2,[3]tert-butyl-4-hydroxyanisole does not reduce SCE induction by benzo(a) pyrene in bone marrow cells of C57BL/6 mice

Recently, a number of publications have suggested that bone marrow cytogenetics may be used to detect anticarcinogenic or antimutagenic activity. In this work, 0.75% 2,[3]-tert-butyl-4-hydroxyanisole (BHA), fed in the diet for 2 weeks, was tested for its ability to reduce the frequency of benzo(a)pyrene (BP)-induced SCE in mouse bone marrow. C57BL/6 male mice, were injected i.p. with BP at 0, 33, 67, and 100 mg/kg body weight. The mean SCE/chromosome +/- s.e.m. for animals on control diet was 0 mg/kg, 0.108 +/- 0.005; 33 mg/kg, 0.225 +/- 0.011; 67 mg/kg, 0.289 +/- 0.012; 100 mg/kg, 0.311 +/- 0.013. The mean SCE/chromosome +/- s.e.m. for animals on the 0.75% BHA diet was 0 mg/kg, 0.105 +/- 0.006; 33 mg/kg, 0.224 +/- 0.009; 67 mg/kg, 0.262 +/- 0.013; 100 mg/kg, 0.326 +/- 0.012. There are no significant differences between animals on the control and BHA diets. Excretion of BP in urine over a 72 hr time period was significantly increased in animals on the BHA diet, at both low and high doses. Water-soluble metabolites accounted for all of this increase. It appears that bone marrow is not a good model for the gastrointestinal tract, and that short-term assays for anticarcinogens or antimutagens are more likely to be predictive if they are done in the target organs.     

Effect of vitamin C or {beta}-carotene on SCE induction by gamma rays in radiosensitized murine bone marrow cells in vivo

The in vivo effect of vitamin C or ß–caroteneon sister chromatid exchange (SCE) radio–induction wasdetermined in murine bone marrow cells sensitized by BrdU incorporation.Pre– or post–treatment with 100 mg/ kg body wt vitaminC did not cause a significant reduction in SCE induced by theexposure to 0.63 Gy     

In vivo persistence of sister chromatid exchanges (SCE) induced by gamma rays in mouse bone marrow cells

The sister chromatid exchange (SCE) frequencies induced in bone marrow cells by in vivo irradiation with gamma rays before or after bromodeoxyuridine (BrdUrd) incorporation were compared. The frequency of SCE at different postirradiation times was also measured in bone marrow cells in vivo, irradiated before BrdUrd incorporation. Increased sensitivity to SCE induction by radiation was found in cells after BrdUrd incorporation for one cycle when compared with cells irradiated before BrdUrd incorporation. The increased SCE frequency persisted for at least 72 hr after the initial irradiation, implying that the gamma ray-induced lesion(s) capable of eliciting an SCE are persistent and cannot be easily repaired.     

Vitamin A can prevent genetic damage induced by benzo(a)pyrene to the bone marrow cells of mice

Vitamin A and its analogues are now known to be of help in the prevention of cancer. One of the mechanisms by which cancer can occur is due to the damage to DNA. Hence, we have investigated the effect of vitamin A on genetic damage induced by benzo(a)pyrene, a known mutagen and co-carcinogen, to the bone marrow cells of mice. The results suggest that vitamin A can prevent genetic damage caused by benzo(a)pyrene.     

Effect of benzo(a)pyrene on monolayer cultures of normal and malignant mouse liver cells

Cells of a monolayer culture of embryonic mouse liver, like cells of a culture of highly malignant hepatoma 22A, maintained by transplantation for 20 years, actively metabolized the carcinogenic hydrocarbon benzo(a)pyrene and are highly sensitive to its toxic action. Considering that liver tissue in vivo is resistant to carcinogenic hydrocarbons, the authors suggest that this resistance is due to factors acting at the organ or organism level but not at the cell level. The problem of the mechanism of preservation of the sensitivity of hepatoma 22A to the toxic action of benzo(a)pyrene also is discussed.Laboratory of Chemical Carcinogenesis, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 9, pp. 346–349, September, 1977.     

The kinetics of in vivo sister chromatid exchange induction in mouse bone marrow cells by alkylating agents: cyclophosphamide

Administration of cyclophosphamide (5, 10, 20 and 25 mg/kg body weight) to male CD-1 mice 2 hr after subcutaneous implantation of a 5-bromo-2'-deoxyuridine (BrdUrd) pellet (55 mg) resulted in a dose-dependent increase in sister chromatid exchanges (SCE) in bone marrow cells. Treatment with cyclophosphamide (15 mg/kg body weight) at the time of BrdUrd implantation and 2, 6.5, and 13 hr post-BrdUrd implantation resulted in the induction of approximately 19 SCE/cell indicating that the bone marrow SCE response was independent of the time of administration. Treatment with cyclophosphamide (15 mg/kg body weight) at 26, 19, 13, and 6 hr prior to BrdUrd implantation resulted in baseline SCE (3.3 SCE/cell) at 26 hr with an increasing number of SCE/cell with decreasing time prior to BrdUrd implantation. These results compare favorably with those obtained by Kram et al [1981] with mitomycin C (MMC) using a similar protocol. The time-dependent induction of SCE is qualitatively similar for CP and MMC, both of which are bifunctional alkylating agents metabolically activated by oxidation and reduction, respectively, and suggests that these two compounds may induce SCE by a similar mechanism.     

Metabolism of aflatoxin B1, benzo[a]pyrene, and 1,2-dimethylhydrazine by cultured rat and human colon

A model system for comparing carcinogen metabolism between human and rat colon has been developed. Tissue explants maintained under chemically defined conditions were treated with radioactively labeled carcinogens. After incubation for 24 hours, the binding of radioactive carcinogen to DNA was quantitated. Further, the carcinogen-DNA adducts and carcinogen metabolites released into the culture media were identified. Both human and rat colon activate benzo[a]pyrene (BP), aflatoxin B1 (AFB), and 1,2-dimethylhydrazine (DMH) into chemical species that reacted with cellular macromolecules. When human and rat colons were compared, the metabolism of AFB and DMH was qualitatively similar - the same major carcinogen-DNA adducts and metabolic profile. However, the mean binding levels of DMH and AFB to colonic DNA were higher in rats than in humans. BP-guanine adducts were the major adducts formed by both rat and human colonic DNA. However, BP-adenine adducts were observed in rat colonic DNA but not in human colonic DNA. A positive correlation for the binding of BP and DMH to human DNA of different individuals was observed, but no correlation was found between BP and AFB. The data suggest that similar enzyme systems may be involved in the metabolism of BP and DMH, whereas different enzymes might be involved in the metabolic activation of AFB.     

Aneuploidy induction by water extract from Tripterygium hypoglaucum (Level) Hutch in mouse bone marrow cells

Hie aneuploidy-inducing activity of a Chinese medicinal herb,Tripterygium hypoglaucum (level) Hutch (THH), was investigatedby means of three cytogenetic end-points, i.e. C-mitotic (CM)effects, micronuclei (MN) and parallel chromosome structuralaberration (CA) analyses in vivo. The CA analysis was expectedto reflect the origins of MN induced by clastogens or aneugens.The experiments were performed on mouse bone marrow cells. Theanimals were treated with the crude water extracts of THH (singlei.p. injection) in the dose range 120–686 mg/kg. Colchicine(COL) was taken as a positive control for its known aneuploidy-inducingeffects. THH showed similar genotoxic effects to COL in CM,MN and CA analyses: positive CM effects were observed accompaniedwith increases of mitotic index and frequencies of CM cellsas well as decreased frequencies of anaphase in all of the THH-treatedgroups. The compound showed a positive MN response in bone marrowpolychromatic erythrocytes but was negative in CA analyses.No sex differences were found in any treated group. The preliminaryresults suggested that THH is an aneuploidy inducer in mousebone marrow cells under the present experimental conditions.     

Effects of black tea theafulvins on aflatoxin B(1) mutagenesis in the Ames test

Black tea theafulvins, a fraction of thearubigins isolated from black tea aqueous infusions, potentiated the mutagenic activity of the mycotoxin aflatoxin B(1) in the Ames test, in the presence of a hepatic S9 activation system derived from Aroclor 1254-treated rats. In contrast, when the S9 activation system was replaced with isolated microsomes, theafulvins suppressed the mutagenicity of the mycotoxin. When microsomal metabolism was terminated after metabolic activation of the mycotoxin, incorporation of the theafulvins into the activation system reduced the mutagenic activity, whereas if it was added before termination of microsomal activity a potentiation of mutagenic response was observed. In in vitro studies, theafulvins inhibited epoxide hydrolase and glutathione S-transferase activities in a concentration-dependent manner. Finally, the mutagenicity of aflatoxin B(1) was much more pronounced in bacteria that were pre-exposed to theafulvins but from which they were subsequently washed off. It may be inferred from the above studies that the genotoxic synergy between aflatoxin B(1) and black tea theafulvins does not occur during the bioactivation of the carcinogen, but may partly be due to decreased deactivation of the reactive intermediate, aflatoxin B(1) 8,9-oxide, by conjugation with glutathione.     

Metabolic differences between whole blood and isolated lymphocyte cultures for micronucleus (MN) induction by cyclophosphamide and benzo[a]pyrene

In order to study the metabolic differences between whole bloodand isolated lymphocyte cultures, two indirectly acting mutagenscyclophosphamide (CP) and benzo[a]pyrene (B[a]P) were assessedfor their potential to induce micronuclei (MN) in the presenceand absence of S9 microsomal fractions. In isolated lymphocytecultures supplemented with S9, CP and B[a]P induced a statisticallysignificant increase in MN which was not observed in whole bloodcultures. However, the directacting agent methyl methanesulphonate(which was used as a positive control) showed an increase inMN frequency in a dose-dependent manner in both culture methods.The effect of erythrocytes was then investigated by treatingisolated lymphocyte cultures simultaneously with CP and S9 mixin the presence of purified erythrocyte concentrate (PEC). Aclear reduction in the MN frequency was observed compared tothe frequencies of MN induced in isolated lymphocyte culturestreated with CP and S9 mix in the absence of PEC. Thus, isolatedlymphocyte cultures may represent a more sensitive test systemfor the evaluation of potential indirectacting mutagens. However,whole blood cultures may reflect the ‘real life’situation more accurately as a consequence of the presence oferythrocytes.     

Infection of rats with Taenia taeniformis metacestodes increases hepatic CYP450, induces the activity of CYP1A1, CYP2B1 and COH isoforms and increases the genotoxicity of the procarcinogens benzo[a]pyrene,cyclophosphamide and aflatoxin B(1)

Infection of rat liver by Taenia taeniformis metacestodes produced an increase in total CYP450 content and induced activity of the CYP1A1, CYP2B1 and COH isoforms. Variations in activity and p450 total content were found with increasing time of infection. During increased activity of p450 isoforms, rats were challenged with carcinogens metabolized by the mentioned isozymes and an increased amount of genotoxic damage was found when benzo[a] pyrene, cyclophosphamide and aflatoxin B(1) were used. No change was seen in CYP2E1 activity. These results support previous findings regarding an increased susceptibility to genotoxic damage of infected organisms.     

Growth rate and sister chromatid exchange (SCE) incidence of bone marrow cells in Acute Myeloblastic Leukemia (AML)

The sister chromatid exchange (SCE) incidence and growth kinetics have been studied by means of an in vitro bromodeoxyuridine (BrdU) chromosome labeling method in the bone marrow cells of 17 acute myeloblastic leukemia (AML) patients with only diploid cells at diagnosis, remission, and relapse of the disease. At diagnosis, the cells tended to exhibit a low SCE frequency as compared to that during remission. An increased SCE frequency was observed after chemotherapy during remission or relapse. At diagnosis and relapse, when leukemic blast cells predominated in the marrow, they were characterized by the predominance of cells that had undergone only one cell cycle after BrdU exposure. In contrast, the marrow cells during remission tended to resemble the control pattern of growth kinetics, with a predominance of cells undergoing second and third cell cycles in the presence of BrdU. These results suggest that the growth rate of leukemic and nonleukemic cells is different, and that chemotherapy can cause an increased SCE frequency in the marrow cells of AML patients irrespective of the state of the disease.     

Modulation of interleukin-1 production by macrophages following benzo(a)pyrene exposure

The effects of benzo(a)pyrene (BaP), a highly prevalent environmental carcinogen, on the ability of peritoneal exudate macrophages to produce the secretory immunomodulatory molecule interleukin-1 (IL-1) in vitro was examined. A dose-dependent increase in lipopolysaccharide stimulated IL-1 production concomitant with decreased cell viabilities was noted in macrophages cultured in the presence of BaP. Antibody responses which are suppressed in BaP dosed mice can be reconstituted in vitro by the addition of exogenous interleukin-1. These results indicate that one of the cellular targets of BaP induced immunosuppression may be cells of the macrophage-monocyte lineage. Furthermore, BaP induced suppression of antibody responsiveness may be a result of alterations in production of IL-1.     

Dose-response studies on neoplastic transformation of BALB/3T3 clone A31-1-1 cells by aflatoxin B1, benzidine, benzo[a]pyrene, 3-methylcholanthrene, and N-methyl-N'-nitro-N-nitrosoguanidine

The BALB/3T3 clone A31-1-1 mouse embryo cell line at passages 7 to 13 was selected for morphologic studies of neoplastic transformation by carcinogens of different chemical classes, in the absence of any added extracellular metabolic activation. Dose-related transforming activity was demonstrated for the carcinogens aflatoxin B1 (AFB) and benzidine (BZ) not previously reported in this system, and was confirmed for benzo[a]pyrene (BP), 3-methylcholanthrene (MCA), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Spontaneous transformation per cells at risk was low (0.14 type III foci x 10(-4), while chemically induced transformation was 2 to 3 orders of magnitude higher with all compounds. The molar concentration of carcinogens in complete medium, required to induce a transformation frequency of 1.0 type III foci x 10(-3) showed the highest level of activity for BP (0.04 microns), an intermediate level for AFB (0.2 to 1.4 microns), MCA (1.1 micron), and MNNG (2.3 microns), and the lowest level of activity for BZ (30.0 microns). The dose-related induction of morphological transformation in this clone by carcinogens of different classes indicates the potential value of this biological system in quantitative studies of carcinogen combinations, especially at low dose levels.     

Inhibitory effects of biochanin A on mouse lung tumor induced by benzo(a)pyrene.

Biochanin A, an isoflavone compound, is reported to have an inhibitory effect on benzo(a)pyrene [B(a)P] metabolism. We examined the modifying effect of biochanin A on in vivo carcinogenesis using a mouse lung tumor model. As carcinogens, a single subcutaneous injection of 0.5mg of B(a)P was given within 24 hours after birth. The test groups were injected with 0.125mg of biochanin A in 0.1ml DMSO by i.p. 3 times a week for 6 weeks after weaning. All mice were sacrificed at week 9 and the incidence and multiplicity of lung tumors were examined. Concomitant administration of biochanin A showed a significant inhibitory effect on the incidence of tumor-bearing mice (12.5%, P < 0.01), as well as the mean number of tumors (0.13, P < 0.001), compared with the group treated with B(a)P alone in which the incidence was 57.1% and the mean number was 1.0. These results suggest that biochanin A has inhibitory potential on the development of mouse lung tumor induced by B(a)P.     

Micronucleus formation by benzene, cyclophosphamide, benzo(a)pyrene, and benzidine in male, female, pregnant female, and fetal mice

Male, female, pregnant female, and fetal ICR mice were compared for their acute sensitivity to four single doses of model carcinogens, as measured by micronucleus formation in polychromatic erythrocytes 24 h after treatment in adult bone marrow and fetal liver at days 17-19 of gestation. Cyclophosphamide caused a dose-responsive increase in micronuclei in all groups, without a consistent difference based on gender or pregnancy. At doses of 50 and 75 mg/kg given orally to the pregnant female, the fetuses were three to six times as sensitive as was the mother. Benzo(a)pyrene showed a similarly increased sensitivity of the fetus relative to the other groups, although it is a much weaker clastogen. Benzidine did not cause an increase in micronuclei in any group, although it was thought that the fetal liver might have been sensitive enough to detect it, relative to adult bone marrow. Benzene caused much less response in females than in males and almost no response in pregnant females and their fetuses, even though pregnant females metabolized at least half as much of the total dose as did the males (as measured by the presence of urinary metabolites of benzene). No single metabolite of benzene in the urine was consistently correlated with micronucleus formation in the bone marrow. Several factors must be interacting in different ways for different chemicals to influence their clastogenicity.     

Activation of benzo[a]pyrene and aflatoxin B1 to mutagenic chemical species by microsomal preparations from rat liver and small intestine in relation to microsomal epoxide hydrolase

Rat small intestinal microsomes have been compared with liverpreparations for their ability to activate promutagens usingthe Salmonella mutagenicity assay. Induced levels of arylhydrocarbonhydroxylase and cytochrome P-450 in intestinal microsomes aresignificantly lower than the corresponding amounts in livermicrosomes. Greater activation of benzo[a]pyrene (BP) by liverextracts would thus be expected. Although this was observedat >1 µg BP/plate, at lower doses comparatively highlevels of activation were obtained with intestinal microsomes.This could be due to preferential formation of the mutagenic4,5-oxide with intestinal microsomes, as opposed to the putativemajor active metabolite, the 7,8-diol-9,10-epoxide. Microsomalepoxide hydrolase inactivates the K-region epoxide by formingthe corresponding dihydro-diol. Differences in the levels ofthese metabolites may thus be a result of higher activity ofthe enzyme in liver extracts. This hypothesis has been studiedusing the epoxide hydrolase inhibitor, 1,2-epoxy-3,3,3-trichloropropyleneoxide (TCPO). Enzyme activity has been measured using [³H]-BP-4,5-oxideas substrate. Since aflatoxin B¹ (AFB) may also be activatedvia analogous epoxide intermediates, the effects of TCPO onactivation of AFB were also investigated. In testinal microsomalexpoxide hydrolase activities were significantly lower thanthose in liver preparations obtained from animals pre-treatedwith enzyme inducers. Enzyme activity and promutagen activationability of intestinal microsomes, respectively, were less susceptibleto and not inhibited by TCPO. However, TCPO strongly inhibitedmicrosomal epoxide hydrolase activity and activation of BP andAFB due to liver microsomes. The differences in dose-responsesfor mutagenicity of BP and AFB obtained are discussed with respectto the relative involvement of epoxide hydrolase in the activationof the two promutagens by the different microsomal preparationsused. ^*To whom reprint requests should be addressed