Effect of Cu supplementation on genomic instability in chemically-induced mammary carcinogenesis in the rat

Backround  

The aim of the present study was to assess the effect of dietary supplementation (copper or copper and resveratrol) on the intensity of carcinogenesis and the frequency of microsatellite instability in a widely used model of mammary carcinogenesis induced in the rat by treatment with 7,12-dimethylbenz[a]anthracene (DMBA).     

High susceptibility of transgenic rats carrying the human c-Ha-ras proto-oncogene to chemically-induced mammary carcinogenesis

A rat line carrying three copies of the human c-Ha-ras proto-oncogenes, including its own promoter region, was established and designated as Hras128. Expression of the transgene was detected in all organs by Northern blot analysis. To examine its influence on susceptibility to mammary carcinogenesis, female rats were treated with N-methyl-N-nitrosourea (MNU) or 7,12-dimethylbenz[a]anthracene (DMBA) at 50 days of age. With MNU, all the transgenic rats rapidly developed multiple mammary carcinomas within as short as 8 weeks (14.1 tumors/rat), in contrast to 0.46 tumors/rat in non-transgenic rats. PCR-RFLP analysis and direct sequencing for the transgene indicated that the large majority of carcinomas (38/44, 86.4%) contained cells with mutations at codon 12 in exon 1. However, comparison of the signal densities of the mutated band to dilution scale bands revealed that the cells with the mutated transgene were not in the majority. By PCR-SSCP analysis for codons 12 and 61 of the rat endogenous c-Ha-ras gene, no mutations were detected. Similarly, with DMBA, almost all (13/14, 92.9%) the transgenic rats developed multiple mammary carcinomas (9.39 tumors/rat) within 16 weeks, and 4 out of 12 (33.3%) non-transgenic rats had only small tumors (0.83 tumors/rat). A lower incidence of mutation of the transgene was found in codon 12 (5/25, 25%) than in MNU-induced tumors, but mutations were detected in codon 61 (7/20, 35%). No mutations were detected in the rat endogenous gene. No mutation was found in the rat endogenous c-Ha-ras gene in non-transgenic rats. As observed in both the MNU- and DMBA-induced tumor cases, the population of cells with the mutated transgene were in the minority. The results thus indicate that rats carrying the transduced human c-Ha-ras proto-oncogene are highly susceptible to MNU- and DMBA-induced mammary carcinogenesis and that this is not primarily due to mutations of the transgene or endogenous c-Ha-ras gene. Furthermore, irrespective of the mechanism of enhanced susceptibility, the Hras128 transgenic rats can be utilized for the screening of mammary carcinogens.     

Synthesis and biological activity of 1alpha-hydroxyvitamin D3

Hydroboration of cholesta-1,5-diene-3β-ol followed by alkaline-peroxide oxidation resulted in the formation of 1α- and 2α-hydroxy derivatives of cholesterol in nearly equal amounts. 1α-Hydroxycholesterol was then transformed to 1α-hydroxyvitamin D₃, via 1α-hydroxycholest-5,7-diene-3β-ol. 1α-Hydroxyvitamin D₃ was as active as 25-hydroxyvitamin D₃ in the stimulation of intestinal calcium transport and bone mineral mobilization in intact rats, and moreover was able to produce both response in anephric rats similar to 1α,25-dihydroxyvitamin D₃, the active metabolite of vitamin D₃, as reported originally by DeLuca's group.     

Chemoprevention of rat liver toxicity and carcinogenesis by Spirulina

Spirulina platensis (SP) is a filamentous cyanobacterium microalgae with potent dietary phyto-antioxidant, anti-inflammatory and anti-cancerous properties. The present study aimed to investigate the chemopreventive effect of SP against rat liver toxicity and carcinogenesis induced by dibutyl nitrosamine (DBN) precursors, and further characterized its underlying mechanisms of action in HepG2 cell line. Investigation by light and electron microscopy showed that DBN treatment induced severe liver injury and histopathological abnormalities, which were prevented by SP supplementation. The incidence of liver tumors was significantly reduced from 80 to 20% by SP. Immunohistochemical results indicated that both PCNA and p53 were highly expressed in the liver of DBN-treated rats, but were significantly reduced by SP supplementation. Molecular analysis indicated that SP treatment inhibited cell proliferation, which was accompanied by increased p21 and decreased Rb expression levels at 48hrs post-treatment. In addition, SP increased Bax and decreased Bcl-2 expression, indicating induction of apoptosis by 48hrs. This is the first report of the in vivo chemopreventive effect of SP against DBN-induced rat liver cytotoxicity and carcinogenesis, suggesting its potential use in chemoprevention of cancer.     

Metabolism of 1alpha-hydroxyvitamin D3 in the chick.

Chicks convert both orally and intravenously administered 1alpha-hydroxy[6-3H]vitamin D3 rapidly to 1alpha,25-dihydroxy[6-3H]vitamin D3. The maximal accumulation of 1alpha,25-dihydroxy[6-3H]vitamin D3 in intestine precedes the intestinal absorption response to 1alpha-hydroxyvitamin D3 by at least 2 hours. Oral administration results in the highest concentrations of 1alpha,25-dihydroxy[6-3H]vitamin D3 in intestine, giving a level about 1.5 times that achieved with an intravenous dose. On the other hand, an oral dose of 1alpha-hydroxy[6-3H]vitaminD3 gives much lower amounts of both 1alpha-hydroxy[6-3H]vitamin D3 and 1alpha,25-dihydroxy[6-3H]vitamin D3 in bone and blood than an intravenous dose, which suggests that the 1alpha-hydroxy[6-3H]vitamin D3 may not be utilized as well by the oral route as by an intravenous route. Liver homogenates from both rat and chick convert 1alpha-hydroxy[6-3H]vitamin D3 to 1alpha,25-dihydroxy[6-3H]vitamin D3. However, intestinal homogenates from chick, but not rat, can also cary out this conversion, which may account for the higher concentration of 1alpha,25-dihydroxy[6-3H]vitamin D3 found in the intestine of chicks given an oral dose of 1alpha-hydroxy[6-3H]vitamin D3.     

Biological activity of 1alpha-hydroxyvitamin D3 in the rat.

The biological activity of 1α-hydroxyvitamin D₃ has been determined in vitamin D-deficient rats. In the accumulation of mineral in bone and cartilage, maintenance of serum calcium, and in efficiency of calcium absorption the 1α-hydroxyvitamin D₃ was approximately two to five times more active than vitamin D₃ or 80–200 units of activity per microgram.     

1 alpha-Hydroxyvitamin D2 is less toxic than 1 alpha-hydroxyvitamin D3 in the rat

An LD50 of 0.2 mg/kg body wt has been determined for 1 alpha-hydroxyvitamin D3 in the rat. In comparison, the LD50 for 1 alpha-hydroxyvitamin D2 is between 3.5 and 6.5 mg/kg. In terms of chronic toxicity, 1 alpha-hydroxyvitamin D3 at a dose of 5 micrograms/kg/day causes death of one-half the animals in a 4-week period. On the other hand, 20 micrograms/kg/day of 1 alpha-hydroxyvitamin D2 is required to induce similar toxicity. The body weight record and renal calcium accumulation during chronic treatment support the above conclusion. It therefore appears that 1 alpha-hydroxyvitamin D2 is between 5 and 15 times less toxic than 1 alpha-hydroxyvitamin D3. This surprising result prompted a reexamination of the relative biological activity of 1 alpha-hydroxyvitamin D2 and 1 alpha-hydroxyvitamin D3. Both compounds are equally potent in the stimulation of intestinal calcium transport, bone calcium mobilization, in the elevation of serum phosphorus, and in the healing of rickets in the rat. The reason for lower toxicity of 1 alpha-hydroxyvitamin D2 is unknown. The results suggest that 1 alpha-hydroxyvitamin D2 might represent a therapeutically superior compound.     

Biological activity of 1 alpha-hydroxyvitamin D2 in the rat.

The biological activity of 1α-hydroxyvitamin D₂ has been determined in vitamin D-deficient rats. In the calcification of the rachitic epiphyseal plate, 1α-hydroxyvitamin D₂ is more active than 25-hydroxyvitamin D₃, while it is equally active in stimulating intestinal calcium absorption. On the other hand, it is much less active (one-third to one-fifth) than 25-hydroxyvitamin D₃ in the mobilization of calcium from bone. In both the intestinal and bone responses, 1α-hydroxyvitamin D₂ (312 pmol) is active in nephrectomized rats while 25-hydroxyvitamin D₃ is not.     

Chemoprevention of precursors to colon cancer by dehydroepiandrosterone (DHEA)

Although dehydroepiandrosterone (DHEA) is recognized as one of the major adrenal androgens, its precise physiological role in the human endocrine system remains to be elucidated. In particular, the effect of DHEA on carcinogenesis has not been fully characterized. We undertook this study to determine whether DHEA has a chemopreventative effect on the precursors of colon cancer in a murine model of azoxymethane (AOM)-induced aberrant crypt foci (ACF). The number of ACF was significantly decreased in mice treated with 0.4% (p < 0.001) and 0.8% DHEA (p < 0.001), but there were no significant differences between DHEA-treated and control mice in terms of the ACF size, 3-catenin expression or level of dysplasia. This is the first study of colon cancer carcinogenesis demonstrating that DHEA treatment can decrease the number of ACF without apparently modifying their malignant potential. These data strongly suggest that DHEA might be a potential chemopreventative agent against human colon cancer.     

Thymosin alpha1 inhibits mammary carcinogenesis in Fisher rats

The effects of thymosin alpha1 (Talpha1) on mammary carcinogenesis was investigated in Fisher rats. Mammary carcinomas were observed 3 months after N-nitrosomethylurea (NMU) injection (10mg, i.p.) into Fisher rats. Daily administration of Talpha1 (10 microg, s.c.) reduced mammary carcinoma incidence and prolonged survival time. Animals treated with exogenous Talpha1 had a significantly greater blood white cell density than control Fisher rats. These results suggest that Talpha1 prevents mammary carcinoma incidence as a result of stimulation of the immune system.     

Transformation of 25- and 1 alpha-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3 by using Streptomyces sp. strains.

To enzymatically synthesize vitamin D derivatives, we screened about 300 Streptomyces sp. strains. Streptomyces sclerotialus FERM BP-1370 and Streptomyces roseoporus FERM BP-1574 were found to have the ability to convert 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3, respectively, to 1 alpha, 25-dihydroxyvitamin D3. The average rates of 1 alpha hydroxylation of 25-hydroxyvitamin D3 were 6.9 micrograms liter-1 min-1 with FERM BP-1370 and 7.0 micrograms liter-1 min-1 with FERM BP-1574. The specific cytochrome P-450 inhibitors carbon monoxide, SKF-525-A, and metyrapone inhibited the hydroxylation of 1 alpha- and 25-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3 by FERM BP-1370 and FERM BP-1574. The cytochromes P-450 of these strains were detected by reduced CO difference spectra in the whole-cell suspensions. The appearance of cytochrome P-450 suggests that the cytochromes P-450 of FERM BP-1370 and FERM BP-1574 carry out the hydroxylation of 25- and 1 alpha-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3.     

Transformation of 25- and 1 alpha-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3 by using Streptomyces sp. strains.

To enzymatically synthesize vitamin D derivatives, we screened about 300 Streptomyces sp. strains. Streptomyces sclerotialus FERM BP-1370 and Streptomyces roseoporus FERM BP-1574 were found to have the ability to convert 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3, respectively, to 1 alpha, 25-dihydroxyvitamin D3. The average rates of 1 alpha hydroxylation of 25-hydroxyvitamin D3 were 6.9 micrograms liter-1 min-1 with FERM BP-1370 and 7.0 micrograms liter-1 min-1 with FERM BP-1574. The specific cytochrome P-450 inhibitors carbon monoxide, SKF-525-A, and metyrapone inhibited the hydroxylation of 1 alpha- and 25-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3 by FERM BP-1370 and FERM BP-1574. The cytochromes P-450 of these strains were detected by reduced CO difference spectra in the whole-cell suspensions. The appearance of cytochrome P-450 suggests that the cytochromes P-450 of FERM BP-1370 and FERM BP-1574 carry out the hydroxylation of 25- and 1 alpha-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3.     

Selenium-mediated inhibition of mammary carcinogenesis

Using the dimethylbenz(a)anthracene-induced mammary tumor model in rats, our studies indicated that there was a dose-response relationship between dietary selenium supplementation and the inhibition of mammary carcinogenesis. The degree of inhibition was proportional to the level of dietary selenium up to 5 ppm, at which point toxicity in the form of a reduction in weight gain was evident. Moreover, it was observed that the chemopreventive efficacy of selenium was influenced by the dose of carcinogen as well as the fat intake of the animals. By supplementing selenium for defined periods of time, we concluded that selenium inhibited both the initiation and the promotion phases of chemical carcinogenesis, and that a continuous intake of selenium was necessary to achieve maximal suppression of tumor growth. In an attempt to improve the efficacy of lower levels of selenium, we conducted another series of experiments in which selenium and vitamin E were tested in combination. Results showed that although vitamin E alone had no prophylactic effect against tumorigenesis, it potentiated the ability of selenium to inhibit the development of mammary tumors. Further investigation suggested that the anticarcinogenic action of selenium could not be explained by its antioxidant function in lipid peroxidation. On the other hand, vitamin E might be able to provide a more favorable climate against oxidant stress to facilitate selenium in exerting its inhibitory effect through some other mechanisms.     

Metabolism of 1alpha-hydroxyvitamin D3 to 1alpha, 25-dihydroxyvitamin D3 in perfused rat liver.

The metabolism of 1α-hydroxyvitamin D₃ (1α-OH-D₃) was studied in rat liver perfused with [³H]-1α-OH-D₃. [³H]-1α-OH-D₃ was converted very rapidly to a more polar metabolite, which was identified as 1α,25-dihydroxy-vitamin D₃ [1α,25-(OH)₂-D₃] by co-chromatography with synthetic 1α,25-(OH)₂-D₃ as well as by gas chromatography-mass spectrometry. [³H]-1α,25-(OH)₂-D₃ appeared in the perfusate as early as 20 min after addition of [³H]-1α-OH-D₃, and its level in the perfusate increased linearly for at least 120 min. These data strongly indicate that 1α-OH-D₃ is metabolized to 1α,25-(OH)₂-D₃, which exerts biological effects on bone and intestine.     

Chemoprevention of DMH-induced rat colon carcinoma initiation by combination administration of piroxicam and C-phycocyanin

Cancer research illustrated that combinatorial studies can provide significant improvement in safety and effectiveness over the monotherapy regimens. A combination of two drugs may restrain precancerous colon polyps, opening a new possible opportunity for chemoprevention of colon cancer. In this context, chemopreventive efficacy of a combination regimen of C-phycocyanin, a biliprotein present in Spirulina platensis, a cyanobacterium, which is a selective cycloxygenase-2 (COX-2) inhibitor and piroxicam, a traditional non-steroidal anti-inflammatory drug was considered in 1,2 dimethylhyadrazine (DMH)-induced colon carcinogenesis in rats. Western blotting, immunohistochemistry, DNA fragmentation, fluorescent staining, PGE(2) enzyme immunoassay, and carrageenan-induced paw edema test were performed along with morphological and histological analysis. DMH treatment showed a rich presence of preneoplastic lesions such as multiple plaque lesions, aberrant crypt foci, and well-characterized dysplasia. These features were reduced with piroxicam and C-phycocyanin administration. The number of apoptotic cells was featured prominently in all the groups compared with DMH. DMH treatment revealed intact high molecular weight genomic DNA with no signs of laddering/DNA fragmentation while it was noticeable significantly in control and DMH + piroxicam + C-phycocyanin. DMH group showed highest COX-2 expression and PGE(2) level in comparison with other groups. Doses of piroxicam and C-phycocyanin used in the present study were established at an anti-inflammatory range. A combination regimen of piroxicam and C-phycocyanin, rather than individually has the much greater potential for reduction of DMH-induced colon cancer development and COX-2 being the prime possible target in such chemoprevention.     

Synthesis of active metabolite(s) from 1 alpha-hydroxyvitamin D3 by human monocytic leukemia cells.

Synthesis of the biologically active metabolite(s) from 1 alpha-hydroxyvitamin D3 (1 alpha(OH)D3) was examined in various types of human leukemia cell lines. Untreated monocytoid leukemia cells (U937 and HEL/S) metabolized 1 alpha (OH)D3 to the active metabolite(s), possibly 1 alpha, 24- and/or 1 alpha, 25-dihydroxyvitamin D3, and these cells were efficiently induced to differentiate by treatment with 1 alpha (OH)D3. However, the other types of leukemia cells did not efficiently metabolize it and were not induced to differentiate by 1 alpha (OH)D3. The possible therapeutic advantage of 1 alpha (OH)D3 in the treatment of monocytic leukemia is discussed.