined the effect of DR on distributions of nuclear ploidy in both sexes and on different age groups of B6C3F1 mice. Differences between young and old male mice and between old male and female mice were also compared. Hepatic nuclear ploidy values were measured by flow cytometry. The DNA histograms were analyzed for the percentage of nuclei having different classes of DNA content by gating channels between the areas under the peaks of diploid, tetraploid, and octaploid. The results indicate that 1 or 26 months of DR started at 4 months of age did not alter hepatic nuclear ploidy distributions in young and old mice. Our data suggest that in the male mouse, polyploidization is established by 5 months of age for hepatic nuclei and that ploidy classes are affected by sex at 30 months of age. For females, effects in the octaploid nuclei are seen as a result of DR.

Spleen cell phosphorylation of salt soluble nuclear protein from isoproterenol treated and sorted nuclei.

The effect of isoproterenol (IPR) on phosphorylation of acidic nuclear proteins was investigated by two-dimensional gel autoradiography. Mouse spleen cells stimulated to divide by the mitogen concanavalin A (Con A) were separated according to cell cycle stage by flow microfluorometric technique. Exposure of cells for 48 h to 4 micrograms IPR/ml culture medium produced no significant change in the proportion of S and G2 phase cells, while a cumulative dose of 8 micrograms IPR/ml caused a significant repression in DNA synthesis and a reduction in the number of nuclei in G2 + M phase. Four micrograms IPR/ml stimulated the greatest amount of G0 + G1 phosphorylation of nuclear protein. Several proteins from G0 + G1 and S nuclei incorporated 32P after Con A + IPR administration, and one protein from S phase nuclei revealed intensified labeling at the 8 micrograms cumulative IPR dose but not at the 4 micrograms dose. The isolated proteins (W, X, Y, and Z) were reassociated with homologous DNA, centrifuged in a sucrose gradient and shown to co-sediment with DNA. S phase nuclear protein X-S, which was found to be a mixture of proteins (X0 and X1), was the only exception. One component of X-S, X0 bound to DNA, while component X1 failed to bind. Chymotryptic and V8 protease digests of all isolated proteins were made and analyzed by autoradiography. Proteins X0 and X1, recovered from the sucrose gradient, possessed dissimilar fragment patterns. It is concluded that protein X-S is composed of two proteins (X0 and X1), one of which (X1) appears during S phase during the 8 micrograms IPR induced nuclear repression.

Contribution of visceral fat mass to the insulin resistance of aging.

Recent studies have shown that central obesity (increased waist to hip ratio [WHR]) is related to insulin resistance and aging. Furthermore, in central-obesity states, the intraabdominal fat (IAF) depot has been postulated to contribute most to the development of insulin resistance. Therefore, the observed insulin resistance of aging may be related more to changes in body composition than to aging per se. The purpose of this study was to explore the association of IAF with age and insulin sensitivity (SI) after controlling for obesity. We examined 60 healthy nondiabetic subjects (normal 75-g oral glucose tolerance test, aged 23 to 83, 15 men and 45 women). We chose subjects so that those < or = 125% and greater than 125% of ideal body weight were equally represented in each age decade. We quantified total and subcutaneous abdominal fat and IAF at the umbilicus using a validated magnetic resonance imaging (MRI) scanning technique and determined SI using a modified minimal model. IAF correlated significantly with age (r = .49, P = .0001) in the group as a whole, as well as in men (r = .58, P = .022) and women (r = .48, P = .0008) separately. In all subjects, SI was significantly related to IAF (r = -.50, P < .0001) but was not related to age (r = .00, P = .98). In multivariate analysis for various combinations of age, sex, and measures of fat distribution, WHR accounted for 28% and IAF for 51% of the variance in SI, whereas age, sex, and interactions of age and sex accounted for only 1%.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin resistance and fat patterning with aging: relationship to metabolic risk factors for cardiovascular disease.

Both insulin resistance and abdominal fat patterning are related to aging, and have been related to cardiovascular disease (CVD) risk factors such as dyslipidemia and hypertension. However, previous studies have not used direct methods to quantify the independent strength of the association of each of these two putative primary factors with metabolic outcomes. We quantified overall obesity by the body mass index (BMI) and used a previously validated magnetic resonance imaging (MRI) method to quantify abdominal fat in 63 healthy nondiabetic individuals aged 22 to 83 years. We also measured the glucose and insulin response to an oral glucose tolerance test and the insulin sensitivity ([SI] by modified minimal model analysis). Body fat patterning was evaluated by the waist to hip ratio (WHR) and by MRI, which allowed direct measurement of subcutaneous (SCF) and intraabdominal (IAF) fat depots at the umbilicus in these subjects. These independent parameters were related to risk factors for CVD (blood pressure, lipids, and lipoproteins) and to plasma concentrations of free fatty acids (FFAs). Measures of overall obesity (BMI), total fat [TF], and/or SCF measured at the abdomen by MRI), glucose/insulin metabolism and SI, and central fat patterning (WHR or IAF measured by MRI) were correlated with mean arterial pressure (MAP), triglyceride (TG), and high-density lipoprotein cholesterol (HDL-C) levels in univariate analysis and after controlling for age and gender. An index of central fat patterning (WHR) added to the informativeness of the insulin area under the curve (IAUC) in explaining 24% of the variability in plasma TG concentration, but measures of overall obesity were not independently related. Both the BMI and TF contributed to the IAUC in explaining 32% to 34% of the variability in MAP, but central fat patterning was not independently related. No index of overall obesity, fat patterning, glucose/insulin metabolism, and/or SI, was independently related to the plasma concentration of HDL-C after controlling for any one of the other two. Direct measurement of glucose/insulin metabolism and SI, as well as fat patterning, provides information on their relative associations with CVD risk factors. The measures of glucose/insulin metabolism and SI were more consistently related to dyslipidemia and hypertension than were the overall obesity and fat patterning in this healthy population.

Prolonged exposure of mice to He-O2 at high pressure: effects on seizure and anesthesia liability.

Multiday exposures of CD-1 mice to He-O2 atmospheres at pressures from 30 to 100 atm result in marked increases of threshold pressures for type I high-pressure neurological syndrome seizures. The effect develops with a half time (t1/2) of 12 h and is reversible (t1/2 = 7 h). The maximum enhancement of Pc is attained at a conditioning pressure of 80 ATA. Pressure conditioning also results in suppression of the compression rate effect on Pc. Furthermore, reserpine blocks the increase in Pc during prolonged pressure exposure. The entire effect thus appears to be an extension in time of the monoaminergic compression rate effect on Pc. Pressure conditioning does not modify anesthesia tolerance, unlike N2 habituation which affects anesthesia threshold pressure as well as Pc. The results are compared with the effects of habituation to inert-gas narcotics and the implications of the data for an understanding of inert-gas high-pressure antagonism in intact animals are discussed.

Effect of habituation to subanesthetic N2 or N2O levels on pressure and anesthesia tolerance.

Exposure of CD-1 mice to subanesthetic partial pressures of N2O (0.5 atm) or N2 (10-20 atm) for periods up to 14 days results in up to 40% decreases in the mean threshold pressure eliciting type I high-pressure neurological syndrome (HPNS) seizures, and in increases up to 38% in the N2 partial pressure producing anesthesia. For all combinations of preexposure time, N2 partial pressure, as well as identity of the conditioning gas the relations between the convulsion threshold pressure (Pc) and the anesthesia N2 pressure (Pa) appear to be uniquely correlated by the equation Pa = 54.5 - 0.2(Pc - 60)1.2. The potency of N2O with respect to these habituation phenomena is between 28 and 33 times higher than that of N2, depending on the aspects compared. Evidence is presented indicating that after 14 days of habituation the animals have attained between 75 and 85% compensation for the anesthetic as well as the anticonvulsant effects of the conditioning gas. The bearing of the results on the problem of the nature of the antagonism between inert gas narcotic agents and high pressure and on the hypothesis that habituation tends toward restoration of isofluidity (or some analogous normalization process) are discussed.

Flow cytometric analysis of bromodeoxyuridine-induced inhibition of cell proliferation in the human teratocarcinoma-derived cell line, P3.

We have developed methods in our laboratory whereby the effects of toxicant exposure on cell proliferation can be evaluated flow cytometrically. We sought to relate the flow cytometric analyses to other biological response measurements. Thus, we exposed P3 cells to increasing concentrations of bromodeoxyuridine (BRdU) and measured sister-chromatid exchange (SCE) frequency, average generation time (AGT), and relative cloning ability. Each of these is well documented (see introduction) to respond to BRdU exposure in a concentration-dependent manner. In this study, SCE frequency remained constant between the concentrations of 2.5 and 10 microM of BRdU. However, a small, but significant, increase in SCE frequency was observed between the concentrations of 10 microM and 50 microM BRdU. A significant increase in AGT was noted in 50 microM BRdU-exposed cells. Relative cloning efficiency decreased in a concentration-dependent manner when cells were cultured for 24, 48, or 72 hours with BRdU. When cell proliferation was assessed by flow cytometric analysis in cells exposed to 0, 10, or 50 microM BRdU, a statistically significant delay in the cell-cycle was observed in BRdU-exposed cells. These results may be interpreted to mean that inhibition of cell proliferation is detected by this type of analysis at toxicant concentrations that induce other biological endpoints. The inclusion of flow cytometric analysis in a test battery to evaluate toxicant effects is warranted.