Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The kidney is a key target tissue in animal and human carcinogenesis, yet there are no established short-term tests for studying the genotoxicity of chemicals in the kidney. We have developed an assay for the measurement of chemically induced DNA repair as unscheduled DNA synthesis (UDS) in isolated rat kidney cells following in vivo treatment. Male Fischer-344 rats were injected intraperitoneally with chemicals dissolved in saline or corn oil. After various treatment times, the kidneys were perfused with a collagenase/trypsin solution (CTS), minced into small pieces, and stirred in CTS at 37 degrees C for 1 hr to dissociate cells. Cultures contain a high proportion of epithelial cells from the proximal and distal tubules. Cultures were incubated for 16-18 hr with 3H-thymidine in Williams' Medium E supplemented with 20% fetal bovine serum. UDS was measured by quantitative autoradiography as net grains/nucleus (NG). The percentage of cells in repair (% IR) was defined as the percentage of cells with greater than or equal to 3 NG. Saline- or corn oil-injected controls consistently produced -3 to -5 NG with less than 1% IR. The time course of DNA repair following treatment with the direct-acting mutagen methylmethane sulfonate (MMS) or the renal carcinogen azaserine showed a peak response at 2 hr after treatment. Azaserine showed a rapid decline in UDS at 12 and 24 hr, whereas MMS exhibited a relatively high UDS level at 24 hr. The renal carcinogens methylazoxymethanol acetate, N-methyl-N-nitrosourea, and streptozotocin all yielded strong positive UDS responses. The liver and intestinal carcinogen 1, 1-dimethylhydrazine at doses up to 50 mg/kg was cytotoxic to kidney cells, but induced less than 0 NG. Treatment with 1,2-dimethylhydrazine, which induces kidney tumors in mice but not rats, also induced less than 0 NG. Treatment with o-anisidine, a weak renal carcinogen, did not induce UDS in the kidney, suggesting that it may be acting as a tumor promoter. These results demonstrate the usefulness of this assay for the detection and study of a variety of genotoxic kidney carcinogens.
Environ Mutagen 1985
PMID:Measurement of unscheduled DNA synthesis in rat kidney cells following in vivo treatment with genotoxic agents. 406 62

The pancreas is a key target tissue in human and animal carcinogenesis, yet few short-term test systems measure genotoxicity in pancreatic cells. A method has been developed for the measurement of DNA repair as unscheduled DNA synthesis (UDS) in primary cultures of rat pancreatic cells (PRP) following in vitro or in vivo exposures to chemicals. PRP were isolated from female Sprague-Dawley (SD) or male Fischer-344 rats by mincing the pancreas in a collagenase solution followed by digestion in dispase. For in vitro exposures, PRP were incubated with 3H-thymidine (3H-TdR) in the presence of genotoxic agents for 18-22 hr. For in vivo exposures, male Fischer-344 rats were treated with genotoxic agents 2 hr prior to sacrifice of the animals, and cells were incubated with 3H-TdR. UDS was measured by quantitative autoradiography as net grains/nucleus (NG). Solvent controls ranged from -1.0 to -2.8 NG. Cells isolated from female SD rats and treated in vitro with methylmethane sulfonate (MMS), ethylmethane sulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and the pancreatic carcinogen azaserine all yielded over 4.0 NG. Cells treated in vitro with the hepatocarcinogens 2-acetylaminofluorene (2-AAF) and dimethylnitrosamine (DMN) and with the multisite carcinogen benzo[a] pyrene (B[a]P) yielded between -1.0 and -3.3 NG. These results are consistent with the lack of carcinogenic activity of 2-AAF, DMN, and B[a]P in the pancreas and indicate that pancreatic cells are incapable of metabolizing these compounds to genotoxic forms. In vivo treatment with MMS at 100 mg/kg yielded 1.9 NG and with azaserine at 100 mg/kg yielded 8.2 NG. This method should be useful in detecting agents that are genotoxic to the pancreas.
Environ Mutagen 1984
PMID:Induction of unscheduled DNA synthesis in primary cultures of rat pancreatic cells following in vivo and in vitro treatment with genotoxic agents. 637 85

A method is described in which primary rat hepatocytes have been cocultured with Chinese hamster ovary (CHO) cells to provide metabolic activation of promutagens in the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) mutational assay. Single cell hepatocyte suspensions were prepared from male Fischer-344 rats using the in situ collagenase perfusion technique. Hepatocytes were allowed to attach for 1.5 hours in tissue culture dishes containing an approximately equal number of CHO cells in log growth. The cocultures were exposed to promutagens for up to 20 hours in serum-free medium. The survival and 6-thioguanine-resistant fraction of treated CHO cells were then determined as in the standard CHO/HGPRT assay. Aflatoxin B1 (AFB1) 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (B(A)P) were found to produce increases in the mutant fractions of treated CHO cells as a function of concentration. The time required for optimum expression of the mutant phenotype following exposure to DMBA and AFB1 was approximately 8 days. Primary cell-mediated mutagenesis may be useful in elucidating metabolic pathways important in the production and detoxification of genotoxic products in vivo.
Environ Mutagen 1982
PMID:The use of primary rat hepatocytes to achieve metabolic activation of promutagens in the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase mutational assay. 680 33

To approximate better the metabolic reactions that take place in vivo yet maintain the simplicity and reproducibility of in vitro systems, we have developed a co-culture system making use of freshly isolated rat hepatocytes and confluent human fibroblasts for the study of SCE induction by genotoxic agents. Hepatocytes were obtained from male rats by reverse collagenase perfusion and plated over low-passage male human fibroblasts. Preliminary studies demonstrated that although the number of hepatocytes plated was not critical, the best attachment to, and coverage of, the fibroblasts occurred when between 10 x 10(6) and 20 x 10(6) hepatocytes were plated/100 mm tissue culture dish. Results with the promutagen cyclophosphamide showed that the hepatocytes could metabolize the compound and deliver active moieties to the fibroblasts resulting in a linear dose-dependent increase in SCE frequencies. Control fibroblast cultures lacking hepatocytes displayed no increase in SEC frequencies. Control fibroblast cultures lacking hepatocytes displayed no increase in SEC frequencies following cyclophosphamide administration.
Environ Mutagen 1980
PMID:Sister chromatid exchange studies in human fibroblast-rat hepatocyte co-cultures: a new in vitro system to study SCEs. 732 65

The unscheduled DNA synthesis (UDS) assay has been used extensively for the in vitro detection of DNA damage caused by compound exposure. However, the in vitro UDS assay has been insensitive for the detection of certain chemicals, particularly nitroaromatic compounds, that are positive in bacterial mutation assays. Recently, studies have been reported which describe alterations in the hepatocyte membrane following collagenase perfusion. Independently, a method for serum-free tissue culture has been developed which results in the up-regulation of cell surface receptors and which may restore membrane functions. Fourteen compounds, including seven nitroaromatics, were evaluated in the in vitro UDS assay employing a serum-free procedure. Five compounds that were previously reported positive in the standard in vitro UDS assay were also found positive using the serum-free method. In addition, five of the nitroaromatic compounds produced positive results with the serum-free method. 1-Methyl-3-nitro-1-nitrosoguanidine and 2-acetylaminofluorene, routinely used as positive controls in the UDS assay, showed greater activity in the serum-free assay. These results suggest that the use of serum-free media improves the sensitivity of the in vitro UDS assay and that the serum-free procedure potentially offers an effective alternative to the more labor intensive and more costly in vivo UDS assay.
Environ Mol Mutagen 1995
PMID:Improved sensitivity of the unscheduled DNA synthesis assay in primary rat hepatocytes following culture in serum-free defined media. 755 11

The bone marrow (BM) micronucleus (MN) test is a sensitive assay for identifying clastogens. However, some clastogenic compounds and metabolites may never reach the BM. The liver has been suggested as an alternative tissue to BM but adult rat liver has a low mitotic index that increases the difficulty of evaluating hepatocytes (HEP) for MN induction. Chemical mitogens and partial hepatectomy have been used to increase HEP proliferation to improve the sensitivity for detection of clastogenic compounds, but these practices raise concerns for the evaluation of drug candidates. The use of 4-wk-old rats provides an alternative to mitogenic stimulation because livers from these animals have approximately 5.4% of their HEP in S-phase. HEP were isolated by collagenase perfusion, or from formalin-fixed tissue, from 4-wk-old treated rats. Six compounds were evaluated for the incidence of MN in HEP that were isolated by both methods. The results for MN induction by these compounds were similar for the two methods and confirmed that formalin-fixed tissue is an acceptable source of cells for evaluating MN induction in HEP. BM polychromatic erythrocytes (PCE) also were harvested at the end of the live phase for each study and then evaluated for the incidence of MN. Diethylnitrosamine and 2-nitrofluorene induced MN in HEP but had no effect in PCE. 2-Acetylaminofluorene, cyclophosphamide and 7,12-dimethylbenz[a]anthracene did not induce MN in HEP but were positive in PCE. The direct-acting clastogen, mitomycin C, was positive in both HEP and PCE. These results indicate that this modified liver micronucleus test, using 4-wk-old rats, offers an alternative to existing methods that use mitogens or partial hepatectomy to stimulate cell replication. Analysis of MN from formalin-fixed tissue provides additional flexibility by allowing the investigator to assess MN induction at a later time.
Environ Mol Mutagen 1997
PMID:An evaluation of micronucleus induction in bone marrow and in hepatocytes isolated from collagenase perfused liver or from formalin-fixed liver using four-week-old rats treated with known clastogens. 921 89

With the aim of explaining a mechanism underlying the dose-dependent change in prevalence of different malformation types (shift in malformation spectra) in a population of chick embryos treated with general cytotoxic agents, we have investigated the effect of cyclophosphamide (CP) on the cell cycle in different organ rudiments. CP was administered intra-amniotically in doses of 2, 4, 8, and 16 micrograms to chick embryos on day 4. Six hours later, the embryos were removed, and limb buds, facial region, brain, and heart rudiments were dissected and treated for isolation of nuclei. The tissues were dissociated mechanically and enzymatically with collagenase-dispase, and suspensions of nuclei were prepared by a detergent and RNAase-mediated cytolysis. Ethidium bromide added to the solution allowed DNA analysis by flow cytometry, which, within the embryotoxic range of doses (4-16 micrograms), revealed a dose-dependent block of cells in the S phase, followed by a decrease of cell numbers in the G2-M phase. The effect of CP on the cell cycle was associated with the degree of damage to the embryo, and dysmorphogenesis appeared proportionate to the magnitude of mitotic inhibition. The results are consistent with the idea that the dose-dependent shift in malformation spectra is causally associated with the dose-dependent and organ-specific depression of mitotic activity.
Teratog Carcinog Mutagen 1998
PMID:Cell-cycle alterations within chick embryonic anlagen after cyclophosphamide treatment. 970 83

Recently we have shown that post-exposure treatment with povidone iodine (PI) protects against nitrogen and sulfur mustard-induced skin lesions. Since proteolytic activity is involved in skin damage caused by chemical irritants, we have studied the effect of iodine on mechlorethamine (HN2)-induced skin collagenolytic activities in the haired guinea pig model. The matrix metalloproteinase-9 (MMP-9) activity increased by 30, 46, 12 and 23% after 3, 24, 48 and 72 h of HN2 exposure, respectively, whereas the MMP-2 was elevated by 8, 65, 8 and 30%, respectively. Topical treatment with PI at 15 and 120 min after HN2 exposure decreased the MMP-9 activity by 67% and 60%, respectively, when skin was analyzed 3 h after exposure. The same trend was observed in the MMP-2 and MMP-1 activities after PI treatment. A stronger effect of PI treatment 15 min following exposure was observed in skin analyzed 24 h after exposure, i.e. a decrease of 83% and 88% in MMP-9 and MMP-2 activities, respectively. Similar findings were observed with an interval of 120 min between HN2 exposure and PI treatment. A much weaker effect was observed on MMP-1 activity. A similar trend of PI-induced reduction in the three types of collagenase activity was found in skin analyzed 48 and 72 h after exposure. Reduced collagenolytic activity may serve as one of the mechanisms by which iodine protects the skin against chemical insult.
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PMID:Topical treatment with povidone iodine reduces nitrogen mustard-induced skin collagenolytic activity. 1191 82

We investigated the action of tissue inhibitor of metalloproteinase-1 (TIMP-1) on apoptosis and differentiation of mouse bone marrow stromal cell line MBA-1. TIMP-1 did not affect alkaline phosphatase (ALP) activity, suggesting that it is not involved in osteoblastic differentiation in MBA-1 cells. However, TIMP-1 inhibited MBA-1 apoptosis induced by serum deprivation in a dose-dependent manner. Our study also showed increased Bcl-2 protein expression and decreased Bax protein expression with TIMP-1 treatment. TIMP-1 decreased cytochrome c release and caspase-3 activation in MBA-1 cells. TIMP-1 activated phosphatidylinositol 3-kinase (PI3-kinase) and c-Jun N-terminal kinase (JNK), and the PI3-kinase inhibitor LY294002 or the JNK inhibitor SP600125 abolished its antiapoptotic activity. To investigate whether antiapoptotic action of TIMP-1 was mediated through its inhibition on MMP activities, we constructed mutant TIMP-1 by side-directed mutagenesis, which abolished the inhibitory activity of MMPs by deletion of Cys1 to Ala4. Wild-type TIMP-1 and mutant TIMP-1 expression plasmids were transfected in MBA-1 cells, and results showed that mutant TIMP-1 still protected the induced MBA-1 cell against apoptosis. These data suggest that TIMP-1 antiapoptotic actions are mediated via the PI3-kinase and JNK signaling pathways and independent of TIMP-1 inhibition of MMP activities.
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PMID:Tissue inhibitor of matrix metalloproteinase-1 suppresses apoptosis of mouse bone marrow stromal cell line MBA-1. 1669 94

High dose glucocorticoid (GC) treatment induces osteoporosis partly via increasing osteoblast apoptosis. However, the mechanism of GC-induced apoptosis has not been fully elucidated. Osteoblast-derived tissue inhibitor of metalloproteinase-1 (TIMP-1) was recently reported to be involved in bone metabolism. Our previous study demonstrated that TIMP-1 suppressed apoptosis of the mouse bone marrow stromal cell line MBA-1 (pre-osteoblast) induced by serum deprivation. Therefore, we tested the effect of the GC dexamethasone (Dex) on TIMP-1 production in murine osteoblastic MC3T3-E1 cells and further determined whether this action is associated with Dex-induced osteoblast apoptosis. Dex decreased TIMP-1 production in MC3T3-E1 cells, and this effect was blocked by the glucocorticoid receptor (GR) antagonists, RU486 and RU40555. Recombinant TIMP-1 protein reduced caspase-3 activation and apoptosis induced by Dex in MC3T3-E1 cells. In addition, the pro-apoptotic effect of the Dex was augmented by suppression of TIMP-1 with siRNA. Furthermore, mutant TIMP-1, which has no inhibitory effects on MMPs, yet protects MC3T3-E1 cells against Dex-induced apoptosis. Our study demonstrates that Dex suppresses TIMP-1 production in osteoblasts through GR, and this effect is associated with its induction of osteoblast apoptosis. The anti-apoptotic action of TIMP-1 is independent of its inhibitory effects on MMPs activities. The decrease in TIMP-1 production caused by Dex may contribute to the mechanisms of Dex-induced bone loss.
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PMID:Suppressive effect of dexamethasone on TIMP-1 production involves murine osteoblastic MC3T3-E1 cell apoptosis. 1962 37


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