Hypersensitive character of Bloom syndrome B-lymphoblastoid cell lines usable for sensitive carcinogen detection

Various carcinogens were tested with regard to the induction of sister-chromatid exchanges (SCEs) and chromosome aberrations using 3 types of Bloom syndrome (BS) B-lymphoblastoid cell lines (LCLs) (type I with normal frequency of SCEs and normal karyotype; type II with high frequency of SCEs and normal karyotype; type III with high frequency of SCEs and abnormal karyotypes) in the presence and absence of S9 mix. Three types of BS B-LCLs and normal cells showed different responses to the various carcinogens in the level of SCE induction. BS type I cells had the same SCE response as normal cells to carcinogens. Some carcinogens that require metabolic activation (S9 mix) had little effect on type II cells without S9 mix but had high SCE levels with S9 mix. BS type III cells were highly susceptible to both direct and indirect carcinogens with respect to high SCE increase without S9 mix (ca. 140 SCEs/cell), though some carcinogens produced SCEs rated in the medium (ca. 120 SCEs/cell) range, and had a high rate (more than 10%) of centromere spreading (CS), in addition to quadriradials. Therefore BS type III is a unique cell line which can be used to detect carcinogens.     

Induction of sister-chromatid exchanges by indirect mutagens/carcinogens in cultured rat hepatoma and esophageal tumor cells and in Chinese hamster Don cells co-cultivated with rat cells

2 rat cell lines originated from ascites hepatoma AH66-B and esophageal tumor R1 were examined for their inducibility of sister-chromatid exchanges (SCEs) after treatment with 14 kinds of indirect mutagens/carcinogens, including 6 amine derivatives, 4 azo compounds, 3 aromatic hydrocarbons and 1 steroid. Of the 14 chemicals tested, 2-acetylaminofluorene (AAF), butylbutanolnitrosamine (BBN), dimethylnitrosamine (DMN), cyclophosphamide (CP), urethane, 2-methyl-4-dimethylaminoazobenzene (2-MeDAB), 3′-methyl-4-dimethylaminoazobenzene (3′-MeDAB), 4-o-tolylazo-o-toluidine (4-TT), benzo[a]pyrene (BP), 7,12-dimethyl-benz[a]anthracene (DMBA) and diethylstilbestrol (DES) were estimated to be effective inducers of SCEs in AH66-B and/or R1 cells, without the use of exogenous activating systems. Cell-mediated SCE tests with 6 selected chemicals, CP, 2-MeDAB, 4-TT, BP, DMBA and DES, showed a significant increase of SCEs in Chinese hamster Don-6 cells co-cultivated with AH66-B or R1 cells, depending on the number and sensitivity of AH66-B or R1 cells, as well as on the dose of chemicals tested, whereas singly cultured Don-6 cells were much less sensitive or almost insensitive to these chemicals. The above findings suggest that AH66-B and R1 cells may retain metabolic activities to convert a wide range of indirect mutagens/carcinogens into their active forms to induce SCEs, and that these cell lines provide simple and reliable screening systems in vitro, including the cell-mediated SCE assay, for detection of genotoxic agents, without the use of exogenous activation systems.     

Induction of sister chromatid exchanges in Chinese hamster cells by carcinogenic mutagens requiring metabolic activation

The induction of SCEs has proven to be the most sensitive mammalian system for detecting the effects of mutagenic carcinogens. Several chemicals that are mutagenic in the exquisitely sensitive Salmonella mutagenesis test have now been tested in Chinese hamster ovary (CHO) cells in culture. Cells were grown for 24 h (two rounds of DNA replication) in the presence of bromodeoxyuridine (Brd Urd) to form harlequin chromosomes in which it is possible to see the SCEs. To test whether the chemicals increase SCEs without metabolic activation, they were added at various concentrations for the entire culture period. To test if they induce SCEs after activation they were added for 30 min along with microsomes from rat liver (S-9 Mix of Ames). After this treatment the cells were cultured with Brd Urd. N-hydrosy-2-acetylamino-fluorene (10^?6?10^?4 M), N-acetoxy-2-acetylaminofluorenee (10)^?9?10^?7 M), and aflatoxin B₁ (10^?6?10^?4 M) all increased the yield of SCEs with increasing concentration. Further, aflatoxin B₁ was dramatically activated by the addition of rat liver microsomes. Benzo(a)pyrene (10^?6?10^?4 M), however, gave an increase only when activated. 2-aminofluorene (10^?6?10^?4 M) gave a slight increase only after long treatments without activation. In no case did 2-acetylamino-fluorene (10^?6?10^?4 M) increase SCE's. It thus appears that some of the chemicals that are positive in the Salmonella system are negative in the mammalian SCE system. Whether this reflects a difference in sensitivity between the two tests or the ability of the SCE test to discriminate between those chemicals that are active in bacteria, but not in mammals, is as yet unknown.     

Cyclophosphamide-Induced IN VIVO Sister Chromatid Exchanges (Sce) in MUS MUSCULUS. III. Quantitative Genetic Analysis

In vivo cyclophosphamide (CP)-induced sister chromatid exchanges (SCEs) were evaluated in females from five genetic strains of mice (C57BL/6J, C3H/S, 129/ReJ, BALB/c and DBA/2) and their F₁ hybrids. Baseline (noninduced) SCE values differ significantly among strains, 129/ReJ having the lowest and DBA/2 having the highest mean SCE per cell values. In general, the baseline SCE of a given F₁ is within the range of its corresponding parental strains or near the lower parental value. Furthermore, there is a genotype-dependent increase in mean SCEs per cell with CP dose. Strain differences in SCE induction are noted particularly at the two higher CP doses (4.50 and 45.0 mg/kg). In general, F₁ hybrids involving a strain with high induced SCEs and a strain with low induced SCEs exhibit mean SCE values that are closer to the value of the lower strain. F₁ s involving two strains with high SCEs or two strains with low SCEs yield SCEs not different from parental strains. The method of diallel cross analysis showed the order of dominance of these strains in SCE induction to be 129/ReJ BALB/c C3H/S DBA/2 C57BL/6J. These results support the involvement of predominantly nonadditive genetic factors as major gene(s) in SCE induction. In addition, involvement of random and independent events in SCE induction is suggested by the distribution of SCEs which follows a Poisson distribution.     

Hepatic microsomal mixed function oxygenase: enzyme multiplicity for the metabolism of carcinogens to DNA-binding metabolites

Microsome-mediated metabolic activation of aflatoxin B₁ (AFB₁) and benzo[a]-pyrene (BP), as determined by the $\text{in vitro}$ formation of DNA binding metabolites, was studied, using hepatic microsomes from untreated, phenobarbital (PB)-treated and 3-methylcholanthrene (MC)-treated male rats. Contrasting results were obtained for the two substrates: in the case of AFB₁, microsomes from PB-treated rats were twice as active as microsomes from untreated and MC-treated rats, whereas, in the case of BP, microsomes from MC-treated rats were several fold more active than microsomes from untreated and PB-treated rats. These data strongly suggests enzyme multiplicity of microsomal mixed function oxygenase for the activation of carcinogens, especially AFB₁ and BP whose reactive metabolites are believed to be epoxides.     

Cell-cycle duration and sister-chromatid exchange frequency in cultured human lymphocytes

Whole heparinized blood samples from normal human donors were grown in culture media containing 10 μg/ml of bromodeoxyuridine. Lymphocytes were harvested after 58, 70, 72 and 80 h and scored for sister-chromatid exchanges (SCEs) under a fluorescence microscope. SCEs which occured during the first and second cell cycles were counted in second or third generation cells selected on the basis of their chromosome fluorescence patterns. The results of a preliminary study showed the mean SCE frequency per cell at 72 h to be 9.0 for second generation cells and 7.8 in third generation cells (P < 0.01). A second study, using culture medium with heat-inactivated fetal-calf serum, gave similar results (9.4 vs. 7.8, P 0.001) at 70 h. Therefore, the difference in SCE frequency between second and third generation cells at 70 or 72 h cannot be attributed to heat-labile substances of serum origin. An additional finding in the second study was that SCE frequencies in third division cells at 70 and80 h were the samee as those of second division cells at 58 h but significantly less (P < 0.001) than the frequency in second division cells at 70 h. These data were interpreted as arising from at least two different lymphocyte populations; one group of cells that is either slower growing or slower in phytohemagglutinin stimulation, with a higher SCE frequency which does not reach second division until 70 or 80 h, and a more rapidly dividing (or more quickly stimulated by phytohemagglutinin) population with a lower SCE frequency which reaches second division at 58 h and third division by 70–80 h. Whether or not this hypothesis is correct, the data show that SCE frequency varies significantly with cell-cycle duration. Since some carcinogens have been shown to alter cell kinetics (Craig-Holmes and Shaw, Mutation Res. 46 (1977) 375), changes in SCE frequency which are caused by a change in cell kinetics must be considered a factor in determining the mutagenicity of an agent by its ability to increase SCE frequency.     

Effect of sodium selenite and methyl methanesulfonate or N-hydroxy-2-acetylaminofluorene co-exposure on sister-chromatid exchange production in human whole blood cultures

Sodium selenite (Na₂Se0₃) was tested for its sister-chromatid exchange (SCE)-inducing ability in human whole blood cultures and for the effect of its co-exposure with methyl methanesulfonate (MMS) or N-hydroxy-2-acetyl aminofluorene (N-OH-AAF) on SCE frequency. Long exposure times (77 h and 96 h) to 3.95 × 10^-6 M Na₂SeO₃ resulted in cell death as measured by mitotic indices, but mitotic figures were present after exposure to higher concentrations for a shorter time (19 h). High Na₂SeO₃ concentrations (7.90 × 10^?6 and 1.19 × 10^?5 M) resulted in a three-fold increase in the SCE frequency above background level (6–7 SCEs/cell). Exposure of lymphocytes to 1 × 10^?4 M MMS for the last 19 h of culture yielded an average SCE frequency of 30.17 ± 0.75 while a similar exposure to 2.7 × 10^?5 M N-OH-AAF resulted in 13.61 ± 0.43 SCEs/cell. Simultaneous addition of the high Na₂Se0₃ concentrations and MMS or N-OH-AAF to the cultures resulted in SCE frequencies that were 25–30% and 11–17%, respectively, below the sum of the SCE frequencies produced by the individual compounds.     

Sister-chromatid exchanges in lymphocytes of workers at a phosphate fertilizer factory

The frequencies of sister-chromatid exchange (SCE) in peripheral blood lymphocytes of 40 workers at a phosphate fertilizer factory in North China were studied. HF and SiF₄ are main air pollutants in the factory, there is also some dust containing fluoride, phosphate fog, NH₃ and SO₂. It was shown that the chemicals caused an increase in SCE, and also induced cell mitotic delays. The mean SCEs/cell of the workers and the non-exposed controls were 7.47 ± 0.31 and 4.94 ± 0.14 (p < 0.01) respectively. SCEs/cell in 75% of 40 workers were higher than 6 while 40 controls all had values lower than 6. SCE frequencies of the workers increased with length of the chemical exposure period up to 10 years. Smoking enhanced the SCE frequencies induced by the chemicals.     

Physical,chemical, and biological factors affecting sister-chromatid exchange induction in human lymphocytes exposed to mitomycin C prior to culture

In experiments to assess the effects of several biological, chemical, and physical variables on sister-chromatid exchange (SCE) induction in cultured lymphocytes exposed to mitomycin C (MMC) before PHA stimulation we observed: (1) high SCE frequencies in female cells, and normal SCE frequencies in Y-bearing metaphases in mixed cultures containing equal numbers of MMC-treated female lymphocytes and untreated male lymphocytes; (2) small, but statistically significant, decreases in SCEs with increasing pH after G₀ exposure in the pH range 6.6–7.6; (3) pronounced reductions in MMC-induced SCEs in lymphocytes exposed at 4°C vs. 37°C. In other studies, SCE induction was evaluated in cultures exposed during G₀ to MMC concentrations ranging from 0.25 to 2.5 μg/ml for varying time intervals ranging from 5 min to 24 h. For all concentrations tested SCE induction varied as a linear function of G₀ exposure time. To compare SCE induction between cultures, we calculated the mean frequencies of SCEs induced per metaphase/unit dose MMC/unit G₀ exposure time (SCE/μg/h). A mean frequency of 20.7 ± 4.8 SCE/μg/h was observed for 41 lymphocyte cultures suggesting that a single term adequately describes the rate of SCE induction following G₀ exposure to a 10-fold range in concentration of MMC for time intervals of 30 min to 24 h.     

Different baseline sister chromatid exchange levels in density fractionated human lymphocytes

Summary Different activation states of B and T lymphocytes, as manifested by differences in cell density, were obtained by Percoll density centrifugation of unstimulated human lymphocytes. Four different density fractions were defined: B cells with low (1.043 g/ml) and high (1.056) density, and T cells with low (1.067) and high (1.077) density, respectively. Sister chromatid exchange (SCE) conditions and proliferation rates were determined. Total B cells, stimulated by the bacterial mitogen Branhamella, had 4.6 SCE per cell, the lowest mean baseline SCE level recorded among lymphocytes. The growth rate was intermediate between that of low and high density T cells. The two T cell fractions stimulated by phytohemagglutinin (PHA) had different baseline SCE frequencies and different growth characteristics: the low density cells had 5.7 SCEs per cell and a short cell cycle, whereas high density cells had 12.5 SCEs per cell and a longer cell cycle. The differences in baseline SCE frequency and growth characteristics between the two T cell fractions seem to be correlated with the differences in the activation state as reflected by the cell density. Both high and low density T cell are G₀ populations which supposedly differ with respect to previous history in vivo such as age and contact with antigens. The reason why these cells react differently to bromodeoxyuridine (BrdU) is unknown, but differences in intracellular DNA precursor pools and enzyme activities might play a role.     

Chromosomal analysis in vinyl chloride exposed workers: Comparison of the standard technique with the sister-chromatid exchange technique

A group of 21 workers occupationally exposed to vinyl chloride and 6 controls were examined for the presence of chromosomal aberrations or sisterchromatid exchanges in their peripheral lymphocytes. These people comprised a second sampling from a group of exposed workers and controls first examined 18 months earlier. The vinyl chloride exposed workers showed levels of chromosomal aberrations elevated above those of the controls, but there was only a slight increase in sister-chromatid exchanges (per cell or per chromosome) and this increase was not statistically significant. Sister-chromatid exchanges (SCEs) were also examined from in vitro cultures of lymphocytes exposed in G₀/early G₁ and late G₁/early S phase to vinyl chloride, both with and without metabolic activation. There was no increase in SCEs in vitro without metabolic activation but there was a marked increase with metabolic activation and this increase was shown to be independent of cell-cylce phase. It thus was apparent that the small increases of SCEs in workers were not due to the inability of vinyl chloride to induce SCEs in human lymphocytes but were probably because of low exposures and SCE levels could have returned to normal relatively quickly after exposure. The present study suggested that the analysis of longer-living conventional chromosomal aberrations appeared to be a more sensitive monitor of exposure to vinyl chloride in exposed workers than the estimation of SCEs; however, it should be noted that in a 3rd sampling taken 24 months later the exposed workers had chromosomal aberration levels similar to the controls.     

Analysis of bromodeoxyuridine-induced single and twin sister chromatid exchanges in tetraploid Chinese hamster ovary cells

Culture of cells in high exogenous levels (>10^–4 M) of bromodeoxyuridine (BrdUrd) or thymidine will increase the baseline sister chromatid exchange (SCE) frequency. The effect is thought to be related to the balance of the DNA precursors thymidine and deoxycytidine. Exogenous addition of deoxycytidine will reverse this effect. Single and twin SCEs were analysed in Colcemid-induced tetraploid Chinese hamster ovary cells exposed to different concentrations of BrdUrd to determine at what stage SCEs are induced by high levels of BrdUrd. In cells exposed to low concentrations of BrdUrd (10^–5 M), equal numbers of SCEs were induced in each of the two cell cycles. With increasing concentrations of BrdUrd (10^–4 to 2×10^–4 M), SCE frequency increased in both cell cycles, but far more SCEs were induced in the second cell cycle. Deoxycytidine (2×10^–4 M) reduced the frequency of SCEs primarily by reducing the frequency of SCEs induced in the second cell cycle. Treatment with 3-aminobenzamide (3AB), a potent inhibitor of poly(ADP-ribose) polymerase, produced effects similar to exposure to high levels of BrdUrd including inducing SCEs in the second replication cycle. This suggests a similar mechanism of action. Deoxycytidine had no effect on 3AB-induced SCEs, however, and there was no interaction between 3AB and high exogenous levels of BrdUrd in SCE induction. Thus these two agents probably act through different mechanisms.     

Methyl methane-sulphonate (MMS) induced SCEs are reduced by the BrdU used to visualise them

SCE induction in synchronised CHO cells treated with methyl methane sulphonate (MMS) in G₁ was studied over successive pairs of cell cycles by introducing bromodeoxyuridine (BrdU) at consecutive G₁ stages. When individual cell cycle SCE values were calculated from the data, anomalous results were obtained with ratios of 1.01.82.1 for the first three cycles but a negative value for the fourth cycle. Further studies using different BrdU concentrations showed that MMS induced SCEs were reduced by values exceeding 50% in DNA containing high levels of incorporated BrdU. This reduction was dose dependent and accounted for the anomalous results obtained over successive cycles. Lesions leading to chromatid exchanges were also reduced by the same mechanism. SCEs induced by UV irradiation were also decreased but those induced by the cross-linking agent nitrogen mustard (HN₂) remained unaffected. The results indicate that not only are SCE lesions induced by MMS, UV or HN₂ expressed independently of the spontaneous SCEs induced by BrdU but that SCE lesions are multiple in nature. Mechanisms by which SCE lesions could be repaired in BrdU containing DNA are discussed. SCE lesions in MMS treated cells arrested in G₁ with arginine deprived medium (ADM) are repaired without the presence of BrdU in the DNA. An opposite effect is seen however in the control cells, where SCEs are increased with time spent in ADM arrest. These interactions between the effects of MMS, BrdU and ADM arrest are discussed.     

Tissue specificity of mitochondrial monooxygenase system for aflatoxin B1 activation

Rat liver mitoplasts free of detectable microsomal contamination can activate significant amounts of aflatoxin B₁ (AFB₁) into electrophilic reactive forms. The activated carcinogen binds to mitochondrial macromolecules and inhibits mitochondrial RNA and protein biosynthesis. The mitochondrial monooxygenase oxygenase system for AFB₁ activation is restricted to liver and kidney as tested by enzyme assays and effects on mitochondrial RNA and protein synthesis.     

Influence of beta-myrcene on sister-chromatid exchanges induced by mutagens in V79 and HTC cells

The influence of beta-myrcene (MC) on sister-chromatid exchanges (SCE) in V79 cells induced by 4 S9 mix-activated indirect mutagens was studied. The mutagens used were cyclophosphamide (CP), benzo[a]pyrene (BP), aflatoxin B1 (AFB) and 9,10-dimethyl-1,2-benz[a]anthracene (DMBA). MC effectively inhibited SCEs induced by CP and AFB in a dose-dependent manner, but it had no effect on SCE induction by BP and DMBA. MC also reduced CP-induced SCE frequencies in a hepatic tumor cell line (HTC). These cells are metabolically competent and activate CP into its biologically active metabolites. Our results support the suggestion that MC modulates the genotoxicity of indirect-acting mutagens by inhibiting certain forms of the cytochrome P-450 enzymes required for activation of premutagens like CP and AFB.     

Differences in murine procarcinogen activation enzymes are not accompanied by parallel differences in procarcinogen-induced sister-chromatid exchange

C57B1/6 and DBA/2 mice, strains in which there is marked induction of hepatic monooxygenase activity by phenobarbital, were tested for in vivo sister-chromatid exchange (SCE) formation in response to cyclophosphamide, an agent metabolized by this inducible enzyme system. Baseline SCE frequencies were between 4 and 6 SCEs/cell in regerating liver and bone marrow of both strains of mice. Administration of cyclophosphamide (5 mg/kg) led to an increase of nearly 8 SCEs/cell in both tissues of C57B1/6 mice and an increase of more than 10 SCEs/cell in DBA/2 mice. Prior exposure to phenobarbital induced p-chloromethylaniline demethylase activity in regenerating liver of both mouse strains approx. 6-fold, but the changes in measured SCE frequencies were not significantly different from those obtained in the absence of enzyme induction. These results, together with our previous observation that induction of by 3-methylcholanthrene of benzo[a]pyrene hydroxylase activity in the same mouse strains was not accompanied by a comparable change in benzo[a]pyrene-induced SCE formation, reinforce the impression that simple assays of differences in mixed function oxidase activities may not necessarily be good predictors of hereditary differences in the response to genetic damage by procarcinogens which are presumed to be metabolized by these enzymes.     

Measurements of chemical carcinogen-induced sister-chromatid exchanges in a whole organ in vitro

Sister-chromatid exchange (SCE) was measured in epithelial cells of mammary organs exposed to 3 different carcinogens in vitro. 7,12-Dimethylbenz[alpha]anthracene (DMBA), N-nitrosodiethylamine (DENA) and N-methyl-N-nitrosourea (MNU) are capable of inducing a significant enhancement of SCE over the basal level in the mammary cells. Analysis of SCE caused by the mutagenic and/or carcinogenic agents in the organ culture presents the advantages that: (1) the culture procedure does not require enzymatic dissociation of the tissue and (2) the medium does not need to be supplemented with serum or exogenous mitogenic agents thereby minimizing the basal level of SCE. Moreover, the assay does not require utilization of exogenous microsomal enzymes needed for activation of certain carcinogenic chemicals. SCE measurements in the organ culture system also should provide clues about the mechanism of action of the chemopreventive agents, particularly the antioxidants, which are under investigation for their possible influence on the processes of neoplastic transformation.     

Inhibition of in vitro aflatoxin B1-DNA binding in rainbow trout by CYP1A inhibitors: α-naphthoflavone, β-naphthoflavone and trout CYP1A1 peptide antibody

Rainbow trout cytochrome P450 (CYP)1A detoxifies aflatoxin B₁ (AFB₁) to aflatoxin M₁ (AFM₁), whereas CYP2K1 activates AFB₁ to AFB₁-8,9-epoxide. We report that α-naphthoflavone (ANF) and β-naphthoflavone (BNF) both strongly inhibit CYP1A-mediated ethoxyresorufin O-deethylase (EROD) activity (K_i = 9.1 ± 0.8 and 7.6 ± 1.1 nM, respectively). These inhibitors (selective for mammalian CYP1A at low concentrations), as well as rabbit polyclonal antibody to a trout CYP1A1 peptide (residues 277–294), also strongly inhibited trout microsome-catalyzed AFB₁-DNA binding and lauric acid (ω-1) hydroxylation in vitro, reactions previously established to be CYP2K1-dependent. ANF at 0.5, 5, 50 and 500 μM inhibited liver microsome-catalyzed AFB₁-DNA binding by 22, 58, 84 and 91%, respectively, whereas BNF at the same concentrations inhibited 22, 74, 78 and 81%, respectively. The CYP1A1 peptide and CYP2K1 polyclonal antibodies (10 mg IgG/mg microsomal protein) inhibited AFB₁-DNA binding by 84 and 66%, respectively, compared with pre-immune IgG. Lauric acid (ω-1) hydroxylation was inhibited 61% by 5 μM ANF, 69% by 5 μM BNF and 100% by either antibody at 12 mg IgG/mg microsomal protein. These results demonstrate that mammalian CYP1A inhibitors also inhibit trout microsomal AFB₁-DNA binding and lauric acid (ω-1) hydroxylation, catalyzed primarily by CYP2K1. In the absence of evidence that trout CYP1A can catalyze AFB₁-DNA binding, the results suggest configuration similarities at, or near, the active sites for these two fish enzymes that result in antibody crossreaction and loss of the inhibitor specificity observed with mammalian CYP1A.     

Influence of low doses of BrdU and estimation of spontaneous SCE in CHO chromosomes: three-way differential staining and an immunoperoxidase method

The influence of low doses of 5-bromodeoxyuridine (BrdU) on the occurrence of sister chromatid exchanges (SCEs) during the first cell cycle, when unsubstituted DNA templates replicate in the presence of the halogenated nucleoside (SCE1) has been assessed in third mitosis (M3) Chinese hamster ovary (CHO) cells showing three-way differential (TWD) staining. In addition, lower concentrations of BrdU, not detectable by Giemsa staining, have been tested by a high resolution immunoperoxidase method (anti-BrdU monoclonal antibody) and SCEs were scored in second mitosis (M2) cells. Our findings was a dose-response curve for SCE1 that allows an estimated mean spontaneous yield of 1.32/cell per cell cycle by extrapolation to zero concentration of BrdU. On the other hand, when the total SCE frequency corresponding to the first and second rounds of replication (SCE1+SCE2) found in M3 chromosomes was compared with the yield of SCEs scored in M2 cells grown in BrdU at doses lower than 1 M no further reduction was achieved. This seems to indicate that SCEs can occur spontaneously in this cell line, though the estimated frequency is higher than that reported in vivo.by S. Wolff     

Interrelationships of dietary protein level,aflatoxin B1 metabolism,and hepatic microsomal epoxide hydrase activity

In a continuation of studies on protein intake and aflatoxin B₁ (AFB₁) metabolism, weanling rats were fed semipurified diets containing either 20% casein or 5% casein for two weeks to determine the effect of dietary protein level on hepatic microsomal epoxide hydrase activity and AFB₁ metabolism in an effort to evaluate the role of protein intake on the formation and degradation of the reactive metabolite of AFB₁. Styrene oxide was used as substrate for epoxide hydrase since the hypothetical AFB₁ 2,3-epoxide (AFB-epox) cannot be synthesized because of its lability. Two groups of animals were fed 20% casein diets; one was fed ad libitum and the second was pair fed to the 5% casein group in order to control the effects of total feed intake. The depression of epoxide hydrase activities caused by the 5% casein diets was approximately equivalent to that previously seen with hepatic microsomal mixed function oxidase (MFO) activities with the identical protocol. Similarly, the metabolism of AFB₁ to AFQ₁ and AFM₁ was depressed by the 5% casein diets, with an increase in the production of chromatographically more polar material. The relationship of the MFO and epoxide hydrase activities to AFB₁ metabolism and formation of macromolecular adducts is discussed.