Validation of an in vivo alkaline elution assay to detect DNA damage in rat testicular cells

An alkaline elution assay was used to measure DNA damage in the rat testis after in vivo treatment with chemicals. All of the chemicals reported to induce heritable mutations in a mammalian assay (mouse specific locus test) were positive in the rat alkaline elution assay. Chemicals that are negative or inconclusive in the specific locus test did not cause detectable DNA damage in rat testes. DNA damage was detected by alkaline elution after either intraperitoneal or oral administration of chemical mutagens. Data from these validation studies were used to establish criteria to be used for evaluation of alkaline elution results with materials of unknown potential for genotoxic effects in germinal tissue.     

Induction of tumoricidal peritoneal exudate cells by administration of Lactobacillus casei

The lifespan of mice into which Meth A fibrosarcoma was transplanted intraperitoneally (i.p.) was prolonged by i.p. treatment with Lactobacillus casei YIT 9018 (LC 9018). Treatment with LC9018 before the tumor inoculation (pre-treatment), was more effective than after the tumor inoculation (post-treatment). When tumor cells were inoculated subcutaneously along with peritoneal exudate cells (PEC) harvested from LC 9018-treated mice in the Winn type test, the tumor growth was significantly inhibited, but it was not inhibited by spleen cells. Adherent and nonadherent cells of LC 9018-induced PEC suppressed tumor growth in the Winn type test. PEC induced with LC 9018 showed significant cytolytic activity for 51Cr-labeled Meth A from 3 days to at least 2 weeks after a single injection of LC 9018. The adherent cells lysed tumor cells in the cytolytic test, though nonadherent cells did not show cytolytic activity. These results show that LC 9018 can induce two cell populations possessing the ability to kill tumor cells in vivo. One may be activated macrophages which directly kill tumor cells and the other may be T-lymphocytes which can induce cytotoxic cells.     

Haemolytic activity of mouse peritoneal exudate cells in vitro

Mouse peritoneal exudate cells, and to a lesser degree spleen cells, can cause haemolysis of both syngeneic and allogeneic mouse red blood cells in vitro whereas lymph node and tumour cells are ineffective. The reaction was clearly detectable after 0.5–1 hour, and is usually complete after 5–6 hours at 37°. An analogous reaction was found also with sheep red blood cell targets, but haemolysis was weaker and a significant effect was not obtained until 24 hours later. The degree of haemolysis did not increase after the addition of complement. Hemolysis was not potentiated when specifically sensitized cells were employed or when PHA was present. Viability and active metabolism of the PE cell was essential for haemolysis. Only red blood cells were affected by the PE cells, nucleated target tumour cells being resistant. It was concluded that macrophages were responsible for haemolysis, which was not mediated by antibody and complement, but represented an active macrophage reaction unrelated to previously known mechanisms of haemolysis in vitro.     

EDTA alkaline elution characteristics and measurement of DNA damage in unlabeled DNA using Hoechst 33258 fluorescence

Hoechst 33258 fluorescence of single stranded DNA has been used to perform alkaline elution with unlabeled DNA. The high background fluorescence of "standard" elution solutions has prompted others to use EDTA but the elution characteristics of DNA in EDTA-containing solutions and the comparability of results with those using "standard" tetrapropyl ammonium hydroxide solutions have not previously been examined. We report here the elution characteristics of DNA in EDTA and the relevant parameters for the successful use of EDTA as an elution solution. An increase in elution pH to 12.4 is required but elution solutions of higher pH cause alkaline hydrolysis of undamaged DNA. Drug-treated DNA from which DNA-protein crosslinks have been removed can be completely removed from the filters at the end of the elution by a Pronase filter digestion. The simplest and most efficient removal of DNA-protein crosslinks is through the inclusion of proteinase-K in an SDS containing lysis solution. EDTA elution can measure interstrand crosslinks and single strand breaks as easily as is performed using radiolabeled DNA under "standard" elution conditions and requires only 1.5-2 x 10(6) cells per elution filter. DNA-protein crosslinking measurements were unsatisfactory, however, since even the Pronase digestion failed to completely remove protein-crosslinked DNA from the elution filters.     

Effect of Corynebacterium acnes on interferon production in mouse peritoneal exudate cells.

Corynebacterium acnes, an organism closely related to C. parvum, has been recognized to have a striking effect on the reticuloendothelial system, as well as on both humoral and cellular immunity. In mice previously exposed to C. acnes, serum interferon levels induced by injection of Newcastle disease virus (NDV), Chikungunya virus (CV), and polyinosinic-polycytidylic acid are suppressed. When peritoneal macrophages and lymphocytes from animals exposed to C. acnes were cultivated in vitro, their capacity to produce interferon in response to NDV and CV was reduced. Furthermore, the interferon-producing capacity of these cells in tissue culture was inhibited after exposure to C. acnes to vitro. Exposure of separated populations of peritoneal macrophages and lymphocytes to C. acnes in vitro demonstrated that the interferon response to NDV by both cell types is inhibited. Peritoneal macrophages appear to be the major contributor to the interferon response in this system. Finally, this inhibitory effect was shown to occur after exposure to a purified cell wall preparation of C. acnes organisms, as well as a lipid extract of this preparation.     

Effect of oltipraz on the susceptibility of adultSchistosoma mansoni to killing by mouse peritoneal exudate cells

Incubation of the adultSchistosoma mansoni with the anti-schistosomal compound oltipraz (OPZ) (40 nM) resulted in a significant decrease in schistosome-reduced glutathione (GSH), a thiol compound which may have a role in protection against oxidant-mediated damage. A significant proportion (20–47%) of worms treated with OPZ became susceptible to in vitro killing by zymosan-stimulated peritoneal exudate cells from mice infected withS. mansoni or inoculated with Bacillus Calmette Guérin (BCG). Killing of the worms was partially inhibited by the addition to the assay system of exogenous glutathione peroxidase with GSH but not by superoxide dismutase. These results suggested that killing of parasites exposed to the drug was partly mediated by cell-generated hydrogen peroxide. They indicate also that depletion of schistosome GSH levels could render the parasites susceptible to killing by oxidative mechanisms, and suggest that there is potential in exploiting schistosome oxidant defense systems and reactive oxygen byproducts in the treatment of schistosomiasis. Inhibition of schistosome oxidant defense systems with drugs may render the parasites susceptible to killing by reactive oxygen byproducts of effector cells.     

Enhanced production of interleukin 1 by mouse peritoneal macrophages after aclacinomycin administration

The peritoneal cells of mice injected with aclacinomycin (ACM), an oncostatic drug of the anthracyclin family, were found to secrete more interleukin (IL-1), after two successive 24-h periods of in vitro LPS stimulation than those of control mice. This measured IL-1 production is one of the signs of enhanced macrophage activity. The cells of ACM-injected mice also contained more intracellular IL-1 than those of controls. In contrast, macrophages from ACM-injected mice only increased their IL-1 production after the first 24-h incubation with PMA, and not after the second 24-h incubation. The response to ACM was dose- and time-dependent. We have also compared the IL-1 production by macrophages from mice injected with other anthracyclins, at doses equimolar to that of 4 mg/kg ACM and we have observed that adriamycin, 4'-epiadriamycin and aclacinomycin had similar activity, while THP-adriamycin an daunorubicine were slightly more active. Exploitation of this increased IL-1 production by macrophages could be beneficial in the design of tumor treatment protocols.     

Phagocytosis and killing of salmonella typhimurium by peritoneal exudate cells.

Normal peritoneal cells from conventional, germfree, or nu/nu mice readily killed opsonized salmonellae, an observation that suggests that this activity in the normal peritoneal cavity may not be dependent on either environmental antigenic stimulation or T-cell mediation. In contrast, peritoneal cells elicited 4 days after injection with thioglycolate medium failed to kill opsonized salmonellae but appeared to be highly phagocytic. Peritoneal cells from thioglycolate-treated mice could be induced to kill opsonized salmonellae by giving the mice a primary footpad injection and a secondary intraperitoneal injection of Corynebacterium parvum. This activation by C. parvum appeared to be thymus dependent, since it did not occur in nu/nu mice.     

Studies on chemiluminescence and protein kinase-C activity of cisplatin treated mouse peritoneal exudate cells.

A single i.p. injection of cisplatin (10 mg/kg body weight) into mice results in a significant increase in chemiluminescence and ATP contents of the peritoneal exudate cells (PEC) than that of PEC from untreated mice. It is also observed that in vitro treatment of macrophages with cisplatin, rIFN-gamma and LPS show increased activity of the protein kinase-C (PK-C). The activation of PK-C could result in stimulation of NADPH-oxidase resulting in increased levels of chemiluminescence. Increased contents of ATP in PEC after cisplatin treatment also suggests that this activation is energy dependent.     

In vivo production of heat shock protein in mouse peritoneal macrophages by administration of lipopolysaccharide.

The in vivo production of heat shock protein was studied by administration of bacterial lipopolysaccharide (LPS) into mice. Heat shock protein 70 was detected in the extract of adherent peritoneal cells from mice injected intraperitoneally with LPS by using the immunoblotting method. The expression of heat shock protein 70 was found 2 days after injection of LPS and reached its peak 4 days after injection. The intraperitoneal injection of LPS induced the expression of heat shock protein 70, whereas its subcutaneous injection did not. The in vivo production of heat shock protein 70 was inhibited by administration of LPS together with quercetin, an inhibitor of accumulation of heat shock protein 70 mRNA. Tumor necrosis factor alpha enhanced LPS-induced heat shock protein production in vivo. There was a decrease of gamma delta T cells in the peritoneal cavity of mice injected intraperitoneally with LPS. It was suggested that bacterial LPS is a stressful agent which induces the in vivo heat shock protein response, and its administration leads to the production of heat shock protein 70 in peritoneal macrophages.     

DNA damage and cytotoxcity induced in mammalian cells by a tetramethylfuroquinolinone derivative

1,4,6,8-Tetramethyl-2H-furo[2,3-h]quinolin-2-one (FQ) is an angelicin isoster characterized by a strong photosensitizing activity. FQ shows a significant antiproliferative activity also in the dark, i.e., without UVA activation. The cytotoxic activity of FQ in the dark was detected in HeLa cells and in normal human lymphocytes; FQ showed notable antiproliferative effects, barely lower in comparison with ellipticine, used as a reference. Similar results were obtained studying the FQ's capacity for forming chromosome aberrations. For both FQ and ellipticine, the chromosomal damage correlated closely with cell killing; when compared with ellipticine at the same levels of survival, FQ appeared to be muchless genotoxic. Using alkaline elution we have investigated the ability of FQ to damage DNA. The formation of equivalent amounts of single-strand breaks (SSB) and DNA-protein cross-links (DPC) was observed; in addition, these lesions appeared to be located at the same sites in DNA. Experiments carried out with neutral elution demonstrated the formation of double-strand breaks (DSB). All these data are consistent with an inhibition of topoisomerase II; this hypothesis was confirmed performing an enzymatic test in vitro using topoisomerase II from Drosophila melanogaster embryos. Environ. Mol. Mutagen. 29:256-264, 1997 © 1997 Wiley-Liss, Inc.     

DNA damage and repair induced by bleomycin in mammalian and insect cells.

We assessed the response of mosquito (ATC-15) and mammalian (CHO) cells to bleomycin (BLM). Comparison of the data obtained in both cell lines indicates that DNA in the chromatin of mosquito cells is 10 to 20 times more resistant to BLM, and that the DNA damage induced by this antibiotic is better repaired in mosquito than in mammalian cells. Permeability of the cell membrane for BLM was found to be the same for both cell lines. Moreover, the time-kinetics of BLM damage to nuclear DNA was similar for ATC-15 and CHO cells. The low sensitivity of mosquito cells to BLM is reflected in better growth efficiency. These cells exhibit a satisfactory growth at BLM doses that produce a permanent arrest of growth in CHO cells. It is proposed that variations in the chromatin structure and in the intracellular free amino acid pool may play an important role in the differential response of insect and mammalian cells to BLM.     

Rapid detection of DNA-interstrand and DNA-protein cross-links in mammalian cells by gravity-flow alkaline elution

Alkaline elution is a sensitive and commonly used technique to detect cellular DNA damage in the form of DNA strand breaks and DNA cross-links. Conventional alkaline elution procedures have extensive equipment requirements and are tedious to perform. Our laboratory recently presented a rapid, simplified, and sensitive modification of the alkaline elution technique to detect carcinogen-induced DNA strand breaks. In the present study, we have further modified this technique to enable the rapid characterization of chemically induced DNA-interstrand and DNA-protein associated cross-links in cultured epithelial cells. Cells were exposed to three known DNA cross-linking agents, nitrogen mustard (HN2), mitomycin C (MMC), or ultraviolet irradiation (UV). One hour exposures of HN2 at 0.25, 1.0, and 4.0 microM or of MMC at 20, 40, and 60 microM produced a dose-dependent induction of total DNA cross-links by these agents. Digestion with proteinase K revealed that HN2 and MMC induced both DNA-protein cross-links and DNA-interstrand cross-links. Ultraviolet irradiation induced both DNA cross-links and DNA strand breaks, the latter of which were either protein and nonprotein associated. The results demonstrate that gravity-flow alkaline elution is a sensitive and accurate method to characterize the molecular events of DNA cross-linking. Using this procedure, elution of DNA from treated cells is completed in 1 hr, and only three fractions per sample are analyzed. This method may be useful as a rapid screening assay for genotoxicity and/or as an adjunct to other predictive assays for potential mutagenic or carcinogenic agents.     

Induction of DNA synthesis in rat peritoneal macrophages in culture by a pleural inflammatory exudate

Peritoneal macrophages in culture are blocked in the G₀ phase of the cell cycle, but retain many of their functional characteristics such as phagocytic ability. Peritoneal macrophages have been thought to be a terminal cell type. It has been investigated whether such properties could be modified by a substance released in acute inflammatory exudates. For this purpose a pleural exudate obtained from rats injected with dextran (40,000) 4 hours before, was centrifuged to eliminate cells, sterilized by filtration on Millipore filter 0.22 m and diluted 50% with 199 medium culture. This medium was used to treat normal and activated peritoneal macrophages in culture. The effects were observed 24, 48, 72, 96 hours after the beginning of treatment. An enhancement of spreading and capacity of phagocytosis was observed 24 hours after the beginning of treatment. After 48 hours, the number of cells incorporating tritiated thymidine increased and became highest 4 days later. These phenomena were also obtained with pleural exudate of inbred rats (Lewis, Wag) treating macrophages of the same strain and with rat pleural exudate treating mouse macrophages. No effects were observed with dextran alone.The chemical nature of the stimulatory factor remains to be elucidated.