Regulation of diapause in carnivores.

Embryonic diapause is an evolutionary strategy to ensure that offspring are born when maternal and environmental conditions are optimal for survival. In many species of carnivores, obligate embryonic diapause occurs in every gestation. In mustelids, the regulation of diapause and reactivation is influenced by photoperiod, which then acts to regulate the secretion of pituitary prolactin. Prolactin in turn regulates ovarian steroid function. Reciprocal embryo transplant studies indicate that this state of embryonic arrest is conferred by uterine conditions and is presumed to be due to a lack of specific factors necessary for continued development. Studies of global gene expression in the mink (Neovison vison) revealed reduced expression of a cluster of genes that regulate the abundance of polyamines in the uterus during diapause, including the rate-limiting enzyme in polyamine production, ornithine decarboxylase (ODC). In addition, in this species, in vivo inhibition of the conversion of ornithine to the polyamine, putrescine, induces a reversible arrest in embryonic development and an arrest in both trophoblast and inner cell mass proliferation in vitro. Putrescine, at 0.5, 2 and 1,000 µM concentrations induced reactivation of mink embryos in culture, indicated by an increase in embryo volume, observed within five days. Further, prolactin induces ODC1 expression in the uterus, thereby regulating uterine polyamine levels. These results indicate that pituitary prolactin acts on ovarian and uterine targets to terminate embryonic diapause. In summary, our findings suggest that the polyamines, with synthesis under the control of pituitary prolactin, are the uterine factor whose absence is responsible for embryonic diapause in mustelid carnivores.

Ubiquitin-independent proteasomal degradation.

Most proteasome substrates are marked for degradation by ubiquitin conjugation, but some are targeted by other means. The properties of these exceptional cases provide insights into the general requirements for proteasomal degradation. Here the focus is on three ubiquitin-independent substrates that have been the subject of detailed study. These are Rpn4, a transcriptional regulator of proteasome homeostasis, thymidylate synthase, an enzyme required for production of DNA precursors and ornithine decarboxylase, the initial enzyme committed to polyamine biosynthesis. It can be inferred from these cases that proteasome association and the presence of an unstructured region are the sole prerequisites for degradation. Based on that inference, artificial substrates have been designed to test the proteasome's capacity for substrate processing and its limitations. Ubiquitin-independent substrates may in some cases be a remnant of the pre-ubiquitome world, but in other cases could provide optimized regulatory solutions. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

A prolonged and exaggerated wound response with elevated ODC activity mimics early tumor development.

Induction of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, in ODC transgenic skin stimulates epidermal proliferation but not hyperplasia, activates underlying stromal cells and promotes skin tumorigenesis following a single subthreshold dose of a carcinogen. Because chronic wounds are a well-recognized risk factor for skin cancer, we investigated the response to a tissue remodeling event in normal skin that is abraded to remove only the epidermal layer in K6/ODC transgenic (follicular ODC expression) and in inducible ODCER transgenic mice (suprabasal ODC expression). When regenerative epidermal hyperplasia was resolved in normal littermates following abrasion, ODC transgenic mice exhibited progressive epidermal hyperplasia with formation of benign tumor growths and maintained an increased epidermal proliferation index and activation of translation-associated proteins at abrasion sites. The epidermal hyperplasia and tumor-like growth was accompanied by activation of underlying stromal cells and prolonged infiltration of inflammatory cells. Treatment with the anti-inflammatory agent dexamethasone did not reduce the high proliferative index in the regenerated epidermis but dramatically reduced the epidermal hyperplasia and prevented the wound-induced tumor growths in abraded ODCER skin. Treatment with α-difluoromethylornithine, a specific inhibitor of ODC activity, normalized the wound response in transgenic mice and decreased wound-induced inflammation if administered from the time of abrasion but not if initiated 4 days following abrasion. These results suggest a role for polyamines in prolonging wound-associated inflammation in addition to stimulating proliferation both of which are sufficient to sustain epidermal hyperplasia and benign tumor growth even in the absence of genetic damage.

Recurrent emergence of catalytically inactive ornithine decarboxylase homologous forms that likely have regulatory function.

Ornithine decarboxylase (ODC) catalyzes the first and rate limiting step in the biosynthesis of polyamines in most eukaryotes. Because polyamines have pleiotropic and often dramatic effects on cellular processes at both high and low concentrations, ODC expression is tightly controlled. ODC is regulated by a family of polyamine-induced proteins, antizymes, which bind to, and inactivate it. In mammals, and apparently most vertebrates, antizymes are in turn antagonized by proteins called antizyme inhibitors. Antizyme inhibitors are homologs of ODC that have lost their decarboxylation activity but have retained their ability to bind antizyme, in most cases even more tightly than ODC. We present a phylogenetic analysis of over 200 eukaryotic homologs of ODC and evaluate their potential to be either true ODCs or catalytically inactive proteins that might be analogs of the previously identified antizyme inhibitors. This analysis yielded several orthologous groups of putative novel antizyme inhibitors each apparently arising independently. In the process we also identify previously unrecognized ODC paralogs in several evolutionary branches, including a previously unrecognized ODC paralog in mammals, and we evaluate their biochemical potential based on their pattern of amino acid conservation.

Ornithine decarboxylase is important in intestinal mucosal maturation and recovery from injury in rats.

A transient increase in ornithine decarboxylase activity and polyamine biosynthesis occurs in the intestinal mucosa of the newborn rat in the third week after birth. During this period, there is a rapid conversion of the mucosa from a fetal to a mature adult status. A similar increase in ornithine decarboxylase activity also accompanies the rapid recovery of the mucosa 1 week after an injury is induced by chemotherapy in adult rats. In vivo, alpha-difluoromethyl ornithine, a highly selective, enzyme-activated, irreversible inhibitor, suppresses these increases in mucosal ornithine decarboxylase and delays both intestinal mucosal maturation and recovery from injury. Thus increased ornithine decarboxylase activity, with the resultant increase in polyamine content, may play an essential role in intestinal mucosal maturation and regeneration in the rat.

Ornithine decarboxylase: essential in proliferation but not differentiation of human promyelocytic leukemia cells.

The ornithine decarboxylase inhibitor DL-alpha-difluoromethyl ornithine inhibited a proliferation-associated increase in ornithine decarboxylase activity in cultured human promyelocytic leukemia cells, resulting in a marked suppression of cell proliferation and subsequent cell loss. It also inhibited increases in ornithine decarboxylase activity associated with the phorbol ester-induced conversion of promyelocytic HL-60 cells to monocyte-like cells and the retinoic acid-induced conversion to granulocyte-like cells. However, the inhibition of ornithine decarboxylase activity did not prevent cellular differentiation. These results suggest that polyamine biosynthesis has a specific role in cell proliferation rather than in inducing differentiation that is not accompanied by proliferation. The data also demonstrate that cessation of proliferation in HL-60 cells is not necessarily associated with differentiation.

L-Ornithine decarboxylase:an essential role in early mammalian embryogenesis.

The highly selective, enzyme-activated, irreversible inhibitor of L-ornithine decarboxylase, DL-alpha-difluoromethylornithine, suppresses the increase in uterine L-ornithine decarboxylase activity associated with early embryogenesis in the mouse and arrests embryonic development at that stage. Contragestational effects were confirmed in the rat and rabbit. An increase in L-ornithine decarboxylase activity that leads to a rapid increase in putrescine concentration appears to be essential during a critical period after implantation for continued mammalian embryonal growth.

Protein meta-functional signatures from combining sequence, structure, evolution, and amino acid property information.

Protein function is mediated by different amino acid residues, both their positions and types, in a protein sequence. Some amino acids are responsible for the stability or overall shape of the protein, playing an indirect role in protein function. Others play a functionally important role as part of active or binding sites of the protein. For a given protein sequence, the residues and their degree of functional importance can be thought of as a signature representing the function of the protein. We have developed a combination of knowledge- and biophysics-based function prediction approaches to elucidate the relationships between the structural and the functional roles of individual residues and positions. Such a meta-functional signature (MFS), which is a collection of continuous values representing the functional significance of each residue in a protein, may be used to study proteins of known function in greater detail and to aid in experimental characterization of proteins of unknown function. We demonstrate the superior performance of MFS in predicting protein functional sites and also present four real-world examples to apply MFS in a wide range of settings to elucidate protein sequence-structure-function relationships. Our results indicate that the MFS approach, which can combine multiple sources of information and also give biological interpretation to each component, greatly facilitates the understanding and characterization of protein function.

Oncogene expression profiles in K6/ODC mouse skin and papillomas following a chronic exposure to monomethylarsonous acid.

We have previously observed that a chronic drinking water exposure to monomethylarsonous acid [MMA(III)], a cellular metabolite of inorganic arsenic, increases tumor frequency in the skin of keratin VI/ornithine decarboxylase (K6/ODC) transgenic mice. To characterize gene expression profiles predictive of MMA(III) exposure and mode of action of carcinogenesis, skin and papilloma RNA was isolated from K6/ODC mice administered 0, 10, 50, and 100 ppm MMA(III) in their drinking water for 26 weeks. Following RNA processing, the resulting cRNA samples were hybridized to Affymetrix Mouse Genome 430A 2.0 GeneChips(R). Micoarray data were normalized using MAS 5.0 software, and statistically significant genes were determined using a regularized t-test. Significant changes in bZIP transcription factors, MAP kinase signaling, chromatin remodeling, and lipid metabolism gene transcripts were observed following MMA(III) exposure as determined using the Database for Annotation, Visualization and Integrated Discovery 2.1 (DAVID) (Dennis et al., Genome Biol 2003;4(5):P3). MMA(III) also caused dose-dependent changes in multiple Rho guanine nucleotide triphosphatase (GTPase) and cell cycle related genes as determined by linear regression analyses. Observed increases in transcript abundance of Fosl1, Myc, and Rac1 oncogenes in mouse skin support previous reports on the inducibility of these oncogenes in response to arsenic and support the relevance of these genomic changes in skin tumor induction in the K6/ODC mouse model.

Human ornithine decarboxylase paralogue (ODCp) is an antizyme inhibitor but not an arginine decarboxylase.

ODC (ornithine decarboxylase), the rate-limiting enzyme in polyamine biosynthesis, is regulated by specific inhibitors, AZs (antizymes), which in turn are inhibited by AZI (AZ inhibitor). We originally identified and cloned the cDNA for a novel human ODC-like protein called ODCp (ODC paralogue). Since ODCp was devoid of ODC catalytic activity, we proposed that ODCp is a novel form of AZI. ODCp has subsequently been suggested to function either as mammalian ADC (arginine decarboxylase) or as AZI in mice. Here, we report that human ODCp is a novel AZI (AZIN2). By using yeast two-hybrid screening and in vitro binding assay, we show that ODCp binds AZ1-3. Measurements of the ODC activity and ODC degradation assay reveal that ODCp inhibits AZ1 function as efficiently as AZI both in vitro and in vivo. We further demonstrate that the degradation of ODCp is ubiquitin-dependent and AZ1-independent similar to the degradation of AZI. We also show that human ODCp has no intrinsic ADC activity.

Polyamines are essential in embryo implantation: expression and function of polyamine-related genes in mouse uterus during peri-implantation period.

Polyamines are key regulators in cell growth and differentiation. It has been shown that ornithine decarboxylase (Odc) was essential for post-implantation embryo development, and overexpression of spermidine/spermine N1-acetyltransferase will lead to ovarian hypofunction and hypoplastic uteri. However, the expression and function of polyamine-related genes in mouse uterus during early pregnancy are still unknown. In this study we investigated the expression, regulation, and function of polyamine-related genes in mouse uterus during the peri-implantation period. Odc expression was strongly detected at implantation sites and stimulated by estrogen treatment. The expression of Odc antizyme 1 and spermidine/spermine N1-acetyltransferase was also highly shown at implantation sites and regulated by Odc or polyamine level in uterine cells. Embryo implantation was significantly inhibited by alpha-difluoromethylornithine, an Odc inhibitor. Moreover, the reduction of Odc activity caused by alpha-difluoromethylornithine treatment was compensated by the up-regulation of S-adenosylmethionine decarboxylase gene expression. Collectively, our results indicated that the coordinated expression of uterine polyamine-related genes may be important for embryo implantation.

Ornithine decarboxylase interferes with macrophage-like differentiation and matrix metalloproteinase-9 expression by tumor necrosis factor alpha via NF-kappaB.

Ornithine decarboxylase (ODC), a tumor promoter, provokes cell proliferation, and inhibits cell death; but the mechanism involved in cell differentiation remains unknown. Herein, we examine whether it functions during macrophage-like differentiation. Previous studies reveal that ODC, a rate-limiting enzyme of polyamine biosynthesis, and polyamines are involved in restraining immune response in activated macrophage. By using 12-O-tetradecanoylphorbol-13-acetate (TPA)-differentiated human promyelocytic HL-60 and promonocytic U-937 cells, we discover that polyamines block the expression, secretion and activation of MMP-9. Meanwhile conventional expression of ODC represses tumor necrosis factor-alpha (TNF-alpha) expression and nuclear factor-kappaB (NF-kappaB) activation as well as MMP-9 enzyme activity. Following stimulation by TNF-alpha, the secretion of MMP-9 is restored in ODC-overexpressed cells. In addition, the NF-kappaB inhibitors (pyrrolidinedithiocarbamate, BAY-11-7082 and lactacystin) suppress the TPA-induced MMP-9 enzyme activity. Concurrently, both the irreversible inhibitor of ODC, alpha-difluoromethylornithine, and TNF-alpha could not recover MMP-9 activation following NF-kappaB inhibitor treatment in parental cells. Furthermore, ODC could directly inhibit and attenuate NF-kappaB DNA binding and transcriptional activation. Therefore, we suggest that ODC inhibits the TNF-alpha-elevated MMP-9 activation via NF-kappaB as TPA-induced macrophage-like differentiation and this interrupting mechanism may provide a new conceivable resolution why leukemia is poorly differentiated besides atypical growth.

The Pseudomonas aeruginosa autoinducer dodecanoyl-homoserine lactone inhibits the putrescine synthesis in human cells.

Pseudomonas aeruginosa uses acyl-homoserine lactones to coordinate gene transcription in a process called quorum sensing (QS). The QS molecules C4-HSL and C12-oxo-HSL are synthesized from the universal precursor S-adenosyl methionine, which is also a precursor of polyamines in human cells. Polyamines are required for mitotic cell division and peak during this phase. The polyamine putrescine is synthesized by ornithine decarboxylase (ODC) as a rate-limiting step. The ODC enzyme concentration also peaks during the mitotic phase. This peak is mediated by translation of ODC mRNA by the ITAF45 protein, which translocates from the nuclear compartment to the cytoplasm in a phosphorylation-dependent manner. We observed that C12-HSL-treated human epidermal cells had a higher cytoplasm-to-nuclear ITAF45 protein concentration and this translocation was dependent on the dephosphorylation of ITAF45. Finally, C12-HSL-treated cells also had a time-course-dependent higher concentration of ODC mRNA. Based on these mitotic markers, more human cells were apparently trapped in the mitotic phase when treated with C12-HSL. This should normally imply higher levels of putrescine. However, C12-HSL-treated human cells had a significantly lower concentration of putrescine and displayed a lower cell proliferation rate. In conclusion, the P. aeruginosa autoinducer C12-oxo-HSL apparently arrests human cells in the mitotic phase by lowering the concentration of putrescine.

Kidney growth, hypertrophy and the unifying mechanism of diabetic complications.

Michael Brownlee has proposed a 'Unifying Mechanism' of hyperglycemia-induced damage in diabetes mellitus. At the crux of this hypothesis is the generation of reactive oxygen species (ROS), and their impact on glycolytic pathways. Diabetes is the leading cause of chronic kidney failure. In the early phase of diabetes, prior to establishment of proteinuria or fibrosis, comes kidney growth and hyperfiltration. This early growth phase consists of an early period of hyperplasia followed by hypertrophy. Hypertrophy also contributes to cellular oxidative stress, and may precede the ROS perturbation of glycolytic pathways described in the Brownlee proposal. This increase in growth promotes hyperfiltration, and along with the hypertrophic phenotype appears required for hyperglycemia-induced cell damage and the progression of downstream diabetic complications. Here we will evaluate this growth phenomenon in the context of diabetes mellitus.

The ornithine decarboxylase gene is essential for cell survival during early murine development.

Overexpression and inhibitor studies have suggested that the c-Myc target gene for ornithine decarboxylase (ODC), the enzyme which converts ornithine to putrescine, plays an important role in diverse biological processes, including cell growth, differentiation, transformation, and apoptosis. To explore the physiological function of ODC in mammalian development, we generated mice harboring a disrupted ODC gene. ODC-heterozygous mice were viable, normal, and fertile. Although zygotic ODC is expressed throughout the embryo prior to implantation, loss of ODC did not block normal development to the blastocyst stage. Embryonic day E3.5 ODC-deficient embryos were capable of uterine implantation and induced maternal decidualization yet failed to develop substantially thereafter. Surprisingly, analysis of ODC-deficient blastocysts suggests that loss of ODC does not affect cell growth per se but rather is required for survival of the pluripotent cells of the inner cell mass. Therefore, ODC plays an essential role in murine development, and proper homeostasis of polyamine pools appears to be required for cell survival prior to gastrulation.

Increased frequency of spontaneous skin tumors in transgenic mice which overexpress ornithine decarboxylase.

Ornithine decarboxylase, a critical regulatory enzyme for polyamine biosynthesis, is highly inducible by growth-promoting stimuli in mouse epidermis but the enzyme level is only transiently elevated due to rapid turnover of the protein. Here we report that constitutive overexpression of the enzyme in the skin of transgenic mice causes several phenotypic abnormalities. Effects observed include development of dermal follicular cysts, excessive skin wrinkling, enhanced nail growth, alopecia, and spontaneous tumor development. These results indicate that up-regulation of polyamine biosynthesis can profoundly disturb skin homeostasis and alter susceptibility to neoplastic development.

Human ornithine decarboxylase-overproducing NIH3T3 cells induce rapidly growing, highly vascularized tumors in nude mice.

Overexpression of human ornithine decarboxylase (ODC) under the control of strong promoters induces morphological transformation of immortalized NIH3T3 and Rat-1 fibroblasts [M. Auvinen et al., Nature (Lond.), 360: 355-358, 1992]. We demonstrate here that ODC-overproducing NIH3T3 cells are tumorigenic in nude mice, giving rise to rapidly growing, large fibrosarcomas at the site of inoculation. The tumors are capable of invading host fat and muscle tissues and are vascularized abundantly. To disclose the molecular mechanism(s) driving the tumorigenic, invasive, and angiogenic phenotype of the tumors, the ODC-overproducing cell lines and tumor tissues were analyzed for the expression of various potential regulators and mediators of cell proliferation, matrix degradation, and angiogenesis. The tumorigenicity of ODC transformants was associated with elevated polyamine levels and down-regulated growth factor receptors. The invasiveness of the ODC-induced tumors could not be attributed to overexpression of various known extracellular matrix-degrading proteases or matrix metalloproteinases. The induction of the tumor neovascularization proved not to be elicited by vascular endothelial growth factor or basic fibroblast growth factor. Instead, the ODC-overexpressing cells appeared to secrete a novel angiogenic factor(s) that was able to promote migration of bovine capillary endothelial cells in collagen gels and increase the proliferation of human endothelial cells in vitro. In parallel, ODC-transformed cells displayed down-regulation of thrombospondin-1 and -2, the negative regulators of angiogenesis. Thus, the induction of the angiogenic phenotype of the ODC transformants is likely due both to increased expression and secretion of the new angiogenesis-stimulating factor(s) and decreased production and release of the antiangiogenic thrombospondins.

Ornithine decarboxylase expression in cutaneous papillomas in SENCAR mice is associated with altered expression of keratins 1 and 10.

The patterns of expression of ornithine decarboxylase (ODC) and a number of keratinocyte differentiation products were examined in early papillomas by immunocytochemistry as an initial effort to develop phenotypic markers of the early stages of mouse skin tumorigenesis. Tumors were induced in SENCAR mice by initiation with 7,12-dimethylbenzanthracene, followed by once or twice weekly promotion treatments with 12-O-tetradecanoylphorbol-13-acetate. The patterns of immunocytochemical staining observed in early papillomas, collected after 7 weeks of promotion, were compared to those obtained with control skin and large, hyperkeratotic papillomas, collected after 23 weeks of promotion. In groups receiving 12-O-tetradecanoylphorbol-13-acetate, constitutive and induced ODC expression were evaluated either several days or 4.5 h after the last treatment, respectively. The major differentiation products in suprabasal keratinocytes are keratins, K1 and K10. These keratins were normally expressed in mildly hyperplastic epidermis, but staining became patchy in markedly hyperplastic epidermis. In early papillomas, K1 staining was patchy, and K10 staining occurred in very limited focal areas or was negative, such that the absence of staining delineated the lesions. Antibodies to the basal cell keratins, K5 and K14, stained both basal and suprabasal cells in hyperplastic epidermis and papillomas. Similarly, an antibody to keratin K6, which is expressed under conditions of hyperproliferation, uniformly stained the basal and suprabasal layers of hyperplastic epidermis and papillomas, demonstrating that the staining patterns observed with the antibodies to K1 and K10 were specific. Expression of ODC in the histologically normal skin of control mice was detected only in the hair follicles and depended on the hair cycle. Staining for induced ODC in mice treated with 12-O-tetradecanoylphorbol-13-acetate once weekly for 7 weeks was intense and diffuse throughout the suprabasal layers of the epidermis. In early and large papillomas, staining for constitutively expressed ODC was intense and diffuse in suprabasal cells. This intense staining for ODC occurred consistently in areas with decreased K1 and K10 expression, and, therefore, was associated with an altered pattern of differentiation. High constitutive ODC expression and decreased K1 and K10 expression will be useful phenotypic markers for studying the early stages of tumorigenesis in mouse skin.

A lack of radiation-induced ornithine decarboxylase activity prevents enhanced reactivation of herpes simplex virus and is linked to non-cancer proneness in xeroderma pigmentosum patients.

Patients with xeroderma pigmentosum (XP), a DNA repair disorder, run a large risk of developing skin cancer in sun-exposed areas. Cancer proneness in these patients correlates with a mammalian SOS-like response, "enhanced reactivation (ER) of viruses." Here, we report that radiation-induced activation of the ornithine decarboxylase (ODC) gene, a putative proto-oncogene, is required for this response. Various diploid fibroblast strains derived from a non-cancer-prone subclass of XP patients, which lack the ER response, were irradiated with 2 J/m2 and assessed for gene induction. In these fibroblasts, an absence of induction of ODC by UV-C was observed at the levels of mRNA, protein, and enzyme activity. This lack of induction is quite specific because the genes for fos and collagenase were induced as they were in normal XP cells. The apparent linkage between non-cancer proneness and a lack of ER and ODC induction was confirmed in a fibroblast strain derived from a patient with another DNA repair disorder, trichothiodystrophy, which does not lead to cancer proneness: in these cells, no induction of the ER response nor of ODC occurs after UV-C irradiation. Repair deficiency, however, is not essential because the simultaneous lack of ODC and ER induction after 10 J/m2 UV-C was found in at least one repair-proficient fibroblast. Next, a specific inhibitor of ODC, difluoromethylornithine, at a dose of 10 mM, completely blocked the ER response in cultured normal skin fibroblasts, suggesting that the ODC enzyme is in fact essential for the ER response. Difluoromethylornithine, although it did not affect other processes such as DNA repair, leads to a block in the cell division cycle at the G1-S transition. Interestingly, other blockers of this transition, wortmannin (500 nM) and mimosine (100 mM), also decreased the ER response. Finally, the ER and ODC responses also seem to be linked after treatment with X-irradiation (3 Gy), suggesting that both are part of a general response to DNA damage, at least in human skin fibroblasts. Apart from the abnormal ER and ODC responses, fibroblasts from non-tumor-prone XP patients react in the same way to radiation as do fibroblasts from tumor-prone XP patients with respect to other parameters. Thus, the lack of ODC induction after radiation may help to protect XP patients against skin carcinogenesis.

Exposure of Sprague-Dawley rats to a 50-Hertz, 100-microTesla magnetic field for 27 weeks facilitates mammary tumorigenesis in the 7,12-dimethylbenz[a]-anthracene model of breast cancer.

We have shown previously (W. Löscher et al., Cancer Lett., 71: 75-81, 1993; M. Mevissen et al., Carcinogenesis (Lond.), 17: 903-910, 1996) that 50-Hz magnetic fields (MFs) of low [50 or 100 microTesla (T)] flux density enhance mammary gland tumor development and growth in the 7,12-dimethylbenz[a]anthracene (DMBA) model of breast cancer in female Sprague Dawley rats. In these previous experiments, groups of rats were given 20 mg of DMBA (four weekly gavage doses of 5 mg each) and were MF- or sham-exposed for 13 weeks. The objective of the present study was to examine whether the use of a lower dose of DMBA (10 instead of 20 mg per rat), MF exposure of the rats before DMBA injection, and the increase of the MF exposure period after DMBA application to 26 weeks enhance the effect of MF on tumor development and growth. A group 99 rats was exposed to a homogeneous, horizontally polarized 100-microT MF of 50-Hz for 24 h/day for 7 days/week; another group of 99 rats was sham-exposed under the same environmental conditions as the MF-exposed rats. The exposure chambers were identical for MF-exposed and sham-exposed animals. The age of the rats was 45-49 days at the onset of exposure; duration of MF or sham exposure was 27 weeks. DMBA was administered p.o. at a dose of 10 mg/rat after 1 week of MF or sham exposure. The animals were palpated once weekly from week 6 onwards to assess the development of mammary tumors. At the end of the exposure period, the animals were killed for the determination of number and volume and histological verification of mammary tumors. All of the recordings were done in a blinded fashion; i.e., the investigators were not aware which animals were MF- or sham-exposed. Mammary tumor development and growth was significantly enhanced by MF exposure, the most marked effect on tumor incidence (190% above sham control) being observed 13 weeks after DMBA administration. At the time of necropsy, i.e., 26 weeks after DMBA administration, the incidence of histologically verified mammary tumors was 50.5% in controls and 64.7% in MF-exposed rats, the difference being statistically significant. More marked intergroup differences were recorded when tumor incidence was separately evaluated for each of the six mammary complexes, the most pronounced MF effect on tumor incidence being seen in the cranial thoracic complex. The data substantiate that, at least under the experimental conditions used in our laboratory, 50-Hz, 100-microT MF exposure significantly facilitates the development and growth of mammary tumors in the DMBA rat model of breast cancer.