Active cell death in hormone-dependent cells was studied using cultured human mammary carcinoma cells (MCF-7) treated with the anti-estrogens (AEs) tamoxifen (TAM), 4-hydroxy-tamoxifen (OH-TAM) or ICI 164 384 (10(-8)-10(-5) M) as a model. The following results were obtained. (i) In untreated MCF-7 cells a wave of replication occurred in the first 5 days of culture. All three AEs caused a dose-dependent inhibition of cell replication. (ii) TAM and OH-TAM at 10(-5) M, but not ICI 164 384, caused lytic cell death (necrosis) within 24 h, which was not inhibited by estradiol (10(-9)-10(-6)M). (iii) Lower concentrations of TAM or OH-TAM (up to 10(-6) M) or ICI 164 384 induced a more gradual appearance of cell death beginning at day 3. This type of cell death was inhibited by estradiol (10(-9) M), indicating its active nature. (iv) Nuclei showed two distinct patterns of alteration: (a) apoptosis-like condensation and fragmentation of chromatin to crescent masses abutting the nuclear envelope; (b) condensation of the chromatin to a single, pyknotic mass in the center of the nucleus, detached from the nuclear envelope. Quantitative histological evaluation revealed the predominance of pyknosis. (v) Biochemical DNA analysis revealed that only a relatively small amount of the total DNA was finally degraded into low molecular weight fragments (20 kb and less). (vi) Active cell death, with both apoptotic and pyknotic nuclear morphology, was associated with extensive formation of autophagic vacuoles (AV).3-Methyladenine, a known inhibitor of AV formation, partially prevented cell death as detected by nuclear changes. (vii) ICI 164 384 was about 10 times more effective than TAM or OH-TAM at inhibiting DNA synthesis, but had equal potency in inducing active cell death. It is concluded that AEs have anti-proliferative and anti-survival effects on MCF-7 human mammary cancer cells in culture. These two effects are under separate control because they differ by kinetics, dose dependence and sensitivity to the various AEs. Active cell death in MCF-7 cells seems to be initiated by autophagy, in contrast to concepts of apoptosis, and thus corresponds to autophagic/ lysosomal or type II death as previously defined. This may be important because of biochemical and molecular differences between these various subtypes of active cell death.
Active cell death, the genetically programmed self-destruction of a cell, is now recognized to be a widespread phenomenon in biology that counterbalances mitosis to preserve tissue homeostasis. It is subject to the control of the growth regulatory networks in tissues. Close examination of the morphology of dying cells in liver and other tissues suggests that there are a number of morphological types of active cell death, ranging from forms dominated by nuclear changes and without signs of autophagy ("classical" apoptosis), e.g., in thymocytes and the liver, to those dominated by autophagic degradation of cytoplasm, e.g., in the mammary gland. The induction of gene expression in these diverse types of cell death is anticipated to be different. Here we review the data regarding the regulation of apoptosis in the liver by liver tumor promoter, transforming growth factor beta-1 and related peptides as well as nutrition. In the course of hepatocarcinogenesis, initiated cells as well as preneoplastic and neoplastic cell populations showed enhanced cell replication, but also enhanced apoptosis. Tumor promoter shift the balance between birth and death by increasing the rate of cell replication and by decreasing the rate of apoptosis. Thereby, liver tumor formation is accelerated. Food restriction exhibits the opposite effect and consequently, provides protection from carcinogenesis.
The induction of organ-specific genotoxic effects of five cooked food mutagens in Swiss albino mice was investigated in microbial animal-mediated assays. The indicator of the induction of DNA damage was a pair of Escherichia coli K12 strains, differing vastly in repair capacity (uvrB/recA versus uvr+/rec+). All compounds gave positive results in the tested dose range between 2.5 and 40 mg/kg body weight (i.p. administration, exposure time 120 min). 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) were slightly more genotoxic than 2-amino-3,8-dimethylimidazo[4,5-f]quinoline (MeIQx), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) which caused similar effects. When the compounds were administered orally, higher doses were required to induce repairable DNA damage. The pattern of organ-specific effects was essentially similar for all compounds; genotoxicity was most pronounced in livers and lungs, whereas in kidneys, spleen and testes comparatively lower effects were measured. The activity of PhIP, MeIQ and IQ in the blood was similar to that observed in the liver. The results obtained in vivo were compared with data gained in vitro with subcellular organ fractions. Our findings indicate the following. (i) The concentrations required to induce repairable DNA damage in microbial animal-mediated assays are substantially higher than might be expected on the basis of the liquid suspension tests. (ii) The ranking order of the genotoxicity of the various compounds in vitro is similar to that measured in vivo, but the differences in genotoxic potencies are less pronounced in the living animal.(ABSTRACT TRUNCATED AT 250 WORDS)
The influence of various dietary constituents--phenethylisothiocyanate (PEITC), oleic acid (OA), triolein (TO), and vitamin A (ROL)--on the genotoxic activity of nitrosamines (NDMA, NDELA, NPYR) was investigated. For this purpose differential DNA repair assays with Escherichia coli K-12 strains were performed in vitro and in vivo with mice. Under in vitro conditions (liquid holding), all compounds reduced nitrosamine induced DNA-damage in the indicator bacteria in the dose range 1-10 micrograms/ml, the ranking order of efficiency being PEITC greater than OA greater than ROL greater than or equal to TO. In animal-mediated assays, acute oral treatment with PEITC (17-150 mg/kg), 2 h before nitrosamine administration, resulted in a marked decrease of nitrosamine genotoxicity in liver, kidneys, lungs and in the blood. Also in other organs (spleen, testes) an increase in differential survival (which serves as a measure for repairable DNA damage) occurred. With ROL only a comparatively moderate antigenotoxic effect was obtained at a high dose level (250 mg/kg) under identical experimental conditions. OA (2000 mg/kg) and TO (16,000 mg/kg) were completely inactive. Upon repeated treatment (consecutive oral administration of the putative antigenotoxins over 4 days, a final treatment 24 h before nitrosamine administration) PEITC (150 mg/kg/day), ROL (80 mg/kg/day) and OA (2000 mg/kg/day) had no influence on the genotoxic effects of the nitrosamines. Repeated treatment with TO (4000-16,000 mg/kg/day) resulted in a moderate dose-dependent reduction of NDMA-induced DNA-damage in the indicator bacteria, whereas in combination with NPYR only a marginal effect was observed. Biochemical experiments indicated that the antigenotoxic effects of PEITC seen under in vivo conditions were due to inhibition of alpha-hydroxylation of the nitrosamines, whereas ROL and TO appeared not to interfere strongly with this metabolic activation step. Our results indicate that in vitro assays do only partly reflect the antigenotoxic properties of the different food constituents in vivo and that animal-mediated DNA repair assays with E. coli strains are an appropriate approach to study the effects of modifiers of nitrosamine genotoxicity in the living animal.
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