Mutagenicity of ptaquiloside, the carcinogen in bracken, and its related illudane-type sesquiterpenes II. Chromosomal aberration tests with cultured mammalian cells

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on ptaquiloside and its related compounds, hypoloside B, hypoloside C, illudin M and illudin S. Ptaquiloside induced chromosomal aberrations at doses as low as 4.5 μg/ml (0.0113 mM). The clastogenic effect was ph-dependent. The same activity was observed at a 90-fold higher dose at pH 5.3 in the culture medium compared with the activity at pH 74. or pH 8.0. Both hypoloside B and hypoloside C were also clastogenic at almost the same dose levels as that of ptaquiloside. Illudin M and illudin S were also potet clastogens and induced aberrations at much lower doses than ptaquiloside. These results suggest that the clastogenic effect is involved in the mechanism of carcinogenic potency of ptaquiloside in animals.     

Evaluation of clastogenicity of formic acid, acetic acid and lactic acid on cultured mammalian cells

Using Chinese hamster ovary K1 cells, chromosomal aberration tests were carried out with formic acid, acetic acid and lactic acid, and the relationship between the pH of the medium and the clastogenic activity was examined. The medium used was Ham's F12 supplemented with 17 mM NaHCO3 and 10% fetal calf serum. All of these acids induced chromosomal aberrations at the initial pH of ca. 6.0 or below (about 10-14 mM of each acid) both with and without S9 mix. Exposure of cells to about pH 5.7 or below (about 12-16 mM of each acid) was found to be toxic. When the culture medium was first acidified with each of these acids and then neutralized to pH 6.4 or pH 7.2 with NaOH, no clastogenic activity was observed. Using F12 medium supplemented with 34 mM NaHCO3 as a buffer, no clastogenic activity was observed at doses up to 25 mM of these acids (initial pH 5.8-6.0). However, it was found that about 10% of the cells had aberrations at pH 5.7 or below (27.5-32.5 mM of each acid). Furthermore, when 30 mM HEPES was used as a buffer, chromosomal aberrations were not induced at doses up to 20 mM formic acid and acetic acid (initial pH 7.0-7.1), and at doses up to 30 mM lactic acid (initial pH 6.6). In the initial pH range of 6.4-6.7 (25-32.5 mM of each acid), chromosomal aberrations were observed. The above results show that these acids themselves are non-clastogenic, and the pseudo-positive reactions attributable to non-physiological pH could be eliminated by either neutralization of the treatment medium or enhancement of the buffering ability.     

Effect of pH on the activity and stability of clastogens in the in vitro chromosomal aberration test with Chinese hamster ovary K1 cells

The effect of the pH of the medium on the clastogenic activity of several direct-acting and indirect clastogens was evaluated in the in vitro chromosomal aberration test with Chinese hamster ovary K1 cells. Furthermore, the stability of the chemicals in the cell culture medium was measured by HPLC over the pH range of 5.0-11.0. The activity of the direct-acting clastogens mitomycin C (MMC), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and 4-nitroquinoline-1-oxide (4NQO) at various pH values depended on their stability. In the case of ENNG, its clastogenic activity decreased to about one-fifth at pH 9 but was about twice as high at acidic pH compared with that at pH 7.4. This is consistent with the observation that ENNG is unstable at basic pH; the residual content of ENNG was 0.5% of the initial amount in cell culture medium at pH 9.0 after a 2-h incubation. 4NQO was unstable at strongly basic pH (pH 10-11), and MMC was unstable at pH 5.0 and pH 11.0. The frequencies of chromosomal aberrations induced by MMC and ENNG were correspondingly decreased at these pH values. On the other hand, the clastogenicities of the indirect clastogens 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (B(a)P) and dimethylnitrosamine (DMN), which require metabolic activation, were reduced at pH 10-11 and pH 5.8. The frequencies of chromosomal aberrations at these pHs were almost equal to negative control values. These chemicals were stable in the medium in the absence of S9 mix over the pH range of 5.0-11.0. Thus clastogenicity of indirect-acting clastogens is reduced under extreme pH conditions, probably because of the instability or nonformation of the active form. The present results indicate that the clastogenic activity of any compound will depend on its stability in the medium irrespective of its direct- or indirect-acting nature. In addition, some of the chemicals that are recognized as clastogens presumably might induce chromosomal aberrations by means of acidic pH itself. It is, therefore, important to take account of the pH of the treatment medium in evaluating the clastogenicity of chemicals.     

Butachlor is cytotoxic and clastogenic and induces apoptosis in mammalian cells

The ability of butachlor to induce cytotoxicity, clastogenicity and DNA damage was assessed using Chinese hamster ovary cells (CHO), Swiss mouse embryo fibroblasts (MEF) and human peripheral blood lymphocytes. A dose and time dependent loss of viability was evident upon treatment of CHO cells with butachlor. Cell killing to an extent of 50% was observed when cells were treated with 16.2 micrograms/ml of butachlor for 24 hr or with 11.5 micrograms/ml for 48 hr. The herbicide induced micronuclei significantly in cultured lymphocytes at 24 and 48 hr of treatment suggesting that it is clastogenic. To understand the mechanism of cell death caused by butachlor, its effect on DNA strand breaks was studied in MEF. A concomitant decrease in cell viability was observed with increase in DNA strand breaks. Agarose gel electrophoresis of DNA from herbicide treated CHO cells and cytochemical staining indicate the induction of apoptosis by butachlor.     

Effects of pH in the in vitro chromosomal aberration test

The relation between the pH of the medium and clastogenic activity was studied in Chinese hamster ovary (CHO) K1 cells in vitro. The pH was adjusted with NaOH, KOH, HCl or H2SO4. No clastogenic activity was observed over the initial pH range of 7.3-10.9 without S9 mix, but a few chromosomal aberrations were induced at pH 10.4 with S9 mix. The frequency of aberrations increased with the increase in amount of S9. At acidic pH, many chromatid breaks were induced at initiatial pH 5.5 or below without S9 mix, and aberrations such as chromatid breaks and chromatid exchanges were induced at initial pH 6.2 or below with S9 mix. Using MES and Bis-Tris as buffers instead of sodium bicarbonate, we observed that aberrations of the chromatid break type were inducible at pH 6.2 or below. These results show that the combination of strong alkalinity and S9 is clastogenic to CHO-K1 cells, and also that weakly acidic media are genetically active. The results indicate that incubations at non-physiological pH might give false-positive responses.     

Salsolinol, a naturally occurring tetrahydroisoquinoline alkaloid, induces DNA damage and chromosomal aberrations in cultured Chinese hamster lung fibroblast cells

Salsolinol (SAL) is a tetrahydroisoquinoline neurotoxin that has been speculated to contribute to pathophysiology of Parkinson's disease and chronic alcoholism. The compound is also found in certain beverages and food stuffs, including soy sauce, beer and bananas. Despite potential human exposure to SAL and its endogenous formation, little is known about the genotoxic or carcinogenic potential of this substance. In the present investigation, SAL induced DNA damage in cultured Chinese hamster lung (CHL) fibroblasts as assessed by single cell gel electrophoresis (Comet). CHL cells treated with SAL also exhibited higher frequencies of chromosomal aberrations than did vehicle-treated controls. Our recent study has revealed that SAL in combination with Cu(II) causes the strand scission in phiX174 supercoiled DNA [Neurosci. Lett. 238 (1997) 95]. In line with this notion, addition of cupric ion potentiated the DNA damaging and clastogenic activity of SAL. Antioxidant vitamins, such as Vitamin C and Vitamin E, and reduced glutathione inhibited clastogenicity of SAL, suggesting the involvement of reactive oxygen species (ROS) in SAL-induced DNA damage and genotoxicity in CHL cells.     

Low pH leads to sister-chromatid exchanges and chromosomal aberrations,and its clastogenicity is S-dependent

The effect of low pH on sister-chromatid exchanges (SCE), chromosomal aberrations (CA), and the cell cycle were investigated in Chinese hamster cells. The cells were treated in media over the pH range 7.2–5.4 during 24-h continuous or 3-h pulse treatments. In Chinese hamster ovary K1 cells, slight increases in SCE frequency were induced by 3-h pulse treatment with a 28-h recovery time. In Chinese hamster V79 379A cells, similar slight increases in SCE frequency were observed with both treatments. A severe delay in the cell cycle was noted in both cell types. DNA analysis with flow cytometry indicated that the cell cycle delay occured in S phase. CA were observed in the first metaphase. Multiple fixation times over a 27-h period were used to determine whether or not CA could be induced in cells exposed to low pH medium in more than one part of the cell cycle. Only a few chromatid gaps were induced when the cells were fixed at 0–9 h after the 3-h treatment, most probably representing cells that were treated in their G2 or late S phase. CA were induced in cells fixed between 12 and 27 h after the 3-h treatment. These cells were most probably treated in early S phase, in G1, or in the previous G2/M. These results suggest that low pH clastogenicity is S-dependent.     

Sodium fluoride-induced chromosome aberrations in different stages of the cell cycle: a proposed mechanism

In an attempt to clarify the controversy about sodium fluoride (NaF) clastogenicity, the induction of chromosome aberrations in Chinese hamster ovary cells (CHO) by NaF was investigated. Following a protocol used for screening chemicals for clastogenic activity, significant increases of aberrant cells were observed when cells were exposed to NaF for 4 h and harvested 8 h later. Cell-cycle kinetic studies demonstrated most cells were exposed in G2 of the cell cycle. Smaller increases in aberrant cells were observed when cells were harvested 20 h later (most cells were exposed in G1/S). The sensitivity of G2 cells to NaF was investigated further, along with the induction of aberrations at low doses. The results indicated that G2 cells are sensitive to NaF and the percent of aberrant cells increased with dose and length of exposure. With a 3-h exposure until harvest, no statistically significant increase in aberrant cells was observed at doses below 10 micrograms/ml NaF. These data are consistent with a threshold for NaF-induced clastogenicity around 10 micrograms/ml, as has been proposed previously (Scott and Roberts, 1987). It thus may be predicted that clastogenic effects would not occur in humans exposed to the levels of fluoride that are present in drinking water or dentifrices. An understanding of the mechanism of NaF-induced clastogenicity would help to clarify this point. It has previously been reported that NaF inhibits DNA synthesis/repair. The types of aberrations, mostly deletions and gaps, the induction of endoreduplicated cells, the cell-cycle delay and the sensitivity of G2 cells to NaF observed are similar to that reported in the literature for DNA synthesis/repair inhibitors like aphidicolin (APC). Similarities in the induction of aberrations by NaF and APC were confirmed in experiments with G2 cells. Based on these results and those previously reported for NaF and APC, it is proposed that NaF-induced aberrations may occur by an indirect mechanism involving the inhibition of DNA synthesis/repair.     

PROPHASING OF INTERPHASE NUCLEI AND INDUCTION OF NUCLEAR ENVELOPES AROUND METAPHASE CHROMOSOMES IN HELA AND CHINESE HAMSTER HOMO- AND HETEROKARYONS

Fusing human HeLa metaphase cells with HeLa interphase cells resulted within 30 min in either of two phenomena in the resultant binucleate cell: either prophasing of the interphase nucleus or formation of a normal-appearing nuclear envelope around the metaphase chromosomes. The frequency of either occurrence was strongly dependent on environmental pH. At pH's of 6.6–8.0, prophasing predominated; at pH 8.5 nuclear envelope formation predominated. Additionally, the frequencies of the two events in multinucleate cells depended on the metaphase/interphase ratio. When the ratio was 0.33 nuclear envelope formation predominated; when it was 2.0 prophasing predominated. In their general features, the results with fused HeLa cells resembled those reported earlier with fused Chinese hamster Don cells. However, the results provided an indication that between pH 6.6 and 8.0 the HeLa metaphase cells possessed a much greater capacity than the Don metaphase cells to induce prophasing. Fusion of Don metaphase cells with HeLa interphase cells or of Don interphase cells with HeLa metaphase cells at pH 8.0 resulted in nuclear envelope formation or prophasing in each kind of heterokaryon. As in the homokaryons, the frequencies of the two events in the heterokaryons depended on the metaphase/interphase ratio. The statistics of prophasing and nuclear envelope formation in the homo- and heterokaryon populations were consistent with the notion that disruption or formation of the nuclear envelope depends on the balance attained between disruptive and formative processes.     

Clastogenic agents in the urine of coffee drinkers and cigarette smokers

Organic material from the urine of smokers, coffee drinkers, and controls was extracted and separated into 3 fractions of differing hydrophobicity using preparative reversed-phase high-pressure liquid chromatography. Fractions were assayed for clastogenic activity using Chinese hamster ovary cells. Smoking, coffee drinking, or both habits together resulted in a substantial increase in the genotoxicity of organic material in all 3 fractions. The clastogenicity of fractions 1 and 2 (the two most hydrophilic) was abolished by the addition of either catalase or superoxide dismutase to the Chinese hamster ovary cell system, suggesting the involvement of active oxygen species in the clastogenic response. Clastogenicity of fraction 3, however, was resistant to the action of catalase and superoxide dismutase. Fractions were tested for their ability to generate hydrogen peroxide in vitro during a 10-min incubation at elevated pH. Fractions 2 and 3, but not fraction 1 from smokers, coffee drinkers, or those with both habits generated significantly more hydrogen peroxide at high pH than did the corresponding fractions from control subjects. For fractions 2 and 3 but not 1, the ability of a sample to generate hydrogen peroxide at high pH was positively correlated with its ability to generate chromosome aberrations at neutral pH in tissue culture. The data indicate that both coffee drinking and cigarette smoking result in the appearance of clastogenic materials in urine, and suggest that such clastogenic agents may produce chromosome aberrations via the production of active oxygen species.     

Mutagenicity of 3-tert-butyl-4-hydroxyanisole (BHA) and its metabolites in short-term tests in vitro

The mutagenicity of 3-tert-butyl-4-hydroxyanisole (BHA) and its metabolites was investigated in the reverse mutation assay using S. typhimurium strains and the chromosomal aberration test in vitro using a Chinese hamster fibroblast cell line, CHL. BHA, tert-butylhydroquinone (BHQ), tert-butylquinone (BQ) and BHA dimer (diBHA) did not show any mutagenic potential with and without S9 mix in the reverse mutation assay. In addition to the above 4 chemicals, 3-tert-butyl-4,5-dihydroxyanisole (BHA-OH), 3-tert-butylanisole-4,5-quinone (BHA-o-Q), and tert-butylquinone oxide (BQO) were tested in the chromosomal aberration test. BHA, BHQ and BQ induced chromosomal aberrations only in the presence of S9 mix, while BHA-OH, BHA-o-Q and BQO induced chromosomal aberrations only without S9 mix. DiBHA, however, showed no clastogenic potential with and without S9 mix. The present findings suggest that BHA-OH, BHA-o-Q or BQO may contribute to the clastogenicity of BHA in the presence of S9 mix.     

Decreased clastogenicity of dinitropyrenes in Chinese hamster lung (CHL) subclone cells with low NADPH-cytochrome P-450 reductase activity

Clastogenic potentials of 1,3-, 1,6- and 1,8-dinitropyrenes (DNPs) were compared between Chinese hamster lung (CHL) cells and its subclone MM1 cells, which were recently isolated as menadione-resistant cells after treatment with MNNG. NADPH-cytochrome P-450 reductase activity of the MM1 cells decreased to 50% of that in the parental CHL cells. All 3 DNPs induced chromosomal aberrations without exogenous metabolic activation systems in the CHL cells. 1,6- and 1,8-DNP showed equivalent clastogenic potency: the maximum frequency of cells with chromosomal aberrations was about 50% for both chemicals. The clastogenic potential of 1,3-DNP was lower than that of 1,6- and 1,8-DNP: the maximum frequency of aberrant cells was 10%. In the MM1 cells, in contrast, the frequencies of aberrant cells decreased to about 30% of those observed for the parental CHL cells after treatment with 1,6- and 1,8-DNP, and to the same level as that of the concurrent control after treatment with 1,3-DNP. These results suggest a possibility that the reduced clastogenic effect of 3 DNPs in MM1 cells may correlate with the reduced activity of NADPH-cytochrome P-450 reductase which is thought to contribute to the metabolic conversion of these DNPs to their clastogenic forms in the CHL cells.     

Clastogenic and Mutagenic Actions of Active Species Generated in the 6-Hydroxydopamine/Oxygen Reaction: Effects of Scavengers of Active Oxygen, Iron, and Metal Chelating Agents

A pro-oxidant triphenol, 6-hydroxydopamine (6-OHDA), induced mutations in the Salmonella typhimurium TA104 tester strain (over the concentration range to 800/JM), and induced chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells at lower concentrations (up to 90 μM). It was however only marginally mutagenic (up to cytotoxic levels of 200 μM) in the TA102 tester strain. Clastogenicity in the more sensitive CHO cell assay was mediated by activated oxygen. Superoxide dismutase decreased the incidence of chromosomal aberrations by 60% and catalase (or superoxide dismutase plus catalase) decreased the incidence to control levels. The clastogenicity of 6-OHDA was dependent upon unsequestered transition metal ions, since addition of EDTA plus desferoxamine decreased chromosomal aberrations by 90%. The simplest explanation of the data is that genotoxicity is mediated by active species generated in a Fenton-type reaction between 6-OHDA and H₂O₂ catalyzed by traces of metals in the medium.     

Clastogenicity, photo-clastogenicity or pseudo-photo-clastogenicity: Genotoxic effects of zinc oxide in the dark, in pre-irradiated or simultaneously irradiated Chinese hamster ovary cells

Zinc oxide (ZnO), a widely used ingredient in dermatological preparations and sunscreens, is clastogenic in vitro, but not in vivo. Given that ZnO has an approximately four-fold greater clastogenic potency in the presence of UV light when compared with that in the dark, it has been suggested to be photo-clastogenic. In order to clarify whether this increased potency is a genuine photo-genotoxic effect, we investigated the clastogenicity of ZnO (mean particle size, 100 nm) in Chinese hamster ovary (CHO) cells in the dark (D), in pre-irradiated (PI, i.e. UV irradiation of cells followed by treatment with ZnO) and in simultaneously irradiated (SI, i.e. ZnO treatment concurrent with UV irradiation) CHO cells at UV doses of 350 and 700 mJ/cm(2). The cytotoxicity of ZnO to CHO cells under the different irradiation conditions was as follows: SI>PI>D. In the dark, ZnO produced a concentration-related increase in chromosome aberrations (CA). In PI or SI CHO cells, ZnO was clastogenic at significantly lower concentrations (approximately two- to four-fold) when compared with effective concentrations in the dark, indicating an increased susceptibility of CHO cells to ZnO-mediated clastogenic effects due to UV irradiation per se. The incidence of CA in SI or PI cells was generally higher than that in the dark. At similar ZnO concentrations, SI conditions generally produced higher CA incidence than PI conditions. However, when ZnO concentrations producing similar cytotoxicity were compared, CA incidences under PI or SI conditions were nearly identical. The modest increase in the clastogenic potency of ZnO following UV irradiation contrasts with the results observed with genuine photo-clastogenic agents, such as 8-MOP, which may produce an increase in clastogenic potency of >15,000-fold under SI conditions. Our results provide evidence that, under conditions of in vitro photo-clastogenicity tests, UV irradiation of the cellular test system per se may produce a slight increase in the genotoxic potency of compounds that are clastogenic in the dark. In conclusion, our data suggest that minor increases in clastogenic potency under conditions of photo-genotoxicity testing do not necessarily represent a photo-genotoxic effect, but may occur due to an increased sensitivity of the test system subsequent to UV irradiation.     

Concurrent detection of gene mutations and chromosome aberrations induced by five chemicals in a CHL/IU cell line incorporating a gpt shuttle vector

We previously established a transgenic Chinese hamster CHL/IU cell line, designated as KN63, for concurrent analysis of gene mutations and chromosome aberrations. The KN63 cell line contains copies of a shuttle vector with the Escherichia coli gpt gene as a mutational target in its chromosome. To evaluate the sensitivity of the cell line to various types of mutagens, methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU), mitomycin C (MMC), vincristine sulfate (VIN) and C.I. basic red 9 hydrochloride (CIB) were assayed. KN63 cells were treated with each test chemical and gene mutations were detected in the gpt gene of the shuttle vector rescued from the KN63 cell genome into an E. coli host. Chromosome aberrations were concurrently evaluated by conventional metaphase analysis. MMS, ENU and MMC induced both gene mutations and structural chromosome aberrations in KN63 cells, with more efficient induction of the latter. VIN, a well-known aneugen, produced only numerical changes to chromosomes, while CIB was negative for both types of alteration. KN63 cells were as sensitive to MMS, ENU, MMC and VIN as Chinese hamster cell lines such as CHL, CHO and V79 cells. The characteristics of test chemicals indicated by this system should be useful for understanding endpoints in chemical mutagenesis.     

In vitro photochemical clastogenicity of quinolone antibacterial agents studied by a chromosomal aberration test with light irradiation

The photochemical clastogenic potential of 12 quinolone antibacterial agents with or without light irradiation was assessed by an in vitro chromosomal aberration test using cultured CHL cells. Exposure to all test compounds, except for DK-507k, increased the incidence of cells with structural aberrations excluding gap (TA) following light irradiation. Test compounds used in the present study under light irradiation were divided into three groups based on their ED(50) values, doses inducing chromosomal aberrations in 50% of cells. The first group with ED(50) values below 30 microg/ml includes sparfloxacin (SPFX), clinafloxacin (CLFX), gemifloxacin (GMFX), lomefloxacin (LFLX), sitafloxacin (STFX), grepafloxacin (GPFX) and fleroxacin (FLRX); the second group with ED(50) values of 100 microg/ml, enoxacin (ENX) and levofloxacin (LVFX); the third group with little or no potency, moxifloxacin (MFLX), trovafloxacin (TVFX) and DK-507k. The photochemical clastogenicity of these compounds correlates well with their reported in vivo phototoxic potentials. In the chemical structure and clastogenicity relationships, substitution of a methoxy group at the C-8 position in the quinolone nucleus was confirmed to reduce not only photochemical clastogenicity, but also the clastogenic potential of quinolone antibacterial agents.     

Pesticide clastogenicity in Chinese hamster ovary cells

Paraquat, alachlor, butachlor, phorate and monocrotophos, several of the most extensively used pesticides in Taiwan, were investigated for their clastogenicity using chromosome aberration (CAb) induction in Chinese hamster ovary (CHO) cells. Significance levels of the binomial trend analysis and binomial mutagenicity data test were two criteria for the summary judgement of the pesticide clastogenicity. Except for phorate, all pesticides tested were clastogenic to CHO cells in the absence of in vitro metabolic activation by S9. 5 microliters/ml rat-liver extract, S9, were used as the source of in vitro metabolic activation. 3 different outcomes were found after the addition of S9. Paraquat: significant decrease in induced CAbs. Monocrotophos: concomitant occurrence of decreased cytotoxicity and increased clastogenicity. Alachlor, butachlor and phorate: increased cytotoxicities with no sign of enhancement in clastogenicity.     

Comparative analysis of the clastogenicity and cytotoxicity of airborne particulate matter generated during the fire at Schweizerhalle on November 1, 1986

Methanol extracts from 4 pairs of airconditioner filters (one fire-exposed and one control) from various locations (A, B, C and D) at various distances from the site of the fire were examined for their capacity to induce structural chromosomal aberrations and/or cytotoxicity in Chinese hamster V79 cells. Extracts from 2 additional sets of 3 filters which were exposed to urban air for 3 consecutive periods of 10 or 11 days some 4 months after the fire were also tested. Chromosomal aberrations were induced by all filter extracts from location B, as well as by an unused (non-exposed) filter, in a dose-dependent manner. Without the addition of metabolizing enzymes, aberrations were induced only at concentrations which caused more than 95% cell killing. This was not taken as an indication for clastogenic activity of the filter extracts, but was assumed to represent the chromosomal expression of metabolic changes in dying cells. Upon the addition of S9, chromosomal aberrations were induced at biologically relevant survival rates. Under metabolizing conditions, the ranking of the potential of the filter extracts from location B to induce chromosomal aberrations and to cause cell killing was identical. The remaining extracts (locations A, C and D) were therefore tested for cytotoxicity only. The toxicity data indicated that, of 3 pairs of filters, the fire-exposed one was not different from the control. Of the fourth pair (location B), the fire-exposed filter was 2.0-2.5 times more cytotoxic and clastogenic than the control. However, extracts of urban air-exposed filters from this location (exposed in March and April 1987) showed a large variation in toxicity and clastogenicity as well. One was clearly more active than the control (but less than the fire-exposed filter), while the other 2 were either somewhat more or less clastogenic than the control filter. In addition, 4 out of 5 filters from this location were more polluted (as indicated by cytotoxicity) than all the filters from the other locations, irrespective of whether they were fire-exposed or not. It is concluded that the results of this V79 cytotoxicity/clastogenicity test did not confirm the hypothesis that the fire at Schweizerhalle produced clastogenic material at quantities detectable under the conditions employed.     

CONTRAST BETWEEN THE ENVIRONMENTAL pH DEPENDENCIES OF PROPHASING AND NUCLEAR MEMBRANE FORMATION IN INTERPHASE-METAPHASE CELLS

In Chinese hamster Don cells, fusion of an interphase cell with a metaphase cell resulted either in prophasing of the interphase nucleus, including loss of the nuclear envelope (NE), or in the formation of a double membrane around the metaphase chromosomes. Only one of these phenomena occurred in a given interphase-metaphase (I–M) binucleate cell. At pH 7.4, there was about an equal probability that either event could occur amongst the population of I–M cells. The effect of pH changes in the medium containing the fused cells was examined. At pH 6.6, prophasing was the predominant event; at pH 8.0, membrane formation predominated. It was found that the rate of progression of a mononucleate cell from G₂ to metaphase was appreciably faster at pH 6.6 than at pH 8.0. Conversely, the progression from metaphase to G₁ was faster at pH 8.0 than at pH 6.6. These results with the mononucleate cells strengthen the hypothesis that structural changes in I–M cells are reflections of normal mitotic phenomena. Additional evidence for this hypothesis was produced by electron microscope examination after direct fixation in chrom-osmium. The double membrane around the chromosomes of the I–M cell was indistinguishable from the normal NE. The results obtained by varying the pH of the medium containing the fused cells provide an indication that disruption or formation of the NE of Don cells depends on the balance reached between disruptive and formative processes.     

Microsome-mediated clastogenicity of butylated hydroxyanisole (BHA) in cultured Chinese hamster ovary cells: the possible role of reactive oxygen species

Butylated hydroxyanisole (BHA) was found to induce chromosome aberrations in Chinese hamster ovary (CHO) cells in the presence of Aroclor-induced rat-liver S9. The effects were more marked when washed microsomes were employed and chromosome damage was considerably reduced in the presence of catalase, suggesting that hydrogen peroxide was involved. Stimulation of H2O2 production by BHA in S9 or microsome incubation mixtures was demonstrated using the catalase-mediated production of formaldehyde from methanol. One of the major microsomal metabolites of BHA, tert.-butyl hydroquinone (t-BHQ), which autoxidises in solution producing H2O2 also induced extensive catalase-sensitive chromosome damage in the absence of metabolic activation. These observations suggest that extracellular generation of reactive oxygen species may be implicated in the mechanism of BHA clastogenicity in vitro. However, chromosome damage was not completely abolished by catalase and the end product of t-BHQ oxidation, tert.-butyl quinone, was also weakly clastogenic, suggesting that intracellular effects of quinone metabolites may also be involved in the clastogenicity of BHA.