Promutagen activation by Vicia faba: an assay based on the induction of sister-chromatid exchanges in Chinese hamster ovary cells

Plant activation of promutagens was studied using Vicia faba S10 (in vitro activation) and the extracts prepared from promutagen-treated roots of Vicia faba (in vivo activation). The induction of sister-chromatid exchanges in Chinese hamster ovary cells was used as an endpoint to evaluate the cytogenetic effects of promutagens activated by Vicia faba. Cyclophosphamide and ethyl alcohol were activated both by Vicia S10 and by the Vicia extracts, and their activation resulted in an increase in SCEs. Benzo[a]pyrene, 2-aminofluorene, and maleic hydrazide were not activated. Aniline was activated, but without effect on the induction of SCEs. The activation capacity in vitro and in vivo of Vicia faba was not very pronounced, except for the activation of ethyl alcohol, when compared with that of rat-liver S9, and showed differences in activation for the 6 chemical agents tested.     

The mouse immune interferon receptor gene is located on chromosome 10

When mouse L cells are incubated with 32P-labeled recombinant murine immune interferon ( [32P]Mu-IFN-gamma) and subsequently cross-linked with disuccinimidyl suberate, a major complex with an apparent molecular mass of 95,000-125,000 daltons can be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The complex was not formed when the binding was performed in the presence of excess unlabeled Mu-IFN-gamma or when Chinese hamster ovary cells were used. This complex therefore represents the Mu-IFN-gamma receptor (or its interferon-binding subunit). The chromosomal location of the Mu-IFN-gamma receptor (or the binding subunit of the receptor) gene, termed Ifgr, was identified by performing the binding and cross-linking reactions on a series of mouse-hamster somatic cell hybrids with different subsets of mouse chromosomes. The presence of mouse chromosome 10 was shown to be necessary and sufficient for the formation of the cross-linked complex. Thus, the gene coding for the binding subunit of the Mu-IFN-gamma receptor was localized to mouse chromosome 10. The presence of this chromosome in the hybrid cells was not sufficient, however, to confer antiviral resistance to the hybrids when they were treated with Mu-IFN-gamma and challenged with encephalomyocarditis virus.     

Induction of sister-chromatid exchanges in Chinese hamster ovary cells treated in vitro with non-k-region dihydrodiols of 7-methylbenz[a]anthracene and benzo[a]pyrene

Studies were carried out on the incidence of sister-chromatid exchanges induced in Chinese hamster ovary cells by in vitro treatment with the polycyclic aromatic hydrocarbons 7-methylbenz[a]anthracene and benzo[a]pyrene and with related K-region and non-K-region dihydrodiols. Appreciable increases in the incidence of sister-chromatid exchanges were apparent in cells treated with non-K-region dihydrodiols: the most active compounds were 3,4-dihydro-3,4-dihydroxy-7-methylbenz[a]anthracene and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene and the effects were dose-dependent. The parent hydrocarbons and the related K-region dihydrodiols induced some sister-chromatid exchanges but they were considerably less active than these two non-K-region diols. The results suggest that this system may usefully be applied to studies aimed at determining which dihydrodiols are important in the metabolic activation of the carcinogenic polycyclic hydrocarbons. These and other results also infer that Chinese hamster ovary cells possess some intrinsic ability to metabolize such compounds in the absence of exogenous activation systems.     

Metabolic activation of polycyclic aromatic hydrocarbons to mutagens in the Ames test by various animal species including man

6 polycyclic aromatic hydrocarbons were assayed for mutagenicity in the Ames test, in the presence of hepatic post-mitochondrial preparations isolated from the mouse, rat, hamster, pig and man. Benzo[a]pyrene, dibenzo[a,i]pyrene and benz[a]anthracene gave a positive mutagenic response only in the presence of activation systems derived from the hamster. With the exception of the pig, activation systems derived from all animal species could convert 3-methylcholanthrene to mutagens, the hamster being the most efficient. With the exception of the rat and pig, all animal species activated 7,12-dimethylbenz[a]-anthracene to mutagens, the human preparation being the most effective followed by the hamster and mouse. Dibenz[a,h]anthracene was not activated by any of the hepatic preparations. It is concluded that, among the animal species studied the hamster is generally the most efficient in activating polycyclic aromatic hydrocarbons to mutagens in the Ames test.     

Endogenous xenobiotic enzyme levels in mammalian cells.

The response of mammalian cell lines to chemicals depends, in part, on the exogenous activation system used for the induction of a biological response. This could be attributed to differences in the expression of enzymes involved in xenobiotic metabolism. We have measured the activities of benzo[a]pyrene hydroxylase, dimethylaminoazobenzene N-demethylase, catalase, superoxide dismutase, peroxidase and glutathione-S-transferase in human lymphoblast TK6, mouse lymphoma L5178Y, Chinese hamster ovary (CHO) and lung (V79) and mouse C3H10T1/2 cell lines as well as in primary hepatocytes and S9 preparations of liver from male F344 rats. Nitroreductase was also measured in some of these preparations. Human lymphoblast TK6 and mouse C3H10T1/2 cells had the capacity to metabolize dimethylaminoazobenzene and the latter cell line also metabolized benzo[a]pyrene, indicating the presence of constitutive mono-oxygenase activity. Cytochrome P450 could not be detected spectrophotometrically in the cell lines. Western blot analysis indicated that P450 from the P450IIA family is expressed in C3H10T1/2 cells. Reactivity was also observed with an antibody to P450IA2; however, the identity of this protein remains uncertain. Superoxide dismutase, catalase and peroxidase, which protect cells against oxygen radical damage, were found in all the cell lines and in rat hepatocytes and S9. The human lymphoblast TK6 cell line, however, had the least of each of these three enzymes. Glutathione-S-transferase activity was detected at varying levels in all cell types. Nitroreductase activity was high in S9 and Chinese hamster ovary cells and lower in mouse lymphoma and Chinese hamster V79 cells.     

Cell and species differences in metabolic activation of chemical carcinogens

Metabolic activation and DNA binding of aflatoxin B1 (AFB1), N-nitrosodimethylamine (DMN) and benzo[a]pyrene (B[a]P) were compared in human, rat and mouse hepatocytes and human pulmonary alveolar macrophages (PAM). The degree of carcinogen activation by hepatocytes and PAM was measured by cell-mediated mutagenesis assays in which co-cultivated Chinese hamster V79 cells were used to monitor mutagenic metabolites. Hepatocytes from human, mouse and rat metabolized DMN and released the active metabolites to induce either ouabain- or 6-thioguanine-resistant mutation. The mutation frequencies mediated by hepatocytes of the 3 animal species were approximately 3-9 mutants/10(5) survivors at a concentration of 0.2 mM DMN. The variations of radioactivity bound to liver cell DNA were relatively small in cultured mouse, rat, and human hepatocytes exposed to 14C label DMN (0.5 mM) and the binding values were in a range of 6-12 X 10(3) pmoles/mg DNA. However, rat hepatocytes were at least 10-fold more effective than either human or mouse hepatocytes in generating mutagenic metabolites of AFB1 and also had a much higher AFB1 metabolite DNA-binding value. The AFB1 DNA-binding levels were 4.1, 12-27 (range), 120 pmoles/mg DNA respectively in mouse, human, and rat liver cells following AFB1 (3.3 microM) exposure for 20 h. Hepatocytes from the 3 animal species were unable to mediate mutation in the presence of 4 microM B[a]P; PAM activated B[a]P and effectively mediated mutation in the co-cultivated V79 cells. In contrast to results with hepatocytes, PAM failed to generate enough mutagenic metabolites of AFB1 (3.3 microM) and the mediation of mutations was seen only at very high concentration of DMN (80 mM). The genotoxic effects of the 3 carcinogens on hepatocytes from different species in vitro were in agreement with the in vivo animal experiments in that mice are relatively resistant to AFB1 carcinogenesis whereas rats are sensitive; B[a]P is not effective as a complete liver carcinogen in adult rat and mouse whereas DMN induces liver cancer.     

Complementation mapping in microcell hybrids: localization of Fpgs and Ak-1 on Mus musculus chromosome 2

The gene encoding folylpolyglutamyl synthetase (FPGS) was assigned to mouse chromosome 2 by complementation mapping. Chinese hamster ovary cells (AuxBl) deficient in FPGS, and consequently auxotrophic for glycine, adenosine, and thymidine (gat-), were employed as recipients in microcell-mediated chromosome transfer experiments. Mouse chromosomes derived from diploid embryo fibroblasts were introduced into hamster AuxBl cells, and gat+ microcell hybrids were selected in medium lacking adenosine and thymidine. Mouse chromosome 2 was the only donor chromosome whose presence correlated with expression of FPGS activity. Furthermore, every gat+ hybrid clone expressed murine AK-1, a marker previously assigned to chromosome 2. Eight of 20 clones analyzed retained deletion chromosomes derived from mouse chromosome 2. These clones were used to localize murine Fpgs and Ak-1 to a region of this chromosome, namely 2 (cen leads to Cl).     

In vitro effects of strontium ranelate on the extracellular calcium-sensing receptor

The extracellular calcium-sensing receptor (CaSR) is activated by divalent cations and might mediate some of the effects of strontium ranelate, a new drug for the prevention and treatment of post-menopausal osteoporosis. Here, we showed that the maximal effect of Sr(2+) was comparable to that observed for Ca(2+) for both the cloned rat CaSR expressed in Chinese hamster ovary [CHO(CaSR)] cells and the mouse CaSR constitutively expressed in AtT-20 cells as measured by the accumulation of [(3)H]inositol phosphates (IP) resulting from CaSR activation. Strontium ranelate also displayed comparable agonist activity for the CaSR in both cell lines. Sodium ranelate did not stimulate the IP response in CHO(CaSR) cells. The IP response resulting from activation of other G-protein-coupled receptors was potentiated by Sr(2+), suggesting that entry of Sr(2+) into the cells might influence phospholipase C activity. Modulation of the CaSR activity in bone cells by strontium ranelate may contribute to its reported antiosteoporotic effects.     

Assignment of A and B Types of Monoamine Oxidase in NCB20 Hybrid Cells to Those of the Parental Cells by Peptide Mapping

The structures of [3H]pargyline-labeled, flavin-containing polypeptides of monoamine oxidase (MAO) from hybrid NCB20 cells, and their parental cells, A/J mouse brain cells and Chinese hamster brain cells, were analyzed and compared by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and limited proteolysis and one-dimensional peptide mapping in SDS gels. After preincubation of mitochondrial preparations with deprenyl or clorgyline, the flavin-containing polypeptide of type A or type B MAO was selectively labeled with [3H]pargyline. SDS-PAGE of [3H]pargyline-labeled mitochondrial samples revealed that the polypeptide with apparent Mr of 62,000 was associated with type A activity in the three types of cells, and that the polypeptide with apparent Mr of 61,000 or 58,000 was associated with type B activity in Chinese hamster brain cells and NCB20 cells or A/J mouse brain cells, respectively. Chymotrypsin digestion of the [3H]pargyline-labeled polypeptides and the peptide mapping in SDS gels from A/J mouse and Chinese hamster brain cells produced identical map patterns between the two type A MAOs, almost the same map patterns (with the exception of one additional peptide fragment) between the two type B MAOs, and different map patterns between type A and type B MAOs. The results of identical treatments of the [3H]pargyline-labeled polypeptides of MAOs in NCB20 cells showed that type A and type B MAO in NCB20 cells were similar to type A MAO of A/J mouse and Chinese hamster brain cells and to type B MAO of Chinese hamster brain cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of the lateral organization of the plasma membrane of Chinese hamster ovary cells by synthetic lipopeptide, Pam3Cys-Ser-Lys4.

The cationic lipohexapeptide (S)-[2, 3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(S)- Lys 4-OH, trihydrochloride (Pam3Cys-Ser-Lys4) is a synthetic analog of the triacylated N-terminal part of bacterial lipoproteins. In this study we addressed the question of whether Pam3Cys-Ser-Lys4 could modify the organization of the plasma membrane of Chinese hamster ovary cells. 1-Acyl-2-[6-(7-nitro-2-1, 3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphocholine (C6-NBD-PC) diffusion was followed by fluorescence recovery after photobleaching experiments carried out on the plasma membrane of Chinese hamster ovary cells. Incubation of cells in the presence of Pam3Cys-Ser-Lys4 induced an increase in the lateral diffusion coefficient and in the immobile fraction of C6-NBD-PC probes. Various control experiments have shown that the increase in the immobile fraction was not due to probe internalization induced by Pam3Cys-Ser-Lys4. Back-exchange experiments showed that a good correlation exists between the fractions of immobilized probes and nonextractable probes in the plasma membrane of Chinese hamster ovary cells. A useful way to analyze the origin of probe immobilization (micrometer-sized domains or aggregated patches of proteins) is to carry out fluorescence recovery after photobleaching experiments at variable observation radii. This type of experiment, carried out on the plasma membrane of Chinese hamster ovary cells incubated with Pam3Cys-Ser-Lys4, confirmed that the lipopeptide induced the aggregation of proteins of Chinese hamster ovary plasma membrane. Lipids which were trapped inside these aggregates were thus prevented from diffusing at long range in the plasma membrane plane and behave as an immobile fraction.     

In vitro co-stimulation of anti-tumor activity by soluble B7 molecules

In order to investigate the anti-tumor activity of a soluble B7-1/immunoglobulin G fusion protein and explore an effective method to eliminate immune escape of tumor cells, a recombinant vector encoding this fusion protein was constructed and constitutively expressed in Chinese hamster ovary cells. After purification with protein G affinity chromatography, the soluble fusion protein was tested for bioactivity. Results showed that the fusion protein could significantly increase the density of B7-1 molecules on WEHI-3 cells, a mouse leukemia cell line. Through allogeneic mixed lymphocyte tumor cultures, it was demonstrated that, with the presence of the first signal, it could also significantly enhance T cell activation and killing activity against WEHI-3 cells and interleukin-2 secretion by activated mouse T lymphocytes. The conclusion can be drawn that the soluble B7-IgG fusion protein has a potent capacity to generate or enhance anti-tumor immune response in vitro, and its clinical value deserves further investigation.     

Reduction of benzo(a)pyrene induced chromosomal aberrations by DL-alpha-tocopherol

The antioxidant, DL-alpha-tocopherol (vitamin E), has been demonstrated to significantly reduce the percentage of benzo(a)pyrene (BP) induced chromosomal aberrations in vitro. Chinese hamster lung (Don) and Chinese hamster ovary (CHO) cells were treated with either 1 microgram/ml or 5 micrograms/ml BP for 4 to 28 h; some cultures were treated with S9 mix activated BP. Additional cultures of Don and CHO were treated simultaneously with 100 micrograms/ml of DL-alpha-tocopherol and BP. In CHO cells 1 microgram/ml non-activated BP significantly increased the chromosomal aberration percentage above the control level. Aberrations observed included breaks, gaps, fusions, rings, dicentrics, and polyploids. Chinese hamster Don cells treated with 1 microgram/ml or 5 micrograms/ml S9 mix activated BP contained significant increases in aberration percentages above the control levels. When Don cells were treated simultaneously with activated BP and DL-alpha-tocopherol for 4 h, there was a slight decrease in the total aberration frequency to less than that of cells treated with activated BP only; however, when Don cells were treated with BP and DL-alpha-tocopherol for 28 h, there was a significant reduction in the aberration percentage below that of BP-treated cells alone. Similar results have been obtained with CHO cells treated with nonactivated BP and DL-alpha-tocopherol. The results reported here provide further evidence that antioxidants may prevent the potential mutagenic and carcinogenic effects of certain polycyclic compounds.     

Cyclic amines are selective cytotoxic agents for pigmented cells

We have shown that morpholine, a cyclic amine, exerts a selective inhibition of growth on melanocytic pigmented cell lines compared to nonpigmented cells. The ID50 of morpholine for the pigmented B-16 cell line HFH was 1200 micrograms/ml, compared to values greater than 2400 micrograms/ml for baby hamster kidney, Chinese hamster ovary and NP, an unpigmented primate cell line. Two other cyclic amines piperazine and piperidine, were similarly found to be selectively toxic to melanocytes. This selective toxicity could be synergistically enhanced by pretreatment of the cells with theophylline, a stimulator of tyrosinase activity, which indicates that the selective toxicity may be associated with melanin synthesis. Low passage HFH, high passage HFH and Syrian hamster melanoma RPMI 1846 cells that were pretreated with theophylline showed between 13 and 29% greater toxicity compared to controls treated with theophylline or morpholine alone. Unpigmented NP primate cells, Chinese hamster ovary and mouse fibroblast L929 remained unaffected. These cyclic amines join a list of other amines that have also been shown to be melanocytotoxic.     

Sphingosine-1-phosphate is a high-affinity ligand for the G protein-coupled receptor GPR6 from mouse and induces intracellular Ca2+ release by activating the sphingosine-kinase pathway

We identified and cloned the mouse orthologue of human GPR6 as a new member of the lysophospholipid-receptor family. Sphingosine-1-phosphate (S1P) activated GPR6, transiently expressed in frog oocytes or in Chinese hamster ovary (CHO) cells, with high specificity and nanomolar affinity. The GPR6 gene was found to be located on chromosome 10B1 and a single exon coded for the entire open-reading frame. Signal transduction of S1P was inhibited by pertussis toxin, suggesting a coupling of GPR6 to an inhibitory G protein. In CHO cells transfected with GPR6, the sphingosine-kinase pathway mediated Ca(2+) mobilization from internal stores. Apoptotic cell death was induced by serum deprivation or H(2)O(2) treatment and was prevented by S1P in GPR6-, but not in vector-transfected CHO cells. The antiapoptotic effect of S1P required activation of sphingosine kinase and was accompanied by an increase in MAP-kinase phosphorylation.     

Genotoxic effects of o-phenylphenol metabolites in CHO-K1 cells

The effects of microsomal activation and/or deactivation on the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) in cultured Chinese hamster ovary cells (CHO-K1 cells) by o-phenylphenol (OPP) were studied, and concurrently the metabolites were determined. After a 3-h incubation in the presence of 15% S9 mix (45 microliters/ml of S9), OPP (25-150 micrograms/ml) dose-independent SCEs and chromosomal aberrations were induced, while the amount of phenylhydroquinone (PHQ) metabolite produced from OPP did not increase linearly in the higher doses. The maximum induction of chromosomal aberrations was 18% at the 150 micrograms/ml dose, and of SCEs 13.8/cell at 75 micrograms/ml. The corresponding control values were 3% and 5.8/cell. The lowest dose required to induce SCEs in the presence of S9 mix was 25 micrograms/ml. Changing the percent of S9 mix (0-50%) while holding the OPP dose constant (100 micrograms/ml) produced a correlation between SCEs and the production of PHQ. PHQ caused cytogenetic effects both with and without S9 mix, however, in the absence of S9 mix it was more lethal and was oxidized to phenylbenzoquinone (PBQ). These results suggest that the enhanced cytogenetic effects of OPP by the addition of S9 mix correlated with the amount of PHQ produced or with the further oxides of PHQ such as phenylsemiquinone and/or PBQ which are capable of being produced from PHQ spontaneously or by the mixed-function oxidase system.     

Evaluation of the genetic and embryotoxic effects of bis(tri-n-butyltin)oxide (TBTO), a broad-spectrum pesticide, in multiple in vivo and in vitro short-term tests

The genetic and embryotoxic effects of bis(tri-n-butyltin)oxide (TBTO) were evaluated in multiple in vivo and in vitro short-term tests preparatory to its potential wide use as a molluscicide in control of schistosomiasis. When tested in the rec assay in Bacillus subtilis, TBTO was not mutagenic and it did not induce reverse mutations in Klebsiella pneumoniae. Neither in the presence nor in the absecne of rat liver activation system did TBTO produce point mutations in Salmonella typhimurium strains TA1530, TA1535, TA1538, TA97, TA98 or TA100. TBTO was matagenic in strain TA100 in a fluctuation test, but only in the presence of rat liver S9 (Aroclor-induced). TBTO did not induce gene mutations in the yeast Schizosaccharomyces pombe, mitotic gene conversions in the yeast Saccharomyces cerevisiae, nor sister-chromatid exchange in Chinese hamster ovary cells in the presence or absence of rat or mouse liver S9. In the latter cells, structural chromosomal aberrations, endoreduplicated and polyploid cells were induced. TBTO did not induce gene mutations in V79 Chinese hamster cells (to 8-azaguanine-, ouabain- or 6-thioguanine-resistance) in the presence of a rat liver postmitochondrial fraction or in cell (hamster embryo cells and human and mouse epidermal keratinocyte)-mediated assays. In mouse lymphoma cells, TBTO did not induce 6-thioguanine- or BUdR-resistant mutations. As many tumour promoters inhibit metabolic cooperation between V79 Chinese hamster 6-thioguanine-resistant/-sensitive cells, TBTO was tested but showed no such activity. TBTO was examined for the induction of recessive lethal mutations in adult Berlin K male Drosophila melanogaster, either by feeding or by injection. Doses of 0.37 or 0.74 mM did not increase the number of X-linked recessive lethal mutations. An increased number of micronuclei was observed in the polychromatic erythrocytes of male BALB/c mice 48 h after a single oral dose of TBTO (60 mg/kg bw), while a lower dose (30 mg/kg bw) was ineffective. Neither of the two doses had induced micronuclei 30 h after treatment. The reproductive toxicity of TBTO was studied in NMRI mice. In a 10-day toxicity study, the LD50 and LD10 were 74 and 34 mg/kg bw, respectively. An increased frequency of cleft palates was seen in the fetuses of mice (compared with controls, 0.7%) treated orally during pregnancy with 11.7 mg/kg TBTO (7%), 23.4 mg/kg (24%) or 35 mg/kg (48%).(ABSTRACT TRUNCATED AT 400 WORDS)     

Perturbation of DNA replication and cell cycle progression by commonly used [3H]thymidine labeling protocols.

The effect of tritiated thymidine incorporation on DNA replication was studied in Chinese hamster ovary cells. Rapidly eluting (small) DNA from cells labeled with 2 microCi of [3H]thymidine per ml (200 microCi/mmol) for 60 min matured to a large nonelutable size within approximately 2 to 4 h, as measured by the alkaline elution technique. However, DNA from cells exposed to 10 microCi of [3H]thymidine per ml (66 microCi/mmol) was more rapidly eluting initially and did not mature to a nonelutable size during subsequent incubation. Semiconservative DNA replication measured by cesium chloride gradient analysis of bromodeoxyuridine-substituted DNA was also found to be affected by the final specific activity of the [3H]thymidine used in the labeling protocol. Dramatic cell cycle perturbations accompanied these effects on DNA replication, suggesting that labeling protocols commonly used to study DNA metabolism produce aberrant DNA replication and subsequent cell cycle perturbations.     

Mutagenicity testing on chinese hamster V79 cells treated in the in vitro liver perfusion system. Comparative investigation of different in vitro metabolising systems with dimethylnitrosamine and benzo[a]pyrene.

A comparative study of three in vitro metabolising systems was performed in combination with Chinese hamster V79 cells, at which point mutation to 6-thioguanine resistance was scored. The three metabolising systems used were: (1) rat liver microsomal fraction (S9-mix); (2) feeder layer of primary embryonic golden hamster cells, according to Hubermann's system; (3) in vitro perfusion of rat liver according to the system of Beije et al. As model substances dimethylnitrosamine (DMN) and benzo[a]pyrene (BP) was used. The liver perfusion was more efficient than S9-mix as an activating system of DMN, while the feeder layer of embryonic cells was unable to activate this compound. The activation of DMN with S9-mix was dependent on the presence of NADP. By exposing the target cells in the liver perfusion at different distances from the liver the biological half life of the active metabolite of DMN could be estimated to less than 5 s. With BP the three metabolising systems showed reversed results as compared with DMN--both the feeder layer cells and S9-mix activated BP, the feeder layer cells being most efficient. With liver perfusion, the perfusate itself was totally negative. Only the bile showed a week mutagenic effect. These results are in accordance with the notion that intact liver cells perform both an activation and a subsequent deactivation of BP. Because of the importance of hepatic bio-transformation in chemical mutagenesis and carcinogenesis it is emphasied that a liver perfusion system could be used in a testing protocol for genotoxic effects as a valuable tool in order to analyse the mechanism of action of mutagenic and carcinogenic compounds detected in other test systems, for instance bacterial/microsomal tests.     

Isolation of rat intestinal crypt cells

Abstract. A technique is presented which yields single cells and intact crypts in suspension from unfixed rat intestinal mucosal epithelium. Everted lengths of intestine were digested by 27 mM sodium citrate in phosphate-buffered saline (pH = 7.3) at 37°C. Mucosal cells were dislodged by vibratory stress (hand vortexing) following incubation for prescribed intervals at 37°C in 1.5 mM ethylenediamine tetraacetic acid (EDTA) and 0.5 mM dithiothreitol (dtt). Alkaline phosphatase determinations, phase microscopy, and in vivo and in vitro evaluations of tritiated thymidine ([³H]TdR) incorporation were performed on isolated intestinal cells. Data indicate that cells were sequentially derived from villus tip to crypt base as judged by cellular morphology, alkaline phosphatase activity/mg protein and radioactivity per μg protein. Upon completion of the intestinal cell isolation assay, scanning electron microscopy of the remaining intestine revealed that approximately 95% of the crypt openings were vacant; the villi were totally denuded; the supporting structures, including the lamina propria, appeared intact. In vitro radiolabelling of intestinal cell fractions enriched with crypts revealed a linear incorporation of [³H]TdR from 0–60 min which was strongly influenced by the presence of foetal calf serum (FCS). Measurements of the compensatory response of the mucosa to resection of 70% of the small bowel indicated that the mucosal cell separation is capable of detecting alterations in crypt cell proliferation. Previously, such alterations were monitored by other methods utilizing microdissection procedures or stathmokinetic agents.