Soy and experimental cancer: animal studies.

Studies are reviewed that report consumption of soy protein diets inhibits the growth of various tumors in rats. The inhibitory effect has been attributed to the phytoestrogens (genistein and diadzein) or protein kinase inhibitor in soy protein products. Recent studies indicate that additional factors in soy protein products may also contribute to the inhibition of tumorigenesis, namely the deficiency of the essential amino acid methionine. Metastatic growth to the lungs of a primary rhabdomyosarcoma tumor was inhibited by feeding a soy protein diet. The effect was reversed by methionine fortification of the diet. Carcinogen-induced mammary tumor development was inhibited during the promotional phase in rats fed soy protein isolate diet and reversed with a methionine-supplemented diet. Additional studies demonstrated that after excision of the primary mammary tumor, growth of additional tumors was inhibited when the diet was changed from casein to soy protein isolate. Histopathologic evaluation of the mammary tumors revealed more benign fibroadenomas and lower-grade adenocarcinomas in the soy protein group. Before carcinogen administration (at 7 weeks of age), ornithine decarboxylase activity and polyamine concentrations in the rat mammary epithelium were significantly lower in the soy protein group. These data suggest an inhibitory effect on mammary epithelial growth in the soy-protein-fed group.

Dietary soybean isolate and methionine supplementation affect mammary tumor progression in rats.

The effect of feeding soybean protein isolate (SBP) diet or soybean protein isolate diet supplemented with 0.7% DL-methionine (SBP + Met) on mammary tumor progression was investigated. Sprague-Dawley female rats were fed from weaning a 20% casein (CAS) diet supplemented with 0.3% DL-methionine (AIN-76) and injected via jugular vein with N-nitrosomethylurea (NMU, 40 mg/kg body weight) at 7 wk of age. Five weeks after NMU treatment, animals were divided into the three isoenergetic, isoprotein diet groups: CAS (25 rats); SBP (26 rats) and SBP + Met (25 rats). First palpable mammary tumors were evident 8, 9 and 13 wk and the mean latency period was 13.30 +/- 1.23, 16.70 +/- 1.32 and 17.82 +/- 1.28 wk after NMU treatment in the CAS, SBP + Met and SBP diet groups, respectively. Tumor incidence was 80% in the CAS group compared with 42.3% in the SBP group (P = 0.01). Methionine supplementation increased tumor incidence to 64%. Total number and total weight of tumors was greater in the CAS group compared with either SBP + Met or SBP groups: 41 vs. 28 or 21 tumors and 97.28 g vs. 27.87 or 32.46 g, respectively. These data indicate that SBP diet, low in methionine content, fed 5 wk after carcinogen exposure significantly repressed mammary tumor progression. Methionine supplementation increased the number of animals with tumors but not the mean tumor weight.

Dietary protein enhanced mammary ornithine decarboxylase activity and tumorigenesis in rats.

Sprague-Dawley rats (F-1) were fed a normal protein (19% casein, NP) or high protein (33% casein, HP) isoenergetic diet containing 15% corn oil prior to conception. Female pups (F-2) were also fed the maternal diet after weaning. At 7 wk of age, before saline or N-nitrosomethylurea (NMU) treatment, ornithine decarboxylase (ODC, EC 4.1.1.17) activity in mammary epithelium and liver tissue was significantly higher in the HP group than in the NP group. Eight weeks after saline treatment ODC activity in mammary tissue decreased in both groups, but remained significantly higher in the HP group. NMU treatment caused a sixfold increase in ODC activity in the mammary tissue in the HP group and a significantly lower response in the NP group. Liver ODC activity had a minimal response to NMU treatment. Changing from the HP to the NP diet 4 wk after NMU treatment reduced mammary ODC induction response but not tumor burden; changing from the NP diet to HP diet produced no change in ODC activity or tumor burden. Mammary tumor burden was positively related to dietary protein and mammary epithelium ODC activity prior to and following NMU treatment.

Restoration of sexual function and fertility by fetal hypothalamic transplant in impotent aged male rats.

The purpose of this study was to observe whether fetal hypothalamic transplant can restore the neuroendocrine and reproductive function in impotent aged male rats. Eighteen to 20 month old impotent male rats received an anterior hypothalamus removed from a 17-19 day old fetus and placed into the anterior third ventricle. Controls were either without surgery (UC) or grafted with cerebral cortex (CG). Before and 2 to 3 months after transplantation, blood samples were collected from the aged rats for testosterone and LH measurement. Before and one to two months after transplantation, each hypothalamic grafted animal (HG) or control rat was put overnight into a cage which contained four, 10 to 12 week old proestrous female rats. Vaginal smear of each female was monitored early the next morning. Sperm seen in the vaginal smear was regarded as copulation and ejaculation. The test was repeated twice, one week apart, and the higher score represented the sexual function and fertility of the males. Seven of 10 HG males restored their sexual function, impregnated 9 females and fathered 106 pups. None of 7 UC restored their reproductive function and only one of 4 CG males impregnated one female which delivered 6 pups. Serum testosterone, LH and pituitary LH in the HG rats, which showed restoration of reproductive function, were significantly higher than those of the controls (UC and CG). These results indicate that the fetal hypothalamic grafts can survive and develop in the brain of impotent aged male rats and restore neuroendorince and reproductive function in senescent rats.

Dietary protein, enhancement of N-nitrosomethylurea-induced mammary carcinogenesis, and their effect on hormone regulation in rats.

The effect of supplemental dietary protein (casein) fed with high fat diets was investigated using the N-nitrosomethylurea-induced mammary tumor model. Isocaloric diets containing casein and corn oil at 19 and 15% (normal protein-high fat) or 33 and 15% (high protein-high fat) were fed ad libitum to Sprague-Dawley mother rats. Female offspring continued on the diet. Food consumption and growth curves were similar over the entire growth period. N-Nitrosomethylurea (50 mg/kg body weight) or saline was administered at 7 and 8 weeks of age via the tail vein. Dietary protein had no effect on serum prolactin or growth hormone throughout the estrous cycle: Prior to carcinogen administration, at 7 weeks old, proestrus at 5 p.m., serum prolactin was 231.6 +/- 141.0 (SE) ng/ml (12 rats) versus 292.2 +/- 141.0 (13 rats) for normal versus high protein diet groups, respectively. No difference was noted after carcinogen injection at 9, 13, 28, and 33 weeks of age. Similarly no effect was noted on serum growth hormone activity. Tumor latency was 7 weeks and incidence was 100% in normal protein (24 rats) and high protein (39 rats) groups 28 weeks after carcinogen treatment. The number of tumors per rat (4.38 +/- 0.37 versus 2.87 +/- 0.35, P less than 0.002) and average tumor weight (17.97 +/- 2.63 versus 9.94 +/- 2.92 g) were significantly greater in the high protein group. Study indicates that diet or carcinogen treatment did not alter hormone regulation during the estrous cycle. However, supplemental dietary protein increased the effect of high fat diets enhancing the mammary tumor burden.

Effect of dietary protein on morphologic development of rat mammary gland.

Isocaloric diets, containing 8% casein [low protein (LP)], 19.5% casein [normal protein (NP)], or 31% casein [high protein (HP)] and 10% corn oil, were fed to adult virgin female Sprague-Dawley rats prior to conception and were continued through mating, gestation, and lactation. Female pups, 8 per litter, were fed on their mother's diet for the duration of the study. The pups' sexual maturation occurred at 33, 35, and 70 days of age for the HP, NP, and LP groups, respectively. At 7 weeks of age, duct development in the mammary gland was markedly delayed in the LP group. No significant differences in the extent of ductal development were noted between the HP and NP groups. Morphologic development into terminal end buds, alveolar buds, lobules, and terminal ducts was determined for each diet group at 7 weeks and at sexual maturity plus 3 weeks (SM+3) of age. At 7 weeks, the terminal end bud was the dominant structure in the LP group and was significantly greater in number than in the HP and NP groups. The latter groups were dominated by the alveolar bud structures. At SM+3, the number of terminal end buds decreased, and the alveolar buds increased in the LP group; however, the numbers were still significantly different than in the HP and NP groups. This study indicates that dietary protein affects mammary duct proliferation and morphologic development.

Fat-protein interaction, defined 2-generation studies.

Mammary tumor burden, in rats fed either normal or high fat diets related positively to the level of protein in the diet. This relationship existed with either a direct (NMU) or indirect carcinogen (DMBA). Significant differences in body growth, sexual maturation, morphologic structures in the mammary duct, and hormone activities during the estrous cycle probably contributed to lower tumor burden in the low protein-normal fat (LP-NF) group. Animals fed a high protein-normal fat (HP-NF) diet throughout their entire life have, with the exception of early sexual maturation, no distinctive characteristics compared to the control group. Inspite of these physical and physiologic similarities, increased dietary protein enhanced the effect of administered carcinogens. Animals fed a high protein-high fat (HP-HF) diet were compared to rats fed a normal protein-high fat (NP-HF) diet. Increased dietary protein further enhanced the effect of the high fat diet resulting in an increased carcinogen-induced tumor burden. These studies indicate that the design of the animal model, i.e. age of initiation of a test diet, appears to have a significant bearing on mammary tumor development. The biologic mechanisms which respond to diet modifications and which may influence breast tumor growth have not been thoroughly elaborated and require additional study.

Development of Na+-K+-ATPase in neonatal rat brain synaptosomes after perinatal protein malnutrition.

Four protein dietary groups of rats were used in this study, namely: 27% casein diet fed throughout gestation and lactation (group C-C); 27% gestation, 10% lactation (group C-D); 10% gestation, 27% lactation (group D-C); 10% throughout gestation and lactation (group D-D). Mean body weight of group D-D at warning was 19 g compared with 59 g for group C-C. The specific activity of Na+-K+-ATPase in isolated synaptosomes from cerebral and cerebellar tissues increases throughout the lactation period. At 1 and 7 days of age no differences in Na+-K+-ATPase activity are noted in the brain regions of Groups C-C and D-D. At 14 and 21 days, however, the specific Na+-K+-ATPase activity was significantly reduced in isolated synaptosomes from both brain regions of the protein-deprived group. ATPase activity in cerebellar synaptosomes from dietary exchange groups (C-D and D-C) developed to the same extent as that of the control group (C-C) after 21 days of lactation. ATPase activity in the cerebral synaptosomes of the dietary exchange group (C-D) did not develop to the same extent as that in the control group (C-C) and the other dietary exchange group (D-C).

Effect of ultraviolet on the survival of bacteria airborne in simulated Martian dust clouds.

A chamber was constructed to create simulated Martian dust storms and thereby study the survival of airborne micro-organisms while exposed to the rigors of the Martian environment, including ultraviolet irradiation. Representative types of sporeforming and non-sporeforming bacteria present in spacecraft assembly areas and indigenous to humans were studied. It was found that daily ultraviolet irradiation of 2 to 9 X 10(7) erg cm-2 was not sufficient to sterilize the dust clouds. The soil particles protected the organisms from ultraviolet irradiation since the numbers of survivors from irradiated environments were similar to those from unirradiated environments. Pending further data of the Martian environment, the contamination and dissemination of Mars with terrestrial micro-organisms is still a distinct possibility.

Use of the scanning electron microscope for viewing bacteria in soil.

Scanning electron microscopy was used for viewing Bacillus cereus and Staphylococcus aureus in three different soils. Both organisms were detected in the test soils at an approximate concentration of 10(7) cells per gram of soil; theoretically, the minimal number of microorganisms required for detection with the scanning electron microscope technique was between 10(7) and 10(10) cells per gram of soil. Because the concentration of cells was critical, the use of scanning electron microscopy as an extraterrestrial life detection instrument would be limited with soils containing more than 10(7) bacteria per gram of soil.

Survival of microorganisms in a simulated Martian environment. II. Moisture and oxygen requirements for germination of Bacillus cereus and Bacillus subtilis var. niger spores.

The effects of moisture and oxygen concentration on germination of Bacillus cereus and B. subtilis var. niger spores were investigated in a simulated Martian environment. Less moisture was required for germination than for vegetative growth of both organisms. A daily freeze-thaw cycle lowered moisture requirements for spore germination and vegetative growth of both organisms, as compared with a constant 35 C environment. Oxygen had a synergistic effect by lowing the moisture requirements for vegetative growth, and possibly germination, of both organisms. Oxygen was not required for spore germination of either organism, but was required for vegetative growth of B. subtilis and for sporulation of both organisms.

Effect of reduced barometric pressure on water availability related to microbial growth.

Data obtained from Mariner IV indicate that the barometric pressure on Mars is considerably lower than previously estimated. Current estimates from Mariner IV indicate a range from 4 to 7 mb and by near infrared spectroscopy 33-56 mb. Inasmuch as the pressure has a marked influence on availability of water, this should affect the existence of Martian life. At the maximum temperatures recorded on Mars, namely 25 degrees C, a barometric pressure of 30 mb is required for the retention of free water. The lower pressure, 4 mb, would suggest that the moisture is present as a vapor above the freezing point and consequently it is not available for utilizing by living cells. The lower estimates of barometric pressure also inversely affect the carbon dioxide concentration in the Martian atmosphere. Our previous studies have demonstrated that spores of Bacillus cereus survive, germinate and grow in a simulated Martian environment (2.4% CO2, 98 mb) supplemented with moisture. The studies described in this paper were designed to determine the effect of low barometric pressures (10 to 98 mb Hg) and high concentrations of carbon dioxide (37 to 100%) in the simulated Martian environment on survival and growth of B cereus. The organism was inoculated into a felsite-limonite soil at 8% moisture level. The temperature cycles used were 8 hr at -65 degrees C and 16 hr at 25 degrees C, or 20 hr at -65 degrees C and 4 hr at 25 degrees C. The data suggest that the organism after achieving maximum growth in the simulated Martian environment (2.4% CO2, 98 mb) immediately enters into the growth phase upon reinoculation into fresh soil. These data reflect upon the possibility of contamination through air movements. Based upon currently available Martian environmental data, the probability of contamination of Mars by terrestrial micro-organisms will be discussed.

Survival and growth of potential microbial contaminants in severe environments.

Studies conducted in our laboratory have established that a number of potential soil microbes could survive a simulated Martian environment. In view of the uncertainty of the Martian environment and the importance of noncontamination of extraterrestrial bodies, studies were performed with common soil microbes to determine the minimal environment necessary to produce a complete growth cycle. The effects of diurnal temperature cycling (+25 degrees C to -65 degrees C) and of limiting concentrations of moisture and oxygen on spore germination, vegetative growth and sporulation of Bacillus cereus and B. subtilis have been determined. The results indicated that diurnally temperature-cycled heat-shocked spores of B. cereus in the simulated Martian atmosphere: 1) survived when the moisture concentration was < or = 4%; 2) germinated but became nonviable when the moisture concentration was > 4 < 8%; 3) germinated with subsequent vegetative growth when a) the moisture concentration was > or = 8%, and b) the concentration was > or = 6% and the partial pressure of oxygen was 15 mm; 4) sporulated when the moisture concentration was > or = 6% and the partial pressure of oxygen was 15 mm. Similar data for heat-shocked spores of B. subtilis in the simulated Martian atmosphere, diurnally temperature-cycled, indicated that the spores: 1) survived when the moisture concentration was > or = 6%; 2) germinated but became nonviable when the moisture concentration was > 1 < 4%; 3) germinated with subsequent vegetative growth when the moisture concentration was > or = 8%; 4) sporulated when the moisture concentration was > or = 6% and the partial pressure of oxygen was > or = 10 mm. Preliminary studies with B. cereus and B. subtilis spores produced in the simulated Martian environment and reintroduced into this environment indicated that vegetative cell growth and sporulation were normal. These data are discussed with regard to the probability of contamination of extraterrestrial bodies.