Mutagenesis, Vol. 15, No. 1, 9-15,
January 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Induction of aberrant mitosis with PCBs: particular efficiency of 2,3,3',4,4'-pentachlorobiphenyl and synergism with triphenyltin
Department of Toxicology, National Institute for Working Life, S-171 84 Solna and 1 Environmental Chemistry and 2 Genetic and Cellular Toxicology, Wallenberg Laboratory, S-106 91 Stockholm, Sweden
 |
Abstract |
|---|
 Top Abstract IntroductionMaterial and methodsResultsDiscussionReferences |
|---|
The polychlorinated biphenyls 2,2',5,5'- and 3,3',4,4'-tetrachlorobiphenyl, 2,3,3',4,4'- and 3,3',4,4',5-pentachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl were tested for spindle-disturbing activity in V79 Chinese hamster cells. Clones lacking endogenous cytochrome P450 activity or expressing rat CYP1A1 or CYP2B1 were used. Induction of abnormal chromosomal arrangements in mitosis were found to be favoured by o-chlorine substitutions, but not by co-planarity giving affinity, for example, for the Ah receptor and CYP1A isoenzymes. Only 2,2',5,5'-tetrachloro- and 2,3,3',4,4'-pentachlorobiphenyl gave dose–response curves similar to many other compounds tested in vitro, showing an increase from the background level of 10 to 100% disturbed mitoses with nominal concentrations >10–6 M, i.e. concentrations far above the total PCB concentrations found in human blood. Cells transfected with rat CYP2B1 were more sensitive to the most active congener, 2,3,3',4,4'-pentachlorobiphenyl, than cells lacking P450 activity or expressing CYP1A1. Induction of abnormal mitosis by PCB metabolites formed by P450 enzymes cannot be excluded, but does not seem likely because of the short treatment time and the reportedly slow metabolism of PCBs. 2,3,3',4,4'-Pentachlorobiphenyl showed synergistic activity with the potent spindle poison triphenyltin. Inactive concentrations of both agents (10 and 50 nM, respectively) caused abnormal configurations when combined. This is an important finding since exposure to mixtures of compounds is common and it motivates further studies of subthreshold activities of highly lipophilic environmental contaminants.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
Disturbance of mitosis by chemicals can be exerted through specific reactions, e.g. binding to microtubules, but there is also a general mechanism based on hydrophobic interactions. Even relatively inert compounds can cause a block in metaphase, shrinkage of the spindle and poor alignment of chromosomes. In general the threshold for abnormal chromosomal arrangements in mitosis appears inversely related to lipophilicity, measured as the partition coefficients in octanol/water (logP) (Önfelt, 1987
). In vitro mitotic cells may survive this type of challenge and form aneuploid/polyploid daughter cells even when thresholds for toxicity and mitotic disturbances are close (Önfelt, 1987), but during culture there is usually prominent selection against cells with abnormal chromosome numbers (Önfelt and Klasterska, 1983; Matsuoka et al., 1998). Here five different PCBs were selected for investigation: 2,2',5,5'- and 3,3',4,4'-tetrachlorobiphenyl (CB-52 and CB-77), 2,3,3',4,4'- and 3,3',4,4',5-pentachlorobiphenyls (CB-105 and CB-126) and 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153). Their partition coefficients in octanol/water are of about the same magnitude as that of benzo[a]pyrene (B[a]P), i.e. in the range 6–8 (De Kock and Lord, 1987; Hawker and Connel, 1988), and they may therefore be expected to be active at similar concentrations. In V79 Chinese hamster cells B[a]P induces c-mitosis at 
10–6 M without metabolic activation, but becomes more active in the presence of CYP1A1 (Ellard et al., 1991), probably due to formation of the highly c-mitotic (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (Jensen et al., 1993).
In this paper we have sought the concentrations required for induction of aberrant chromosomal arrangements in mitosis and the relative importance of co-planarity of the PCBs, as well as the influence of in situ expressed cytochrome P450 enzymes. CB-77 and CB-126 can attain a coplanar configuration, show affinity for the Ah receptor (Rannug et al., 1991) and induce the CYP1A isozyme (Bandiera et al., 1982). Chlorine atoms in more than one ortho position, as in CB-52 and CB-153, leads to restrictions in orientation of the phenyl rings and the entire molecule therefore attains a more globular structure, which precludes binding to the Ah receptor (Bandiera et al., 1982; Rannug et al., 1991), but favours induction of the CYP2B isozyme (Allen and Abrahamson, 1979). The mono-substituted congener CB-105 induces both CYP1A and CYP2B isozymes (reviewed in Safe, 1994) and may show intermediate activity if either ortho substitution or coplanarity is most important for induction of mitotic disturbances. Hydroxylated metabolites of all the selected PCB congeners have been identified in vitro and in vivo (Jensen and Sundström, 1974; Klasson Wehler et al., 1989; Koga et al., 1990; Ariyoshi et al., 1992; Borlakoglu and Wilkins, 1993; Klasson Wehler et al., 1993), but CB-153 is considered a model for non-metabolizable compounds due to its extremely slow metabolic conversion (Mühlebach et al., 1991; Ariyoshi et al., 1997).
Besides V79 cells lacking CYP-activity, V79 cell lines transfected with genes for rat CYP1A1 (Dogra et al., 1990) and CYP2B1 (Doehmer et al., 1988) were used in these tests. During the short incubations used in the experiments described here only limited metabolic conversion of the compounds was expected. Still, if any very active metabolite can be formed by the CYP enzymes, enhanced c-mitotic activity in transfected compared with non-transfected cells might be observed. To investigate possible subthreshold activities, one of the active congeners was combined with triphenyltin, previously found to cause abnormal mitosis in V79 cells and human lymphocytes at low concentrations (Jensen et al., 1991a).
|
Material and methods |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
Chemicals
The PCBs (numbered according to Ballschmiter et al., 1993) CB-52 (2,2',5,5'-tetrachlorobiphenyl), CB-77 (3,3',4,4'-tetrachlorobiphenyl), CB-105 (2,3, 3',4,4'-pentachlorobiphenyl), 14C-labelled CB-105, CB-126 (3,3',4,4',5-penta-chlorobiphenyl) and CB-153 (2,2',4,4',5,5'-hexachlorobiphenyl) (all PCB congeners were of 98% purity or higher) were synthesized as described elsewhere (Sundström, 1973
; Bergman et al., 1990); dichloromethane and hexane were of pesticide grade and were obtained from Riedel de Haen; sulphuric acid (98%) and 2-propanol (p.a.) were from Merck; 7-ethoxyresorufin and 7-penthoxyresorufin were from Molecular Probes and Geneticin (G 418) was from Gibco. Triphenyltin chloride (95% purity) was from Aldrich.
Cell lines
The transfected cell lines were a gift from Dr J.Doehmer and have been described in detail elsewhere (Doehmer et al., 1988; Dogra et al., 1990). Briefly, full-length cDNAs were inserted into the expression vector pSV2 and placed under the control of the SV40 early promoter. These pSV450 plasmids were co-transfected with plasmid pdBPV-MTneo, which carries the neomycin phosphotransferase gene and thus confers resistance to the drug G418.
The cell line SD-1 expresses rat CYP2B1 and the XEM-2 cell line expresses rat CYP1A1 (nomenclature according to Nelson et al., 1993). The non-transfected V79 cells lack measurable CYP activity. They are deficient in p53 (C.Arnaudeau, personal communication) with the same point mutations as published by Chaung et al. (1997).
Culture conditions
The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 7.5% heat-inactivated mycoplasma and virus screened foetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin. Selection of plasmid-containing cells was performed by addition of 400 µg/ml G418. Cells were kept from reaching confluence at any time to ascertain high CYP expression (Jensen et al., 1993). All incubations were performed at 37°C in 5% CO2 in air.
Treatment
PCBs were dissolved and diluted in acetone before addition to complete medium. Cells were exposed in this medium for 4 or 24 h. The concentration of acetone used was 0.5–1%. Some cultures were treated with CB-105 at the indicated concentrations for 1 or 2 weeks before being reseeded on coverslips, incubated for 24 h in the presence of CB-105 (the same concentration as before) and then briefly (30 min) treated with triphenyltin dissolved in dimethylsulphoxide in Hank's balanced salt solution (HBSS). The concentration of dimethylsulphoxide was 0.2%.
Metabolic activity measurements
7-Ethoxyresorufin O-deethylation activity (EROD) and 7-penthoxyresorufin O-dealkylation (PROD) were measured by the appearance of resorufin in the medium of intact cell cultures as described by Wortelboer et al. (1990). The monolayers were rinsed twice with 5 ml HBSS at 37°C and then incubated with 5 µM of one of the reagents and 10 µM dicoumarol for inhibition of diaphorase. The samples were diluted (1:1) with incubation mixture (in 0.1 M phosphate buffer, pH 7.4) and the pH was brought to >7.5 by addition of 100 µl 0.1 M NaOH to 2 ml of diluted medium samples. Fluorescence was determined with a Perkin-Elmer LS-5 fluorimeter (excitation 530 nm, emission 585 nm, slit width 5 nm).
Uptake of CB-105
Radioactive CB-105 was used to measure uptake by the cells. Cells (2x106/culture) were exposed to 10–5 M CB-105 in 4 ml complete medium for 4 or 24 h. The medium was removed and the cell layers were rinsed with 2x5 ml ice-cold HBSS. Thereafter the cells were rinsed with 2x1 ml serum (Mangelsdorf et al., 1987). The cells were finally removed with trypsin and the dishes were rinsed with 2x5 ml HBSS. Cells and the latter HBSS rinses were pooled. This fraction was extracted with 1 ml n-hexane and radioactivity in a 200 µl aliquot was determined by scintillation counting.
Losses of the compound to the Petri dish and coverslip were estimated for both CB-77 and CB-105 in separate experiments by determining the amount of compound (not labelled) remaining in the medium after incubation (4 h) without cells. Before extraction an internal standard, 2,3,3',4,4',5,5'-heptachlorobiphenyl, was added. 2-Propanol (2 ml) and 6 M hydrochloric acid (1 ml) were added to the samples before extraction with hexane:dichloromethane (5:1, 5 ml). The samples were gently rocked for 1.5 h. The phases were separated by centrifugation and the organic phase was removed. The extraction was repeated twice with hexane (3 ml). The combined organic phases were concentrated to dryness in order to remove all dichloromethane, redissolved into hexane (2 ml) and partitioned with concentrated sulphuric acid (1 ml) to remove lipids. The volume of the hexane phase was adjusted before analysis by gas chromatography (GC). The GC, a Varian 3400, was equipped with an electron capture detector (ECD) kept at 350°C, a split/splitless detector operated in the splitless mode and kept at 260°C and a fused silica column DB5 30 mx0.22 mm i.d. and of 0.25 µm film thickness from Resteck Ltd. The carrier was helium and the make-up gas nitrogen. The GC oven temperature programme was 80°C for 2 min and then increasing at 10°C/min up to 300°C, where it was kept for 10 min. The data were recorded by ELDS software (Kungshög, Sweden).
Effects on mitosis
Cells (1.5x105) were seeded on plastic coverslips in plastic Petri dishes and incubated for 24 h, giving asynchronously growing populations. Cultures were rinsed twice with HBSS at 37°C and then exposed to PCB in 2 ml complete medium without G418 at pH 7.2–7.4 for 4 h. After treatment the cells were fixed in situ with methanol:acetic acid (3:1) and stained with 4% Giemsa (Gurr) in phosphate buffer.
c-mitosis
Scoring included normal metaphase, anaphase and telophase, as well as abnormal configurations; defined as partial c-mitosis representing weak disturbances of the spindle, e.g tripolar spindles, lagging of chromosomes in anaphase, `ball' metaphase, poor alignment of chromosomes in metaphase, and finally the configuration associated with complete depolymerization of spindle microtubules and defined as full c-mitosis, i.e total scattering of chromosomes in the cytoplasm. One hundred mitotic cells/slide and 3 slides/concentration were examined in each experiment. Two or three experiments/cell line were performed. All slides were coded before being scored by one person.
Frequency of mitoses and frequency of anaphases among mitotic cells
The frequency of mitoses was determined by scoring 1000 cells/slide and the frequency of anaphases was determined by scoring 100 mitotic cells/slide.
Immunofluorescent and phalloidin staining
Cells grown on coverslips were fixed with 3.5% formaldehyde for 10 min, rinsed with phosphate-buffered saline (PBS) several times, incubated with anti-
-tubulin antibody (mouse IgG; Amersham) at 4°C overnight, rinsed extensively, incubated with biotinylated rabbit anti-mouse antibodies (rabbit IgG; Zymed) for 1.5 h, rinsed, incubated with streptavidin–Cy3 for 10 min and rinsed, and then TO-PRO (Molecular Probes) was applied for 20 min before final quick rinses and mounting. Phalloidin staining was performed on fixed and rinsed cells by incubating them with phalloidin–Oregon Green (Molecular Probes) for 20 min, followed by rinses, TO-PRO staining as above and mounting. Images were obtained with a Zeiss 510 confocal laser scanning microscope using a 488 nm laser line for Oregon Green, a 543 nm laser line for Cy3 and a 633 nm laser line for TO-PRO. Each frame was scanned with alternating single laser lines to avoid bleed-through.
Single cell survival
After 4 h treatment the cells were rinsed, trypsinized and resuspended in medium. From the suspension 100 cells were seeded per Petri dish. There were 2 cultures/concentration and 3 dishes/treated culture in an experiment. Two experiments/cell line were performed. After 1 week the colonies were fixed with methanol and counted.
Statistics
Concentration responses with individual congeners were evaluated with Spearman's rank correlation test. Comparisons between cell lines were made by use of analysis of variance or the Kruskall–Wallis or the Mann–Whitney U-test. Effects of the combined treatments with CB-105 and triphenyltin were evaluated by factorial analysis of variance after logarithmic transformation of variates.
|
Results |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
Metabolic activity
The EROD activity for the CYP1A1-expressing cell line XEM-2 was on average 12.7 ± 2.7 pmol/min/106 cells and the PROD activity for the CYP2B1-expressing cell line SD-1 was on average 1.6 ± 0.2 pmol/min/106 cells. The metabolic activities were found to be comparable in all experimental series and formation of product was linear with time up to 4 h (data not shown). EROD activity in SD-1 and PROD activity in XEM-2 were negligible, with only trace amounts of products (data not shown).
Uptake of CB-105
Non-transfected cells exposed in complete medium for 4 h accumulated the same amount of labelled CB-105 as cells exposed for 24 h. The uptake of CB-105 was estimated to be 3x10–16 mol/cell at a nominal concentration of 10–5 M.
The other measurements performed with non-labelled compound showed that ~30% of the amount added in medium was lost to the plastic Petri dishes during incubation. Similar losses to those for CB-105 were recorded for the coplanar CB-77. Thus the concentrations shown in the figures and tables are nominal.
Single cell survival
CB-52 and CB-105 appeared most cytotoxic, followed by CB-153 (Figure 1a–c). A significantly higher toxicity of CB-52 and CB-153 was seen with the CYP2B1-transfected cells, at 10–4 M, while the reverse was found for CB-105 under similar conditions (P < 0.001; Figure 1c).
|
The coplanar CB-77 and CB-126 were less toxic than the o-substituted congeners and the transfected cell lines showed higher survival ratios than non-transfected V79 cells (P = 0.0001; Figure 1a–c
).
Effects on mitosis
The non-transfected cells were tested at both 4 and 24 h of treatment. The results were virtually identical (not shown) and only the 4 h treatment series is presented below.
The coplanar congeners CB-77 and CB-126 and the most lipophilic and o-substituted CB-153 were inefficient, while the o-substituted CB-52 and CB-105 caused high frequencies of c-mitosis in all three cell lines (Figure 2a–c). Although weak, the effects of CB-126 were significant; the CYP2B1-expressing SD-1 cells seemed most sensitive (Figure 2d). CB-52 gave virtually the same c-mitotic response in all three cell lines (Figure 2b) while the effect of CB-105 (Figure 2c) appeared more pronounced in SD-1 cells (combined probability 5x10–6 –2x10–5M, 0.025 < P < 0.05).
|
c-mitotic effects (compare Figure 3c and d with a and b
) coincided with an increased mitotic index, decrease in anaphases and displacement of chromosomes to the cell periphery (Figure 3g and h). The displacement reaction could be seen concomitant with cytoplasmic changes typical of anaphase/telophase (compare Figure 3g and h with e and f).
|
Treatment with CB-105 for 1 or 2 weeks followed by a brief treatment with triphenyltin
Non-transfected V79 cells grown in the presence of CB-105 showed a significant increase in c-mitoses at 4x10–6 M but not at the lower concentrations applied (Figure 4a and b
). The response was slightly higher after 1 week compared with 2 weeks (P = 0.0209).
|
The applied concentrations of triphenyltin (10–8 and 5x10–8 M) did not alone increase the frequency of c-mitosis significantly. Yet, pretreatment with CB-105 rendered the cells more sensitive to triphenyltin in a concentration-dependent manner (Figure 4a and b
). Variation in both factors had a significant influence (P < 0.0001) and the analysis showed significance for interaction (P = 0.024) between the two. Thus, their combined effect was not simply additive, but synergistic. There was a tendency for decreased response to triphenyltin after 2 weeks compared with 1 week of pretreatment with CB-105. The cultures exposed to 4x10–6 M CB-105 for 1 or 2 weeks showed reduced growth rates compared with controls (density 75 and 85% of control density, respectively) and many cells with high chromosome numbers were observed (not counted).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
In the present study, five different PCBs were tested under the assumption that most compounds can cause c-mitotic effects by hydrophobic interactions, but still show structure-dependent differences in activity thresholds. The PCB congeners with their extreme lipophilicity and low chemical reactivity (Rapaport and Eisenriech, 1984
) did show differences in their toxicity and c-mitotic activity, and there were differences between cell lines (Figures 1 and 2). We used three closely related cell lines, two originating from one derivative used for transfection with rat CYP genes (Doehmer et al., 1988), while the third, which lacked CYP activity, has been cultured in our laboratory for several years (see for example Jenssen and Ramel, 1980; Önfelt and Klasterska, 1983).
The o-substituted biphenyls displayed more cytotoxicity (Figure 1a–c), an effect possibly related to the more prominent enhancement of membrane fluidity with such congeners (Reich et al., 1981). Differences between the cell lines were indicated, but there are arguments against metabolites as the source of these differences. The metabolic conversion of PCBs is expected to be very slow (reviewed by Safe, 1994) and the incubation time applied here was comparatively short. Thus, any metabolites formed would have to be very potent to cause the differences seen (Figures 1 and 2). Additionally, the congener CB-153, expected to be metabolized very slowly, if at all (Mühlebach et al., 1991; Ariyoshi et al., 1997), did cause different survival ratios among cell lines (100 µM, V79 versus SD1, P < 0.05; Figure 1). Thus, other traits acquired during culture and transfection could have been more important for the responses than CYP enzyme content. However, we have not attempted to identify metabolites, which is the only way to elucidate this point.
Of the five PCBs tested only the o-substituted CB-52 and CB-105 were found to induce high frequencies of abnormal mitosis. The low efficacy of CB-77 and CB-126 compared with CB-52 and CB-105 implies that lipophilicity and coplanarity, favouring Ah receptor binding and induction of CYP1A isozymes, are not sufficient or not the most important features for an effect on mitosis. CB-126 has the highest affinity for the Ah receptor and is comparable with B[a]P in this respect (Rannug et al., 1991). c-mitosis was not secondary to toxicity and o-substitution did not appear sufficient to cause induction of c-mitosis. CB-153, o-substituted and more lipophilic than CB-52 and CB-105, showed no increase in mitotic disturbances at equally toxic concentrations (Figures 1a–c and 2a). Further, the cell line showing the highest c-mitotic response also showed the highest survival ratios (Figure 2b–d), suggesting that the formation of c-mitotic configurations was in fact hampered by cytotoxicity.
The very low activity of CB-153 with regard to c-mitosis is not likely to have been due to poor uptake of the compound. Firstly, 24 h incubation gave no response either and, secondly, the amount of CB-105 taken up by the V79 cells corresponded well with the amount of CB-153 taken up by Chang liver cells under similar conditions (Mangelsdorf et al., 1987). Therefore, the difference between CB-52 and CB-153 is more likely to be due to differences in distribution within the cell and inability of CB-153 to either reach or bind to the target(s). A cut-off in the series due to the molecular dimensions of CB-153 cannot be excluded. This in turn would imply a certain degree of selectivity of CB-52 and CB-105.
The difference seen between CB-52 and CB-105 with regard to lowest active concentration cannot be explained as long as the most significant target(s) is unknown, but the displacement of chromosomes and aberrant cell elongation and furrowing observed may be of interest here (Figure 3g and h). Similar effects have been reported to be induced by diethylstilboestrol in human fibroblasts (Danford and Parry, 1982), 7,12-dimethylbenz[a]anthracene (Matsuoka et al., 1998) and carbaryl, 1-naphthol and tyrphostins in V79 cells. The latter three compounds inhibit tyrosine kinase activity in vitro and enhance cellular serine/threonine protein phosphatase activity (Renglin et al., 1999). It could be speculated that one target site for the very lipophilic PCBs could be directly or indirectly related to membrane-bound tyrosine kinase activity.
Synergistic effects of CB-105 and triphenyltin
Exposure to PCBs in the environment is among the highest observed for pollutants (Jones et al., 1992; Safe, 1994; cf. Borlakoglu and Haegele, 1991) but the actual concentration within cells may still not be sufficient for the biological effects observed when using very high doses in experimental systems. A question of general interest is therefore whether they have any subthreshold activity. To test this aspect we combined `chronic' treatment with CB-105 and pulse treatment with triphenyltin in V79 cells lacking CYP activity. Triphenyltin efficiently interferes with microtubule polymerization in the test tube and is an efficient inducer of c-mitosis and aneuploidy at low concentrations in vitro (Jensen et al., 1991a,b). Triphenyltin is also an ionophore and inhibitor of ion translocating ATPases (Selwyn, 1976), making it particularly attractive as a test substance with CB-105; hypothetically triphenyltin could add to the putative effects of CB-105 on the membrane level as well as directly on microtubules.
Combinations of CB-105 and triphenyltin acted synergistically, i.e. the responses were not simply additive (Figure 4). There were also weak, but significant, signs of acquired resistance to CB-105 (compare Figure 4a and b, CB-105 5x10–6 M), but the molecular background of the adaptation can only be speculated on. One possibility could be an altered incorporation of saturated/unsaturated lipids (cf. Borakoglu et al., 1990; Keweloh et al., 1991), antagonizing the negative consequences of intercalation of CB-105 in lipid bilayers (cf. Ingram 1976, 1977). Although CB-105 rendered cells more sensitive to the organometal spindle poison the concentrations of CB-105 and triphenyltin required for an effect must be considered very high in comparison with levels likely to be common in vivo (Manzo et al., 1981; Mussalo-Rauhamaa, 1991). Yet, from a toxicological point of view, this is perhaps the most interesting result of our study.
Conclusions
CB-105 was found to be the most active of the tested congeners and showed high survival ratios concomitant with abnormal spindle function. The poor activity of the coplanar CB-77 and CB-126 indicates that structural characteristics giving affinity for the Ah receptor are of minor importance for induction of c-mitosis. Instead, o-chlorine substitution appears crucial for prominent c-mitotic activity and cytotoxicity, just as it is for a reduction in dye transfer between rat liver WB cells (Hemming et al., 1991), a reduction in dopamine content in PC12 cells (Shain et al., 1991) and binding to glucocorticoid (Schuetz et al., 1986) and oestrogen receptors (Korach et al., 1988; Krishnan and Safe, 1993). The poor response with the o-substituted CB-153 further suggests that CB-52 and CB-105 bind selectively to a target of importance for proper mitotic performance. This appears mainly of theoretical interest, because nominal concentrations required were high in relation to the suggested partition coefficients (6–8; see De Kock and Lord, 1987). In view of the synergism between CB-105 and triphenyltin it seems important to investigate subthreshold activities of highly lipophilic environmental contaminants further. Abnormal chromosomal arrangements in mitosis due to aberrant function of the spindle is a sensitive and easily applied end-point relevant to induction of aneuploidy.
|
Acknowledgments |
|---|
This work was financed in part by the EEC, contract no CT91-0159 (A.Ö.), National Institute for Working Life and the Swedish Environmental Protection Agency. We gratefully acknowledge that the confocal microscope was donated by the Knut and Alice Wallenberg Foundation.
|
Notes |
|---|
3 To whom correspondence should be addressed. Tel: +46 8 162 914; Fax: +46 8 612 4004; Email: onfelt{at}genetics.su.se
4 Present address: Department of Drug Metabolism, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark
|
References |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
-
Allen,J.R. and Abrahamson,L.J. (1979) Responses of rats exposed to polychlorinated biphenyls for 52 weeks. II. Composition and enzymatic changes in the liver. Arch. Environ. Contam. Toxicol., 8, 191–200.[ISI][Medline]
Ariyoshi,N., Koga,N., Oguri,K. and Yoshimura,H. (1992) Metabolism of 2,4,5,2',4',5'-hexachlorobiphenyl with liver microsomes of phenobarbital-treated dog; the possible formation of PCB 2,3-arene oxide intermediate. Xenobiotica, 22, 1275–1290.[ISI][Medline]
Ariyoshi,N., Koga,N., Yoshimura,H. and Oguri,K. (1997) Metabolism of 2,4,5,2',4',5'-hexachlorobiphenyl (PCB153) in guinea pig. Xenobiotica, 27, 973–983.[ISI][Medline]
Ballschmiter,K.H., Mennel,A. and Buyten,J. (1993) Long chain alkyl-polysiloxanes as non-polar stationary phases in capillary gas chromatography. Fres. J. Anal. Chem., 346, 396–402.
Bandiera,S., Safe,S. and Okey,A.B. (1982) Binding of polychlorinated biphenyls classified as either phenobarbitone-, 3-methylcholantrene-, or mixed-type inducers to cytosolic Ah receptor. Chem. Biol. Interact., 39, 259–277.[ISI][Medline]
Bergman,Å., Nilsson,A., Riego,J. and Örn,U. (1990) Synthesis of 14C-labelled and unlabelled coplanar polychlorinated biphenyls (PCBs). Acta Chem. Scand., 44, 1071–1076.
Borlakoglu,J.T. and Haegele,K.D. (1991) Comparative aspects on the bioaccumulation, metabolism and toxicity with PCBs. Comp. Biochem. Physiol., 100C, 327–338.
Borlakoglu,J.T. and Wilkins,J.P.G. (1993) Metabolism of di-, tri-, tetra-, penta- and hexachlorobiphenyls by hepatic microsomes isolated from control animals and animals treated with Arochlor 1254, a commercial mixture of polychlorinated biphenyls. Comp. Biochem. Physiol., 105C, 95–106.
Borlakoglu,J.T., Edwards-Webb,J.D. and Dils,R.R. (1990) Polychlorinated biphenyls increase fatty acid desaturation in the proliferating endoplasmic reticulum of pigeon and rat livers. Eur. J. Biochem., 188, 327–332.[ISI][Medline]
Chaung,W., Mi,L.J. and Boorstein,R. (1997) The p53 status of Chinese hamster V79 cells frequently used for studies on DNA damage and DNA repair. Nucleic Acids Res., 25, 992–994.
Danford,N. and Parry,J.M. (1982) Abnormal cell division in cultured human fibroblasts after exposure to diethylstilboestrol. Mutat. Res., 103, 379–383.[ISI][Medline]
De Kock,A.C. and Lord,D.A. (1987) A simple procedure for determining octanol-water partition coefficients using reverse phase high performance liquid chromatography (RPHPLC). Chemosphere, 16, 133–142.
Doehmer,J., Dogra,S., Friedberg,T., Monier,S., Adesnik,M., Glatt,H. and Oesch,F. (1988) Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1. Proc. Natl Acad. Sci. USA, 85, 5769–5773.
Dogra,S., Doehmer,J., Glatt,H., Mölders,H., Siegert,P., Friedberg,T., Seidel,A. and Oesch,F. (1990) Stable expression of rat cytochrome P-450IA1 cDNA in V79 Chinese hamster cells and their use in mutagenicity testing. Mol. Pharmacol., 37, 608–613.[Abstract]
Ellard,S., Mohammed,Y., Dogra,S., Wolfel,C., Doehmer,J. and Parry,J.M. (1991) The use of genetically engineered V79 Chinese hamster cultures expressing rat liver CYP1A1, 1A2 and 2B1 cDNAs in micronucleus assays. Mutagenesis, 6, 461–470.
Hawker,D.W. and Connell,D.W. (1988) Octanol-water partition coefficients of polychlorinated biphenyl congeners. Environ. Sci. Technol., 22, 382–387.
Hemming,H., Wärngård,L. and Ahlborg,U.G. (1991) Inhibition of transfer in rat liver WB cell culture by polychlorinated biphenyls. Pharmacol. Toxicol., 69, 416–420.[ISI][Medline]
Ingram,L.O. (1976) Adaptation of membrane lipids to alcohols. J. Bacteriol., 125, 670–678.
Ingram,L.O. (1977) Changes in lipid composition of Escherichia coli resulting from growth with organic solvents and food additives. Appl. Environ. Microbiol., 33, 1233–1236.
Jensen,K.G., Andersen,O. and Rønne,M. (1991a) Organotin compounds induce aneuploidy in human peripheral lymphocytes in vitro. Mutat. Res., 246, 109–112.[ISI][Medline]
Jensen,K.G., Önfelt,A., Wallin,M., Lidums,V. and Andersen,O. (1991b) Effects of organotin compounds on mitosis, spindle structure, toxicity and in vitro microtubule assembly. Mutagenesis, 6, 409–416.
Jensen,K.G., Önfelt,A., Poulsen,H.E., Doehmer,J. and Loft,S. (1993) Effects of benzo[a]pyrene and (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene on mitosis in Chinese hamster V79 cells with stable expression of rat cytochrome P4501A1 or 1A2. Carcinogenesis, 14, 2115–2118.
Jensen,S. and Sundström,G. (1974) Metabolic hydroxylation of a chlorobiphenyl containing only isolated unsubstituted positions—2,2',4,4',5,5'-hexachlorobiphenyl. Nature, 251, 219–220.[Medline]
Jenssen,D. and Ramel,C. (1980) Relationship between chemical damage of DNA and mutations in mammalian cells. I. Dose-response curves for the induction of 6-thioguanine-resistant mutants by low doses of monofunctional alkylating agents, X-ray and UV radiation in V79 Chinese hamster cells. Mutat. Res., 73, 339–347.[ISI][Medline]
Jones,K.C., Sanders,G., Wilds,S.R., Burnett,V. and Johnston,A.E. (1992) Evidence for a decline of PCBs and PAHs in rural vegetation and air in the United Kingdom. Nature, 356, 137–140.
Keweloh,H., Diefenbach,R. and Rehm,H.-J. (1991) Increase of phenol tolerance of Escherichia coli by alterations of the fatty acid composition of the membrane lipids. Arch. Microbiol., 157, 49–53.[ISI][Medline]
Klasson Wehler,E., Bergman,Å., Darnerud,P.O. and Wachtmeister,C.A. (1989) 3,3',4,4'-Tetrachlorobiphenyl: excretion and tissue retention of hydroxylated metabolites in the mouse. Drug Metab. Dispos., 17, 441–448.[Abstract]
Klasson Wehler,E., Lindberg,L., Jönsson,C.-J. and Bergman,Å. (1993) Tissue retention and metabolism of 2,3,4,3',4'-pentachlorobiphenyl. Chemosphere, 27, 2397–2412.
Korach,K.S., Sarver,P., Chae,K., McLachlan,J.A. and McKinney,J.D. (1988) Estrogen receptor-binding activity of polychlorinated hydroxybiphenyls: conformationally restricted structural probes. Mol. Pharmacol., 33, 120–126.[Abstract]
Koga,N., Beppu,M. and Yoshimura,H. (1990) Metabolism in vivo of 3,4,3',4',5-pentachlorobiphenyl and toxicological assessment of the metabolite in rats. J. Pharmaco-Bio Dyn., 13, 499–506.
Krishnan,V. and Safe,S. (1993) Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) as antioestrogens in MCF-7 human breast cancer cells: quantitative structure-activity relationships. Toxicol. Appl. Pharmacol., 120, 55–61.[ISI][Medline]
Mangelsdorf,I., Buff,K. and Berndt,J. (1987) Uptake of persistent environmental chemicals by cultured human cells. Biochem. Pharmacol., 36, 2071–2078.[ISI][Medline]
Manzo,L., Richelmi,P., Sabbioni,E., Pietra,R., Bono,F. and Guardia,L. (1981) Poisoning by triphenyltin acetate. Report of two cases and determination of tin in blood and urine by neutrone activation analysis. Clin. Toxicol., 18, 1343–1353.[ISI][Medline]
Matsuoka,A., Matsuura,K., Sakamoto,H., Hayashi,M. and Sofuni,T. (1998) Spindle disturbances induced by benzo[a]pyrene and 7,12-dimethylbenz[a]antracene in a Chinese hamster cell line (V79-MZ) and the stability of the numerical chromosome aberrations that follow. Mutat. Res., 419, 1–12.[ISI][Medline]
Mussalo-Rauhamaa,H. (1991) Partitioning and levels of neutral organochlorine compounds in human serum, blood cells and adipose and liver tissue. Sci. Total Environ., 103, 159–175.[Medline]
Mühlebach,S., Wyss,P.A. and Bickel,M.H. (1991) The use of 2,4,5,2',4',5'-(6-CB) as an unmetabolizable lipophilic compound. Pharmacol. Toxicol., 69, 410–415.[ISI][Medline]
Nelson,D.R., Kamataki,T., Waxman,D.J., Guengerich,F.P., Estabrook,R.W., Feyereisen,R., Gonzalez,F.J., Coon,M.J., Gunsalus,I.C., Gotoh,O., Okuda,K. and Nebert,D. (1993) The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes and nomenclature. DNA Cell Biol., 12, 1–51.[ISI][Medline]
Önfelt,A. (1987) Spindle disturbances in mammalian cells III. Toxicity, c-mitosis and aneuploidy with 22 different compounds. Specific and unspecific mechanisms. Mutat. Res., 182, 135–154.[ISI][Medline]
Önfelt,A. and Klasterska,I. (1983) Spindle disturbances in mammalian cells. II. Induction of viable aneuploid/polyploid cells and multiple chromatid exchanges after treatment of V79 Chinese hamster cells with carbaryl. Mutat. Res., 119, 319–330.[ISI][Medline]
Rannug,U., Sjögren,M., Rannug,A., Gillner,M., Toftgård,R., Gustafsson,J.-Å., Rosenkranz,H. and Klopman,G. (1991) Use of artificial intelligence in structure–affinity correlations of 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) receptor ligands. Carcinogenesis, 12, 2007–2015.
Rapaport,R.A. and Eisenriech,S.J. (1984) Chromatographic partition coefficients (Kow's) for 58 polychlorinated biphenyl congeners. Environ. Sci. Technol., 18, 163–170.
Reich,T., Depew,M.C., Marks,G.S., Singer,M.A. and Wan,J.K.S. (1981) Effect of polychlorinated biphenyls (PCBs) on phospholipid membrane fluidity. J. Environ. Sci. Health, A16, 65–71.
Renglin,A., Hämäla-Brasken,A.-S., Eriksson,J.E. and Önfelt,A. (1999) Mitotic aberrations induced by carbaryl reflect tyrosine kinase inhibition with coincident upregulation of serine/threonine protein phosphatase activity—implications for coordination of karyokinesis and cytokinesis. Mutagenesis, 14, 327–333.
Safe,S.H. (1994) Polychlorinated biphenyls (PCBs): environmental impact, biochemical and toxic responses and implications for risk assessment. Crit. Rev. Toxicol., 24, 87–149.[ISI][Medline]
Schuetz,E.G., Wrighton,S.A., Safe,S.H. and Guzelian,P.S. (1986) Regulation of cytochrome P-450p by phenobarbital and phenobarbital-like inducers in adult rat hepatocytes in primary monolayer culture and in vivo. Biochemistry, 25, 1124–1133.[Medline]
Selwyn,M.J. (1976) Triorganotin compounds as ionophores and inhibitors of ion translocating ATPases. In Zuckerman,J.J. (ed.) Organotin Compounds: New Chemistry and Applications. American Chemical Society, Washington, DC, pp. 204–226.
Shain,W., Bush,B. and Seegal,R. (1991) Neurotoxicity of polychlorinated biphenyls: structure-activity relationship of individual congeners. Toxicol. Appl. Pharmacol., 111, 33–42.[ISI][Medline]
Sundström,G. (1973) Polychlorinated biphenyls II. Synthesis of some tetra- and pentachlorobiphenyls. Acta Chem. Scand., 27, 600–604.
Wortelboer,H.M., Kruif,C.A.D. and van Iersel,A.A.J. (1990) The isoenzyme pattern of cytochrome P450 in rat hepatocytes in primary culture, comparing different enzyme activities in microsomal incubations and in intact monolayers. Biochem. Pharmacol., 40, 2525–2534.[ISI][Medline]
Received on February 17, 1999; accepted on September 10, 1999.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  K.-J. Shin, S.-H. Kim, D. Kim, Y.-H. Kim, H.-W. Lee, Y.-S. Chang, M.-B. Gu, S. H. Ryu, and P.-G. Suh 2,2',4,6,6'-Pentachlorobiphenyl Induces Mitotic Arrest and p53 Activation Toxicol. Sci., April 1, 2004; 78(2): 215 - 221. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Desaulniers, K. Leingartner, B. Musicki, A. Yagminas, G.-H. Xiao, J. Cole, L. Marro, M. Charbonneau, and B. K. Tsang Effects of Postnatal Exposure to Mixtures of Non-ortho-PCBs, PCDDs, and PCDFs in Prepubertal Female Rats Toxicol. Sci., October 1, 2003; 75(2): 468 - 480. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||