Protein synthesis and oxygen consumption in fish cells

Oxygen consumption and protein synthesis were measured concurently in four fish cell types: BF-2 and TRG-2 cell lines, rainbow trout macrophages and scale cells. The fractional rates of protein synthesis (percentage of the protein mass synthesised per day) were ranked: BF-2 cells > macrophages > RTG-2 cells > scale cells. Oxygen consumption rates were ranked BF-2 cells = macrophages = RTG-2 cells > scale cells. Within three of the cell types (BF-2, RTG-2 and scale cells) oxygen consumption and protein synthesis were linearly correlated, whereas comparison between the four cell types gave rise to an exponential relationship between fractional rates of protein synthesis and oxygen consumption. Inhibition of protein synthesis with cycloheximide by 41–65% resulted in a 62–89% reduction in oxygen consumption depending on cell type. Calculations of the aerobic cost of protein synthesis using the cycloheximide-sensitive protein synthesis and oxygen consumption rates resulted in estimates ranging from 11 to 217 mol O₂·mg protein^-1 synthesised depending on the cell type. The lowest net protein synthesis costs, which are close to theoretical values for peptide bond formation, were associated with the most rapid rates of protein synthesis.Abbreviations ANOV A analysis of variance - BDH British Drug Housing - BOC British Oxygen Company - ADT A ethylenediaminetetra-acetic acid - FCS foetal calf serum - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethane-sulphonic acid] - LCD least square differnece - MEM munimum essential media - PBS phosphate-buffered saline - PCA perchloric acid - SIS spectral index of the sample - tSIE transformed spectral index of the external standard spectrum     

Inter-laboratory comparison of cell lines for susceptibility to three viruses: VHSV, IHNV and IPNV.

Eleven European National Reference Laboratories participated in an inter-laboratory comparison of the susceptibility of 5 selected cell lines to 3 fish pathogenic viruses. The test included viral hemorrhagic septicaemia virus (VHSV); infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV), and the cell lines derived from bluegill fry (BF-2), chinook salmon embryo (CHSE-214), epithelioma papulosum cyprini (EPC), fathead minnow (FHM) and rainbow trout gonad (RTG-2). The results showed that for isolation of VHSV, BF-2 and RTG-2 cells performed equally well and had higher sensitivity compared to the other cell lines. For IHNV, EPC and FHM cells gave the best results, and for IPNV it was BF-2 and CHSE-214 cells. FHM cells showed the largest variability among laboratories, whereas EPC was the cell line showing the smallest variability.     

Assessment of in vitro cytotoxic and genotoxic activities of some trimethoprim conjugates

Trimethoprim, a commonly used antibacterial agent, is widely applied in the treatment of variety of infections in human. A few studies have demonstrated an extensive exposure of man to antibiotics, but there is still a lack of data for cytotoxic effects including nephrotoxicity, gastrointestinal toxicity, hematotoxicity, neurotoxicity and ototoxicity. The main purpose behind this study was to determine cytotoxic and genotoxic activities of trimethoprim (1), trimethoprim with maleic acid (2) and trimethoprim in conjugation with oxalic acid dihydrate (3). The cytotoxic effects of these three conjugates were elucidated by employing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoium bromide (MTT) assay using embryonic rat fibroblast-like cell line (F2408) and H-ras oncogene activated embryonic rat fibroblast-like cancer cell line (5RP7). Additionally, determination of genotoxic activity of these three compounds were studied by using cytokinesis blocked micronucleus assay (CBMN) in human lymphocytes. The results demonstrated that trimethoprim alone and its combination with other compounds are able to induce both cytotoxic and genotoxic damage on cultured cells (F2408, 5RP7, human lymphocytes).     

Flow cytometric detection of micronuclei and cell cycle alterations in fish-derived cells after exposure to three model genotoxic agents: mitomycin C, vincristine sulfate and benzo(a)pyrene

The measurement of cytogenetic alterations in vitro is considered an initial step in the risk assessment procedures for genotoxic agents. The concern about genotoxic pollutants in natural fish population makes the use of fish-derived cells an useful tool for these purposes. The technological improvements in well-established cytogenetic endpoints, such as micronuclei (MN) estimations by means of flow cytometry, have been proposed in the later years using mammalian cells. In this work, we test the capability of flow cytometry to evaluate MN induction and cell cycle alterations in an established fish cell line (RTG-2) using three agent-inductor models at different concentrations and exposure periods. For mitomycin C, an inverse relationship between length of exposure period and concentrations was observed. A dose-response relationship was observed after exposing RTG-2 cells to vincristine sulfate and benzo(a)pyrene. As this study shows, RTG-2 cells respond to clastogenic and aneugenic effects of the tested chemicals through the induction of MN at similar doses to mammalian cells and without the addition of exogenous metabolic activity. The possibility to check cell cycle alterations, in the same sample, gives the opportunity to evaluate early signals of cytotoxicity. The use of flow cytometry improves the assay by means of its speed and objectivity, which makes the assay very useful for genotoxicity assessment of aquatic chemicals.     

Culture conditions for arresting and stimulating the proliferation of a rainbow trout fibroblast cell line,RTG-2

Summary Conditions for arresting and stimulating the proliferation of the rainbow trout fibroblast cell line RTG-2 have been examined and the time course of events after stimulation determined. Quiescent populations were achieved in two ways. Cultures grown to confluency without a medium change for at least 7 d had fewer than 5% of the cells in S phase and few mitotic figures. Cultures deprived of serum, which could be done for up to 3 d without a loss in cell number, also achieved quiescence. After 3 d without serum, less than 1% of cells were in S phase and mitotic figures were infrequent. Addition to these cultures of fresh serum-containing medium brought about the synchronous entry of cells into S phase and mitosis. For cultures in which either the medium had been changed after 7 d without a change or serum-containing medium had been added after 3 d of serum deprivation, DNA synthesis increased after a lag period of 20 to 24 h, was pronounced between 30 and 45 h, and then declined. This was followed by a peak in the mitotic index. These protocols for arresting and subsequently stimulating RTG-2 proliferation should allow the G₁-S transition to be studied in a representative of teleosts. This research was supported by Natural Sciences and Engineering Research Council of Canada grant to N. C. B.     

A comparative study of the cytotoxic and genotoxic effects of ICRF-154 and bimolane,two catalytic inhibitors of topoisomerase II

ICRF-154 and bimolane have been used for the treatment of cancer, psoriasis, and uveitis in humans. Previous reports have revealed that the two drugs are topoisomerase II catalytic inhibitors, and patients treated with these agents have developed unique types of secondary leukemia. A study published in 1984 by Camerman and colleagues proposed that the therapeutic effects of bimolane could be due to ICRF-154, an impurity present within the bimolane samples that may also be responsible for the toxic effects attributed to bimolane. To date, this hypothesis has not been evaluated. In addition, little is known about the potential cytotoxic and genotoxic effects of ICRF-154. In this study, a combination of in vitro tests in human TK6 lymphoblastoid cells has been used to characterize the cytotoxic and genotoxic effects of ICRF-154 and bimolane as well as to compare the results for the two chemicals. ICRF-154 and bimolane were both cytotoxic, exhibiting very similar effects in three measures of cytotoxicity and cell proliferation. In the cytokinesis-block micronucleus assay with CREST-antibody staining, the two agents similarly induced chromosome breakage and, to a lesser extent, chromosome loss. Intriguingly, both drugs resulted in the formation of binucleated cells, perhaps as a consequence of an interference with cytokinesis. To further investigate their aneugenic effects, flow cytometry and fluorescence in situ hybridization analyses revealed that both compounds also produced similar levels of non-disjunction and polyploidy. In each of the cellular and cytogenetic assays employed, the responses of the ICRF-154-treated cells were very similar to those observed with the bimolane, and generally occurred at equimolar test concentrations. Our results, combined with those from previous studies, strongly suggest that bimolane degrades to ICRF-154, and that ICRF-154 is most likely the chemical species responsible for the cytotoxic, genotoxic, and leukemogenic effects exerted by bimolane.     

2-Dodecylcyclobutanone, a radiolytic product of palmitic acid, is genotoxic in primary human colon cells and in cells from preneoplastic lesions

The irradiation of fat results in the formation of 2-alkylcyclobutanones, a new class of food contaminants. Results of previous in vitro studies with primary human colon cells and in vivo experiments with rats fed with 2-alkylcyclobutanones indicated that these radiolytic derivatives may be genotoxic and enhance the progression of colon tumors. The underlying mechanisms of these effects, however, are not clearly understood. Therefore we performed additional investigations to elucidate the genotoxic potential of 2-dodecylcyclobutanone (2dDCB) that is generated from palmitic acid. In particular, we explored the relative sensitivities of human colon cells, representing different stages of tumor development and healthy colon tissues, respectively. HT29clone19A cells, LT97 adenoma cells and primary human epithelial cells were exposed to 2dDCB (150-2097 microM). We determined cytotoxic effects using trypan blue exclusion. Genotoxicity, reflected as strand breaks, was assessed using the alkaline version of the comet assay and chromosomal abnormalities were investigated by 24-color fluorescence-in-situ-hybridization. 2dDCB was cytotoxic in a time- and dose-dependent manner in LT97 adenoma cells and in freshly isolated primary cells but not in the human colon tumor cell line. Associated with this was a marked induction of DNA damage by 2dDCB in LT97 adenoma cells and in freshly isolated colonocytes, whereas in the HT29clone19A cells no strand breaks were detectable. A long-term incubation of LT97 adenoma cells with lower concentrations of 2dDCB revealed cytogenetic effects. In summary, 2dDCB was clearly genotoxic in healthy human colon epithelial cells and in cells representing preneoplastic colon adenoma. These findings provide additional evidence that this compound may be regarded as a possible risk factor for processes in colon carcinogenesis related to initiation and progression.     

JNK and p38 MAPK are independently involved in tributyltin-mediated cell death in rainbow trout (Oncorhynchus mykiss) RTG-2 cells

Mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases that transmit various extracellular signals to the nucleus inducing gene expression, cell proliferation, and apoptosis. Recent studies have revealed that organotin compounds induce apoptosis and MAPK phosphorylation/activation in mammal cells. In this study, we elucidated the cytotoxic mechanism of tributyltin (TBT), a representative organotin compound, in rainbow trout (Oncorhynchus mykiss) RTG-2 cells. TBT treatment resulted in significant caspase activation, characteristic morphological changes, DNA fragmentation, and consequent apoptotic cell death in RTG-2 cells. TBT exposure induced the rapid and sustained accumulation of phosphorylated MAPKs, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase (p38 MAPK). Further analysis using pharmacological inhibitors against caspases and MAPKs showed that TBT also induced cell death in a caspase-independent manner and that p38 MAPK is involved in TBT-induced caspase-independent cell death, whereas JNK is involved in the caspase-dependent apoptotic pathway. Thus, TBT employs at least two independent signaling cascades to mediate cell death in RTG-2 cells. To our knowledge, this is the first study revealing the relationship between MAPK activation and TBT cytotoxicity in RTG-2 cells.     

A Cytochemical Study of the Stem Cell Concept in Specimens of a Human Ovarian Tumor

The amounts of DNA in interphase nuclei were compared with the amounts of DNA in metaphase and anaphase figures in Feulgen-stained tissue sections of 5 specimens of the human ovarian papillary serous adenocarcinoma. The relative amounts of DNA per cell were determined by cytophotometric measurements of interphase nuclei at a single wavelength and of mitotic figures by the two wavelength method. The 5 specimens conformed to the stem cell concept of cell proliferation since anaphase distributions of amounts of DNA were restricted to a narrow range of DNA values indicating the successful mitosis of a single cell type (stem cell) out of several cell types whose presence were suggested by the wide spread of interphase and metaphase values. In addition, the data indicated that, in some instances, only the amounts of DNA in anaphase figures can reliably identify the stem cell. Changes in the frequency of dividing cells having doubled amounts of DNA, and/or the presence of cells resulting from endoreduplication can distort the interphase distribution of amounts of DNA and thus give rise to a modal DNA interphase value which is not the same as the DNA value of the stem cell (anaphase figures).     

Effects of 5-fluorouracil on cell cycle arrest and toxicity induced by X-irradiation in normal mammalian cells

From clinical studies in cancer patients and experimental in vitro studies, there is evidence of an increased cytotoxic effect, and even synergy, when irradiation is combined with 5-fluorouracil (5-FU). The mechanism for this is unclear.  

Materials and Methods :

Mouse fetuses (C₃H) have been exposed in vivo to X-irradiation and 5-fluorouracil (5-FU) as single agents or in combination. Cell proliferation, cell cycle progression, fetal survival and incidence of fetal malformations have been studied.  

Purpose :

The aim of this study was to determine possible synergistic cytotoxic effects when 5-FU and ionizing radiation were combined, particularly concerning the regulation of cell cycle progression in proliferating, non malignant mammalian cells in vivo .  

Results :

The combination of low-toxic doses of X- irradiation and 5-FU had a synergistic toxic effect in nonmalignant mouse fetuses in vivo . The cell cycle regulation was perturbed and the radiation-induced G₂-arrest was eradicated by 5-FU during the initial hours.  

Conclusions :

The time for repair of radiation induced DNA-damage is probably reduced, which may explain the increased toxicity of this combination.     

In vitro assessment of DNA damage after short- and long-term exposure to benzo(a)pyrene using RAPD and the RTG-2 fish cell line

Genotoxins present in the aquatic environment are often associated with the decline or disappearance of many wild populations. The hazard assessment of chemicals requires sensitive and specific tests to study the genotoxic effects in order to establish the maximum allowable chemical concentrations prior to the release to the environment. We have previously shown that an established fish cell line (RTG-2) together with the random amplified polymorphic DNA (RAPD) technique, can be used to detect alterations in the DNA caused by direct acting genotoxins. The current study takes this a step further and examines in the same system the effect of a pro-mutagen benzo(a)pyrene (B(a)P) at different concentrations (0.05, 0.1, and 0.5 microg/ml) and at different exposure periods (1, 2, 3, 15, and 30 days). After comparing DNA fingerprints from control and exposed cells, both qualitative and quantitative analysis show an increase in the instability in the DNA fingerprint of exposed cells over a time- and concentration-dependent manner. At the higher concentration (0.5 microg/ml) three out the four primers showed altered bands after 1 day of exposure, while after 3 days all used primers showed an altered pattern. At the lower concentration of B(a)P (0.05 microg/ml) the appearance of new bands was observed with a 100% level of reproducibility after 30 days of exposure suggesting an inheritance of the altered DNA. We conclude that this in vitro system is useful to evaluate genotoxic effects, both after acute and chronic exposures and of direct and non-direct acting genotoxins. Cultured cells can be considered as genetically homogenous populations. Therefore, in vitro systems permits us to undertake mechanistic studies avoiding the interference of polymorphisms inherent in the in vivo systems. Furthermore, the RTG-2 fish cell line combined with a RAPD assay could be used in studies of hazard identification in risk assessment protocols of chemicals.     

Genotoxicity Induced by Foetal and Infant Exposure to Magnetic Fields and Modulation of Ionising Radiation Effects

Background

Few studies have investigated the toxicity and genotoxicity of extremely low frequency magnetic fields (ELF-MF) during prenatal and neonatal development. These phases of life are characterized by cell proliferation and differentiation, which might make them sensitive to environmental stressors. Although in vitro evidences suggest that ELF-MF may modify the effects of ionizing radiation, no research has been conducted so far in vivo on the genotoxic effects of ELF-MF combined with X-rays.

Aim and methods

Aim of this study was to investigate in somatic and germ cells the effects of chronic ELF-MF exposure from mid gestation until weaning, and any possible modulation produced by ELF-MF exposure on ionizing radiation-induced damage. Mice were exposed to 50 Hz, 65 μT magnetic field, 24 hours/day, for a total of 30 days, starting from 12 days post-conception. Another group was irradiated with 1 Gy X-rays immediately before ELF-MF exposure, other groups were only X-irradiated or sham-exposed. Micronucleus test on blood erythrocytes was performed at multiple times from 1 to 140 days after birth. Additionally, 42 days after birth, genotoxic and cytotoxic effects on male germ cells were assessed by comet assay and flow cytometric analysis.

Results

ELF-MF exposure had no teratogenic effect and did not affect survival, growth and development. The micronucleus test indicated that ELF-MF induced a slight genotoxic damage only after the maximum exposure time and that this effect faded away in the months following the end of exposure. ELF-MF had no effects on ionizing radiation (IR)-induced genotoxicity in erythrocytes. Differently, ELF–MF appeared to modulate the response of male germ cells to X-rays with an impact on proliferation/differentiation processes. These results point to the importance of tissue specificity and development on the impact of ELF-MF on the early stages of life and indicate the need of further research on the molecular mechanisms underlying ELF-MF biological effects.     

Tests for genotoxicity and mutagenicity of furan and its metabolite cis-2-butene-1,4-dial in L5178Y tk+/- mouse lymphoma cells

Furan is found in various food items and is cytotoxic and carcinogenic in the liver of rats and mice. Metabolism of furan includes the formation of an unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). In view of the multifunctional electrophilic reactivity of BDA, adduct formation with protein and DNA may explain some of the toxic effects. Short-term tests for genotoxicity of furan in mammalian cells are inconclusive, little is known for BDA. We investigated BDA generated by hydrolysis of 2,5-diacetoxy-2,5-dihydrofuran for genotoxicity in L5178Y tk+/- mouse lymphoma cells using standard procedures for the comet assay, the micronucleus test, and the mouse lymphoma thymidine kinase gene mutation assay, using 4-h incubation periods. Cytotoxicity was remarkable: cell viability at concentrations>or=50 microM was reduced to <50%. In the dose range up to 25 microM, viability was >90%. Measures of comet-tail length and thymidine-kinase mutant frequency were increased 1.6- and 2.4-fold above control, respectively. Analysis of three fully independent replicates with a linear mixed-effects model showed a highly significant increase with concentration for both endpoints. Compared to methyl methanesulfonate used as a positive control, BDA was of similar potency with respect to genotoxicity, but it was much more cytotoxic. Furan added to cell cultures at doses that resulted in time-averaged effective concentrations of up to 3100 microM was neither cytotoxic nor genotoxic. A potential cross-linking activity of BDA was investigated by checking whether gamma radiation-induced DNA migration in the comet assay could be reduced by pre-treatment with BDA. In contrast to the effect of the positive control glutaraldehyde, BDA treatment did not reduce the comet tail length. On the contrary, an increase was observed at >or=100 microM BDA, which was attributable to early apoptotic cells. Although BDA was found to be a relatively potent genotoxic agent in terms of the concentration necessary to double the background measures, cytotoxicity strongly limited the concentration range that produced interpretable results. This may explain some of the inconclusive results and indicates that non-genotoxic effects must be taken into account in the discussion of the modes of toxic and carcinogenic action of furan.     

Triflumuron induces cytotoxic effects on hepatic and renal human cell lines

Insect growth regulator insecticides are a new class of pesticides, commonly used around the world to control insect damages. Among those compounds, we focused our interest on triflumuron (TFM), which is less toxic than other conventional insecticides. However, not much is known about its toxic effects on mammalian systems. Therefore, our study aimed toward evaluating the cytotoxic and genotoxic effects of TFM using two different cell lines, the human renal embryonic cells (HEK 293) and hepatocytes (Hep G2). We showed, according to the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay, that TFM reduced significantly the cell viability and increased the reactive oxygen species generation, malondialdehyde levels, and mitochondrial membrane potential in both cell lines. The antioxidant system was disturbed as assessed by the increased activities in both catalase and superoxide dismutase. We demonstrated also, that TFM is an inductor of DNA damages quantified by the comet assay. Moreover, we showed an overexpression of proapoptotic Bax and a decrease in antiapoptotic Bcl‐2 expression. As a conclusion, we demonstrate that the liver presents the major target organ to TFM, in which the cytotoxicity and the genotoxic effects were significantly higher in hepatic cells than in renal cells and by consequence its uses must be controlled.     

A garlic derivative, S-allylcysteine (SAC), suppresses proliferation and metastasis of hepatocellular carcinoma

Background

Hepatocellular carcinoma (HCC) is highly malignant and metastatic. Currently, there is no effective chemotherapy for patients with advanced HCC leading to an urgent need to seek for novel therapeutic options. We aimed to investigate the effect of a garlic derivative, S-allylcysteine (SAC), on the proliferation and metastasis of HCC.

Methodology/Principal Findings

A series of in vitro experiments including MTT, colony-forming, wound-healing, invasion, apoptosis and cell cycle assays were performed to examine the anti-proliferative and anti-metastatic effects of SAC on a metastatic HCC cell line MHCC97L. The therapeutic values of SAC single and combined with cisplatin treatments were examined in an in vivo orthotopic xenograft liver tumor model. The result showed that the proliferation rate and colony-forming abilities of MHCC97L cells were suppressed by SAC together with significant suppression of the expressions of proliferation markers, Ki-67 and proliferating cell nuclear antigen (PCNA). Moreover, SAC hindered the migration and invasion of MHCC97L cells corresponding with up-regulation of E-cadherin and down-regulation of VEGF. Furthermore, SAC significantly induced apoptosis and necrosis of MHCC97L cells through suppressing Bcl-xL and Bcl-2 as well as activating caspase-3 and caspase-9. In addition, SAC could significantly induce the S phase arrest of MHCC97L cells together with down-regulation of cdc25c, cdc2 and cyclin B1. In vivo xenograft liver tumor model demonstrated that SAC single or combined with cisplatin treatment inhibited the progression and metastasis of HCC tumor.

Conclusions/Significance

Our data demonstrate the anti-proliferative and anti-metastatic effects of SAC on HCC cells and suggest that SAC may be a potential therapeutic agent for the treatment of HCC patients.     

Chemically induced aneuploidy in mammalian cells in culture

Our objectives were to assess whether there exist useful aneuploidy tests in vitro, to identify chemicals that showed potential for mitotic aneuploidy induction, and to recommend some features of suitable protocols for such testing. From over 100 papers we selected 24 for review. The acceptable studies examined hyperdiploidy at metaphase, had concurrent negative controls with low background rates of hyperdiploidy, used a fixation time sufficient for cells to complete more than one cell cycle after treatment and had multiple dose levels with at least 100 cells scored per point. We judged that 12 compounds were positive, 7 inconclusive, and 4 negative with the reservation that 2 of the 4 compounds had not been tested up to toxic doses. Many of the positive compounds are also known to cause structural chromosome aberrations. We separately reviewed qualitative reports of 'C-mitotic' effects, anaphase lagging, multipolar mitoses, or altered DNA content, since these effects may sometimes by associated with aneuploidy induction. No well-validated in vitro aneuploidy assay exists, and much research is required to develop tests, perhaps using chromosome counts, DNA content, or effects on cell organelles necessary for mitosis. In test protocol development we should carefully consider choice of cell sample size, use of in vitro metabolic activation systems, and selection of doses, especially with regard to the problem of whether cytotoxic concentrations should be used.     

Effects of Interferon and PKC Modulators on Human Glioma Protein Kinase C,Cell Proliferation,and Cell Cycle

The in-vitro effects of human interferon -2b (HuIFN -2b), protein kinase C (PKC) agonist [TPA (12-0-tetra-decanoyl-phorbol-13-acetate)] and PKC inhibitor (calphostin C) on human glioma (U-373MG) PKC activity, cell proliferation and cell cycle were compared. HuIFN -2b and TPA increased PKC activity, elevated the number of cells in DNA synthesis (S) phase and decreased cell proliferation by similar magnitudes. Calphostin C inhibited PKC activity, increased the number of cells in S phase and produced strong cytotoxic effects (IC₅₀ 150 nM). Higher concentrations of calphostin C with or without serum induced an additional block in gap2 and mitosis. We conclude that HuIFN -2b's mode of action may be directly or indirectly affecting PKC. The response produced by HuIFN -2b is similar to TPA (potent PKC activation and S phase arrest).     

Differential effects of human blood monocytes on the growth of human tumour cell lines in vitro

Summary Monocytes and macrophages have been shown to be cytotoxic towards tumour cells in vitro. However, although tumour-associated monocytes and macrophages are now widely accepted to contribute a relatively high proportion of the cellular infiltrate of experimental and human solid carcinomas, a cytotoxic/cytostatic effector function for these cells in vitro or in vivo has yet to be conclusively demonstrated. In the present study, we show that non-activated peripheral blood monocytes co-cultured with tumour cells across a semi-permeable membrane release soluble factors that modulate the growth of tumour cells in contrasting ways. After Nycoprep 1.068 separation, non-activated peripheral blood monocytes enhanced the in vitro proliferation of HT29 colon adenocarcinoma cells but inhibited T47D breast carcinoma cell replication; peripheral blood lymphocytes were incapable of mediating these effects. In contrast, peripheral blood monocytes activated by interferon caused a pronounced inhibition of both HT29 and T47D cell proliferation.     

The comet assay differentiates efficiently and rapidly between genotoxins and cytotoxins in quiescent cells

Our main aim was to establish the efficiency of the single cell electrophoresis technique for differentiating between drugs that bind DNA and those that do not. The alkaline comet assay was used to test the responses of human leukocytes (quiescent cells) to damage induced by reportedly genotoxic and reportedly cytotoxic agents. Incubation of G0 leukocytes for 1 h with the genotoxic agents camptothecin and actinomycin C provoked DNA migration, observed as comet figures. On the other hand, when cells were treated with the cytotoxic agents cordycepin, fluorodeoxyuridine and puromycin, the leukocyte nuclei were indistinguishable from those of untreated cells. In addition, we have developed a rapid method using non-proliferating cells that requires neither culture nor lymphocyte isolation. This method promises to be useful as a rapid in vitro screening assay.     

Species differences in cytotoxic and genotoxic effects of phenacetin and paracetamol in primary monolayer cultures of hepatocytes

The cytotoxic and genotoxic effects of phenacetin and paracetamol were examined in monolayer cultures of hepatocytes isolated from the mouse, hamster, rat and guinea pig. No marked increase in unscheduled DNA synthesis (UDS) after exposing hepatocytes from any of the species to phenacetin was observed. At cytotoxic concentrations of paracetamol, an increased UDS in mouse hepatocytes in vitro was observed. Pretreatment of the mice by inducers of drug-metabolizing enzymes, such as 3-methylcholanthrene and Aroclor 1254, lowered the concentration threshold for the toxic responses. With rat hepatocytes only a minor increase in UDS was noted, while with hepatocytes from hamsters and guinea pigs in fact a decrease was seen. The narrow range observed between the cytotoxic and genotoxic effects of paracetamol makes it difficult to predict whether the initial DNA damage could lead to a mutation or whether the cells will die before the mutation is expressed. With respect to the cytotoxic effects, hamster hepatocytes were found to be most susceptible to paracetamol, followed by mouse, while rat and guinea pig were less affected. These data were in accordance with in vivo findings (Davis et al., 1974), indicating the potential value of hepatocyte culture when screening for possible liver toxic substances.