Estrogen effects on sister chromatid exchanges in mouse uterine cervical and kidney cells

Uterine cervical fibroblasts and kidney cells from 3-to 4-day-old, previously untreated or phenobarbital [(PBA) CAS: 50-06-6]-treated female NMRI mice were cultured in vitro in the presence of diethylstilbestrol [(DES) CAS: 56-53-1] or 17 beta-estradiol [(E2) CAS: 50-28-2]. DES at 10(-7) M in the culture medium increased the number of sister chromatid exchanges (SCE); no further increases was obtained with higher concentrations of DES. Pretreatment of the females with PBA resulted in an increase in SCE when DES was used in the range of 10(-7) to 10(-5) M. The level of this increase was significantly higher than that in fibroblasts from untreated females. E2 had no effect. DES had no effect on SCE in kidney cells from PBA-treated females. Indomethacin and alpha-naphthoflavone reduced the number of SCE in PBA-exposed fibroblasts to an intermediate level. The presence of both enzyme inhibitors in the medium depressed the number of SCE to the control level.     

Induction of sister chromatid exchanges by carcinogens mediated through cultured rat liver epithelial cells

A cloned, untransformed rat liver cell-line, LNRL (with the normal diploid karyotype and epithelial morphology) was tested for use as the metabolizing component in a co-cultivation system for carcinogen-screening using sister chromatid exchanges (SCE) as the criterion. The well characterized early cultures of LNRL cells were co-cultivated with Chinese hamster ovary (CHO) cells, the target commonly used for the induction of SCE, and exposed to different concentrations of various chemicals for 24 h. Two polycyclic hydrocarbons, 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene did not produce any SCE in CHO cells cultured alone, but produced a significant number of SCE in CHO co-cultivated with LNRL. The aromatic amine, 2-acetylaminofluorene, and the potent carcinogenic mycotoxin, aflatoxin B1, induced SCE to a limited extent even in CHO cells cultivated alone, but in the presence of liver cells the SCE frequency was greatly increased. Aflatoxin G2, the least potent of the aflatoxins, also produced a response in the co-cultivation system. These results indicate that cultured liver cells can be used as the metabolizing cells in co-cultivation systems for carcinogen-screening. The advantages of this assay over those employing liver microsomal fractions are discussed.     

Influence of spermidine on sister chromatid exchanges induced by alkylating agents in mammalian cells in vitro

Sister chromatid exchanges (SCEs) are a very sensitive genetic end-point for in vitro identification of presumed carcinogenic and mutagenic agents, although the mechanism of their formation is still to be elucidated. The present work shows the influence of spermidine on SCE induction by two different DNA damaging agents: Mitomycin-C (MMC) and N-Methyl-N'-Nitro-N-Nitrosoguanidine (MNNG). The SCE level induced by MMC was significantly decreased by spermidine. On the contrary, MNNG-induced SCEs were not affected. It has recently been suggested that MMC, via its reduced metabolite mitosene, produces bulky mono-and bi-adducts in DNA, mainly located in the minor groove of the double helix. MNNG, instead, directly methylates several electrophilic sites of DNA bases, such as the N7 and the O6 of guanines and the N3 of adenines. Both MMC and MNNG, despite their different mechanism of action, are potent SCE inducers. Spermidine, similarly to its structural analogue Spermine, is known to interact with DNA phosphate groups and to bind reversibly to the minor groove, thus stabilizing the double helix structure. Spermidine, being therefore ineffective on the MNNG-mediated DNA methylation, might affect DNA, making it structurally unavailable for MMC binding.     

Cumulative effects of chromosome aberrations and sister chromatid exchanges in rat liver induced in vivo by heterocyclic amines

Cumulative effects of chromosome aberrations and sister chromatidexchanges (SCEs) were studied in hepatocytes of F344 rats exposedin vivo to 2-amino-3-methylimidazo[4,5-fIquinoline (IQ) at dosesof 12.5,25 or 50 mg/kg body wt/day or 2-nitro-3-methylimidazo[4,5-f quinoline (nitro-IQ) at doses of 12.5, 25 or 50 mg/kg bodywt/day. Hepatocytes were isolated 24 h after 1, 7,14 or 28 repeateddoses (once a day) by gastric intubation and allowed to proliferatein Williams' medium E supplemented with epidermal growth factor.Cells were fixed after a culture period of 48 h. Multiple treatmentwith IQ or nitro-IQ induced significant chromosome aberrationstime- and dose-dependently, the maximum frequency of chromosomeaberrations in metaphase cells being 39 and 33% respectively,while that in controls was 1.1%. Single treatment with IQ ornitro-IQ induced significant SCEs dose-dependently, the maximumfrequency being 0.83 and 0.79 per chromosome respectively, whilethe control value was 0.51. Multiple treatment with nitro-IQinduced significant SCEs to a plateau level of 0.90 per chromosome.Cytogenetic damage in the liver by IQ was greater than thatby nitro-IQ. These results show that this assay of chromosomeaberrations and SCEs in rat liver in vivo without partial hepatectomyor mitogen treatment in vivo is a sensitive method for evaluatingthe cumulative tumor-initiating activities of carcinogenic heterocyclicamines at low doses and should be useful for the detection ofunknown hepatocarcinogens.     

Incidence and localization of sister chromatid exchanges induced by nickel and chromium compounds

Carcinogenic nickel compounds enhanced the incidence of sisterchromatid exchanges (SCEs) in a concentration-dependent fashionin intact Chinese hamster ovary cells. There was a preferentialinduction of these exchanges in the heterochro-matic regionsof the chromosomes. CaCrO₄ also caused a dose-dependent inductionof SCEs. However, in contrast to NiCl₂, the exchanges inducedby CaCrO₄ were not localized in any particular chromosomal region.The total incidence of exchanges was higher with CaCrO₄ thanwith NICI₂. CaCrO₄, crystalline NiS and NiCI₂ enhanced the incidenceof SCEs at concentrations below the threshold of DNA damageas detected by the technique of alkaline elution. Additionally,following treatment time intervals of 24–48 h, there wasan increase in SCEs at concentrations of NiCl₂ or CaCrO₄ thatproduced little disruption of cell cycle progression. Theseresults are consistent with the hypothesis that potently carcinogenicnickel compounds which are not very mutagenic exert selectiveeffects on genetically inactive heterochromatin, while potentlymutagenic and carcinogenic chromate do not appear to producea similar predominance of SCEs in hetero-chromatk regions.     

Increased fragile sites and sister chromatid exchanges in bone marrow and peripheral blood of young cigarette smokers

Cigarette smoking is considered to be the single most important acquired cause of cancer mortality. Studies of chromosome aberrations, sister chromatid exchanges, and fragile sites in peripheral blood or bone marrow are useful methods to detect the effects of the environmental mutagens or carcinogens found in cigarette smoke. The effects of smoking on the immature cells in the bone marrow have not been studied. Here, we examine the peripheral blood and bone marrow in 18 smokers (15 females and 3 males) with a median age of 25 years (range, 21-40) and an average cigarette use corresponding to 6 pack years. In both bone marrow cells and peripheral blood lymphocytes, we were able to show a significantly increased frequency of sister chromatid exchanges in smokers with a 5 or more cigarette pack year history, but not in those who smoked less than 5 pack years. We also found a higher frequency of sister chromatid exchanges in peripheral blood lymphocytes than in bone marrow cells. In addition, the peripheral lymphocytes of smokers demonstrated (a) a significantly higher frequency of fragile sites, (b) an increased number of metaphases with extensive breakage; and (c) elevated expression of fragile sites at the cancer breakpoints 3p14.2, 11q13.3, 22q12.2, and 11p13-p14.2 and at the oncogene sites bcl 1, erb B, erb A, and sis. Our results suggest that chromosomal DNA of peripheral blood lymphocytes is sensitive to cigarette smoking. Studies of the chromosomal changes in these cells provide an index of the mutagenic damage caused by these exogenous agents in individual patients and the ability of individuals to repair that damage, and might predict susceptibility to malignant events.     

Effect of beta-carotene on sister chromatid exchanges induced by MNNG and aflatoxin B1 in V79 cells

In order to elucidate the mechanism of tumor prevention of beta-carotene, its effect on sister chromatid exchanges (SCE) induced by MNNG in cultured V79 cells, under condition free of the enzyme system to convert beta-carotene into vitamin A, was studied. It was found that SCE level was significantly increased by high doses beta-carotene (10(-5)-10(-4) M) and the enhancement of SCE was restored to its original level by addition of alpha-tocopherol (final concentration 2 micrograms/ml). This may be due to the latter inhibiting the oxidation of beta-carotene and reducing the amount of oxidated carotene, which is toxic for cultured cells. Combination of beta-carotene and alpha-tocopherol at low doses inhibited SCE induced by MNNG (P less than 0.05) but no protective activity was observed when used separately. It was also found that beta-carotene (2 x 10(-7) M) and retinol (16 micrograms/ml) inhibited SCE induced by aflatoxin B1, which is activated by S-9 mixture. The present data clearly show that the antitumor activity of beta-carotene may be attributed to both itself and its degraded compound vitamin A, and may take part in the initiation of carcinogenesis. Combination of beta-carotene and other cancer preventive drugs is more effective and safer than it used individually.     

Persistence of sister chromatid exchanges and in vitro morphological transformation of Syrian hamster fetal cells by chemical and physical carcinogens

The induction of neoplastic cell transformation is closely associatedwith DNA alterations which occur shortly after carcinogen exposure.Sister chromatid exchange (SCE) formation is a sensitive indicatorof carcinogen-DNA interaction and correlates with the inductionof morphological cell transformation. The persistence of lesionsgenerating SCE produced by chemical and physical carcinogensand its relevance to the induction of morphologic transformationwas evaluated in coordinated experiments with cultured Syrianhamster fetal cells (HFC). Exponentially growing HFC were exposedfor 1 h to benzo[a]pyrene (BP), methyl-methanesulfonate (MMS),cis-platinum (II) diaminedichloride (cis Pt II), N-methyl-N'-nitrosourea(MNU), mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine(MNNG), N-acetoxy-2-fluorenyl-acetamide (AcAAF) or u.v. lightirradiated. Cells were incubated for 24 h with 5-bromodeoxyuridine(BrdUrd) required for SCE visualization at 1, 24 and 48 h aftercarcinogen exposure. The induction of morphological transformationwas determined on the quantitative colony assay at 6 days aftercarcinogen treatment. SCE analysis demonstrates that for a periodof 48 h after carcinogen exposure, during which time the cellsundergo at least four replicative cycles, DNA damage generatingSCE induced by all chemical carcinogens either persisted orwas partially removed, whereas u.v.-induced lesions were completelyremoved. An elevated SCE frequency persisted after two additionalcell cycles after treatment with BP, AcAAF or MMC without increasedcell lethality as compared to other carcinogens whose lesionswere completely eliminated during the same period. Althougha correlation between the persistence of SCE and the inductionof transformation was not observed for all carcinogens, thisstudy illustrates that DNA damage generating SCE can persistover several replicative cycles, thus raising the possibilitythat lasting DNA alterations are important for the inductionof neoplastic cell transformation.     

Enhancement of benzo[a]pyrene-induced sister chromatid exchanges in lymphocytes from cigarette smokers occupationally exposed to asbestos

The frequencies of base-line and benzo[a]pyrene [(BP) CAS 50-38-8]-induced sister chromatid exchanges (SCE) were measured in peripheral blood lymphocytes from 22 male asbestos-exposed workers and 10 nonexposed workers of comparable age. A clear association between cigarette smoking and asbestos exposure in the sensitivity of lymphocytes to BP was observed. Among asbestos-exposed workers, lymphocytes from those who smoked cigarettes were significantly more susceptible to the induction of SCE by in vitro exposure to BP (P = .01) than were lymphocytes from nonsmokers. Active smoking elevated the base-line SCE frequency in both asbestos-exposed and nonexposed workers (P = .001), and an interaction between smoking and asbestos in the production of base-line SCE was suggested (P = .07). Asbestos exposure alone was not associated with an enhanced susceptibility to the induction of SCE by BP or with an elevation of base-line SCE. Increased age was associated with an increase in SCE inducibility by BP (P = .01), and a history of smoking was marginally associated with SCE inducibility by BP (P = .07). These findings support the hypothesis that an increased susceptibility of asbestos-exposed individuals to polyaromatic hydrocarbon-induced cancer results from an enhanced sensitivity to the induction of genetic damage rather than to an asbestos-induced differential cellular metabolic capacity.     

Nitrosourea-induced sister chromatid exchanges and correlation to cell survival in 9L rat brain tumor cells

The ability of various nitrosoureas to induce sister chromatid exchanges (SCEs) in 9L rat brain tumor cells was investigated. Treatment of cells for 1 hr with the alkylating and cross-linking agents 1,3-bis(2-chloroethyl)-1-nitrosourea or chlorozotocin produced concentration-dependent increases in SCEs; elevations above controls were detected at concentrations of 1 microM or more. Above 0.25 mM, the alkylating agent ethylnitrosourea produced a dose-dependent increase in SCEs. Treatment with the carbamoylating agent 1,3-bis(trans-4-hydroxycyclohexyl)-1-nitrosourea did not induce SCEs. The maximum drug concentration at which SCEs are readily scored kills approximately 50% of cells. When accurate cell survival data in this dose range were obtained, a direct correlation between nitrosourea-induced cell kill, measured by a colony-forming efficiency assay, and SCE induction was found. Thus, analysis of the levels of SCE production may provide information about the efficacy of antineoplastic drugs.     

Induction of sister chromatid exchanges and chromosome aberrations in cultured mammalian cells by N-Nitrosocimetidine

N-Nitrosocimetidine (NC) induces significant numbers of sister chromatid exchanges (SCE) and chromosome aberrations in cultured Chinese hamster ovary (CHO) cells even at a concentration of 1.2 × 10^?7 M. Its effectiveness in SCE induction is about two thirds that of the gastric carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). These results constitute further evidence that NC possesses carcinogenic activity.     

Induction of in vivo sister chromatid exchanges by arecaidine, a betel nut alkaloid, in mouse bone-marrow cells

The genotoxicity of arecaidine, an alkaloid of betel nut, was studied on mouse bone marrow cells in vivo by sister chromatid exchange (SCE) method. Arecaidine was administered intraperitoneally to mice at the dose levels of 2.5 mg, 5 mg and 7.5 mg to each mouse weighing 25 +/- 1 g for 5, 10 and 15 days. Significant increase in the number of SCEs was observed in the treated groups, and this increase, although dose-dependent, was not dependent upon the duration of exposure.     

Cordycepin reduces the sensitivity of BALB/Mo mouse lymphocytes to the induction of sister chromatid exchanges

Lymphocytes from the spleen of BALB/Mo mice, which carry endogenousMoloney murine leukemia virus (M-MuLV), show in vitro frequenciesof sister chromatid exchanges (SCEs) significantly higher thanlymphocytes from control (M-MuLV free) BALB/c mice. In vitrotreatment of lymphocytes with the antiviral antibiotic cordycepin(10 µg/ml) lowers the level of SCEs in BALB/Mo cells tothe same value of BALB/c cells. M-MuLV yield is also markedlyreduced in BALB/Mo lymphocytes cultured in the presence of cordycepin.The drug also abolishes the increased sensitivity of BALB/Molymphocytes to the induction of SCEs by mitomydn C (MMC) eitherin vitro (3 ? 10^–8/10^–7M) or in vivo (0.3/3 mg/kg).Since cordycepin is known to inhibit pohy(A)synthesis thus blockingRNA maturation, it is suggested that M-MuLV provlral integrationis not per se the sole factor responsible for the more pronouncedsusceptibility of BALB/Mo lymphocytes to SCE induction, butmost likely viral gene expression and amplification are neededfor this effect to occur.     

Increased frequencies of alpha-naphthoflavone induced sister chromatid exchanges in long-term survivors of childhood acute lymphoblastic leukemia

Cultures of whole-blood were established from 12 children (mean age = 13.5 years) surviving acute lymphoblastic leukemia (mean time of discontinued therapy = 4.5 years) and 10 age-matched controls were assessed for the level of baseline, alpha-naphthoflavone (ANF) and methyl-methane-sulphonate (MMS) induced sister chromatid exchanges (SCEs). The average baseline levels of SCEs in the patient group (7.65) and in the control (6.72) were different at a statistical marginal level (P = 0.05). No statistical difference was observed in the levels of MMS-induced SCEs between the two groups (P greater than 0.1). In contrast, highly significant differences (P = 0.003) were observed in the level of ANF-induced SCE-levels between the patients (10.78) and the controls (8.76).     

Potentiation of BCNU-induced cytotoxicity and sister chromatid exchanges by dibromodulcitol in vitro

Dibromodulcitol (DBD) is an anticancer agent that is cytotoxic against animal and human brain tumors in vivo. Clinical trials of combination therapy with radiation, DBD, and a nitrosourea have shown some efficacy, but the mechanisms that lead to enhanced cytotoxicity are poorly understood. We investigated the effects of pretreatment with DBD on cell survival and sister chromatid exchange (SCE) caused by subsequent treatment with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) in 9L rat brain tumor cells. Pretreatment of 9L cells for 24 hr with 5 microM DBD potentiated the cell kill produced by a 1-hr treatment with BCNU; the dose enhancement ratio was 1.7 at the 10% survival level. Treatment of 9L cells for 24 hr with 1 microM DBD induced 39 +/- 12 SCEs/metaphase. There was a 50-75% increase in BCNU-induced SCEs, compared with BCNU alone, after a 24 hr pretreatment with DBD. Thus DBD potentiation of BCNU cytotoxicity appears to be related to increased DNA damage.     

Cytotoxicity and sister chromatid exchanges in 9L cells treated with monofunctional and bifunctional nitrogen mustards

To investigate the role of DNA interstrand crosslink formationon cytotoxicity and the induction of sister chromatid exchanges(SCEs), we treated 9L cells with the bifunctional mustard bis(2-chloroethyl)methylamine(HN2), which can alkylate DNA and form DNA interstrand crosslinks,or with two monofunctional mustards, bis(ethyl)-2-chloroethylamineand bis(methyl)-2-chloroethylamine, which can only alkylateDNA. On a molar basis, HN2 was 900—2400 times more cyto-toxicand 471—686 times more effective at inducing SCEs thanthe monofunctional mustards. HN2 induced high levels of DNAinterstrand crosslinks in 9L cells; no interstrand crosslinkswere detected in cells treated with the monofunctional mustards.Comparison of the alkylating activity of each of the mustardsby 4-(p-nitrobenzyl)pyridine reactivity showed only a 4-folddifference, which is not sufficient to account for the largedifferences in cell survival and induction of SCEs. We concludethat the effectiveness of HN2 at inducing cytotoxicity and SCEsresults from the formation of DNA interstrand crosslinks.     

Correlation of morphological transformation to sister chromatid exchanges induced by split doses of chemical or physical carcinogens on cultured Syrian hamster cells

The relationship between the induction of DNA damage as reflected by sister chromatid exchange (SCE) formation and morphological transformation in exponentially growing Syrian hamster embryo cells was determined quantitatively after split doses of chemical or physical carcinogens. With split doses of carcinogen separated by 2 to 24 hr, only N-acetoxy-2-fluorenyl-acetamide (0.50 microgram/ml) enhanced both SCE induction and transformation when compared to single exposure. Split doses of N-methyl-N'-nitro-N- nitrosoguanidine (0.20 microgram/ml), mitomycin C (50 ng/ml), or ultraviolet light (3.0 J/sq m) were less effective than single exposures, while split doses of methyl methanesulfonate (40 micrograms/ml) caused transformation frequencies similar to a single treatment and decreased SCE frequencies with time intervals greater than 4 hr. Split or single exposures of X-irradiation (200 R) resulted in similar low frequencies of transformation and SCE. Contrasting with these results, a significant potentiation of SCE occurred after split doses of N-methyl-N'-nitro-N-nitrosoguanidine in cultures arrested in G1 with arginine-glutamine-deficient medium or by contact inhibition compared to a single treatment. This response was attributed to the interaction of carcinogen with DNA containing unrepaired damage and demonstrates the importance of the cell cycle phase of the target cell during carcinogen exposure for the induction of SCE by split doses of N-methyl-N'-nitro-N-nitrosoguanidine. The similarity of responses for transformation and SCE induction with split doses of carcinogens suggests that DNA lesions involved in SCE are essential for the initiation of neoplastic development.     

Role of the maternal environment in determining susceptibility to transplacentally induced chemical carcinogenesis in mouse fetuses.

Treatment of pregnant mice with 3-methylcholanthrene (MC) causes lung and liver tumors in the offspring, the incidences of which are greatly influenced by the Ah locus regulated induction phenotype for aryl hydrocarbon hydroxylase activity (AHH) in both the mother and fetuses. In order to examine the biochemical and molecular mechanisms responsible for the modulating effect of maternal environment on tumor susceptibility, reciprocal crosses between responsive C57BL/6 and non-responsive DBA/2 mice were made and the pregnant mothers were treated i.p. on the 17th day of gestation with either olive oil alone, 30 mg/kg of MC, or 30 mg/kg of beta-naphthoflavone (beta NF). At various times after injection, the mothers were killed and the fetuses removed for enzymatic and molecular blot analysis. In fetal lung tissues, the absolute levels and relative induction ratios of AHH activity from D2B6F1 fetuses were very similar to those obtained in B6D2F1 fetuses during the first 24 h following a transplacental exposure to either inducing agent. This was also the case 48 h after an injection of beta NF. However, 48 h after exposure to MC, the AHH activity in fetal lungs from B6 mothers had declined to practically control values, whereas fetal lungs from D2 mothers still exhibited a high level of AHH activity. Similar induction kinetics for the CYPIA1 gene were obtained in fetal livers. These results were confirmed at the RNA level by quantitative slot-blot analysis of fetal RNA preparations. In both organs, treatment with inducing agents for the P450IA1 gene resulted in a rapid and early induction of CYPIA1 RNA by 4 h. Fetuses from D2 mothers, however, showed a more sustained induction of CYPIA1 RNA following exposure to MC than did fetuses from B6 mothers. These results suggest that the observed increase in tumor susceptibility observed in the offspring of D2 mothers compared to the offspring of B6 mothers was due, at least in part, to the differences in the persistence of induction of the CYPIA1 gene locus, and may be the result of differences in the clearance rates of MC from the fetal and maternal compartments or its pharmacokinetic distribution in the two types of maternal environments.     

Factors influencing the induction of sister chromatid exchanges in mammalian cells by 12-O-tetradecanoyl-phorbol-13-acetate

The induction of sister chromatid exchanges (SCE) by 12-O-tetradecanoyl-phorbol-13-acetate(TPA) was measured in mouse IOT and 3T3 cells released fromdensity inhibition, and Chinese hamster CHO cells synchronizedby mitotic selection. The induced frequency of SCE was similarin each cell type, but depended upon the concentration of TPAemployed, its source, and the particular lot from the same source.Most SCE occurred in the first two rounds of cell replicationafter addition of TPA. The induction of SCE by TPA was markedlysuppressed if the fetal calf serum in the incubation mediumwas not heat-inactivated. The addition of superoxide dismutaseto medium with heat-inactivated serum also suppressed the inductionof SCE. These results suggest that free radicals, particularlythe super-oxide anion, may be important intermediates in someof the biologic effects of TPA.     

Cytotoxicity and sister chromatid exchanges induced in vitro by six anticancer drugs developed in the People's Republic of China

Growth inhibition in the Chinese hamster cell line V79 and in the human lymphoid cell line Raji and induction of sister chromatid exchange(s) (SCE) in V79 cells after treatment with six anticancer drugs [harringtonine (HRT), homoharringtonine (HHRT), camptothecin (CPT), hydroxycamptothecin (HCPT), lycobetaine (LBT), and oxalysine (OXL)] developed in the People's Republic of China were studied. OXL is a new antibiotic; all other drugs are plant extracts. All drugs caused a dose-dependent growth inhibition in both cell types, as evidenced by decreases in plating efficiencies of V79 cells and in viable cell counts of Raji. However, the degree of inhibition differed widely among the drugs. HRT, HHRT, CPT, and HCPT were the most potent growth inhibitors, LBT was next, and OXL was the least effective inhibitor. SCE analyses were made in V79 cells treated with a drug in the presence or absence of the metabolic activation system S9 mixture (S9 mix), except for the HRT assay in which the S9 mix was not used. CPT, HCPT, and LBT induced a dose-dependent increase in SCE frequencies, while HRT, HHRT, and OXL caused no SCE induction at any dose level used. CPT was the most powerful SCE inducer. HCPT induced SCE but at a much reduced rate when compared to that of CPT. LBT was a weak SCE inducer; SCE induction was seen only in cultures treated with 40 micrograms or more LBT/ml. Addition of the S9 mix did not alter SCE frequencies, indicating that the drugs were direct-acting agents. HRT and HHRT were highly toxic, but they induced no increases in SCE frequency, indicating that cytotoxicity of a compound does not necessarily correlate with SCE induction.