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We hypothesized that the pathological effects on the neonatal rat heart could be aggravated by Cu deficiency due to the combined effects of caffeine exposure and malnutrition. Upon birth, pups were mixed and randomly picked; 8 pups were assigned to each dam and then divided into 4 groups. Group 1 dams received a normal diet containing 20% protein. Group 2 dams were fed 20% protein diet supplemented with caffeine (4 mg/100 g BW). Group 3 dams received 6% protein diet as a malnourished group, and group 4 dams received 6% protein diet supplemented with caffeine (4 mg/100 g BW). On postnatal day 10, dams and pups were killed. Group 2 tended to have a decrease in the Cu levels of dams' plasma and milk and in pups' plasma and heart tissue compared to those of group 1. This pattern was not observed consistently between groups 3 and 4. Transmission electron microscopy of group 2 pups' hearts revealed a degree of disruption in the mitochondria compared to normal mitochondria seen in group 1. There was no consistent change in the mitochondria of group 4 compared to group 3. The caffeine level observed in all categories of group 4 (dams' plasma and milk, pups' plasma and heart tissue) was lower than those in group 2. Although malnutrition affected body weight and heart weight, combined effects of caffeine and malnutrition on Cu content in the neonatal heart was relatively minor compared to the well nourished group. This well nourished group showed that the effects of caffeine on Cu were more consistent, resulting the changes of mitochondria.

Caffeine (1,3,7-trimethylxanthine) is one of the most commonly consumed drugs in our daily life, and its use is increasing. However, very little attention has been paid to its potential effects on early growth and development. Because of the steady increase in breast feeding of infants and because caffeine diffuses readily into breast milk, the present study examined if caffeine intake by newborn rats during lactation would affect the saturated and monounsaturated fatty acids in the growing cerebellum. A total of 10 timed pregnant rats were purchased from the breeder. At birth litters were combined, and 8 pups were randomly assigned to each dam without regard to the sex of the pups. Dams with litters were divided into 2 groups. Dams of group 1 received a 20% protein diet as a control, and dams of group 2 received a 20% protein diet plus caffeine (4 mg/100 g BW). Pups were killed at day 10. The cerebellums were removed, weighed and homogenized. Gas chromatograph-mass spectrometry was used to identify and quantify free fatty acids. Chronic caffeine exposure from birth to day 10 in pups through the maternal milk resulted in a decrease in cerebellum weight, a significant increase in the saturated fatty acids, and a tendency toward an increase of monounsaturated fatty acids. In addition, there was a slight increase of some of the polyunsaturated fatty acids. However, there was no difference in food intake of the lactating dams and weight gain of the pups between the groups. These data indicate that early caffeine intake by the suckling pups alters the composition of fatty acids of the cerebellum; thus, avoidance of caffeine during lactation is critical. The risks and benefits of caffeine administration in premature infants must be carefully evaluated during this rapid period of brain growth., (Copyright 2004 S. Karger AG, Basel)

Although chronic caffeine exposure during pregnancy has been shown to affect fetal growth, adverse effects of caffeine on embryogenesis are not only well understood, but also controversial. We have used gene chip technology in an attempt to identify to what extent, if any, caffeine could possibly alter gene expressions in the cytotrophoblast-like cell line BeWo. Few down-regulated genes were found; most of the genes were up-regulated, suggesting that chronic caffeine exposure during the gestational period could exert certain influences on embryogenesis. The highest up-regulated gene expression of BeWo cells by caffeine was angiotensin II type 2 (AT(2)) receptor gene. We focused the genes of the renin-angiotensin system (RAS), angiotensin II type 1 (AT(1)) and AT(2)receptors and angiotensin I converting enzyme, for study on caffeine's responsive gene expression in BeWo cells and in the placentae of pregnant rats that were fed a diet supplemented with caffeine (2 mg/100 g body weight) during gestation, and analysed the gene expressions using RT-PCR and LightCycler system. A significantly increased AT(2)receptor gene expression and a slight decreased AT(1)receptor gene expression demonstrated the caffeine's effect to the placental RAS.

Because caffeine and tissue levels of Zn are closely related, the objectives of this study were to determine the changes in plasma caffeine levels over a period of 5 h when different concentrations of Zn combined with a fixed concentration of caffeine were injected into the femoral vein of rats and to determine the relationship between tissue levels of caffeine and Zn at 5 h postinjection. Rats were divided into three groups: group 1, 220 microg caffeine; group 2, 220 microg caffeine + 8 microg Zn/g body weight (BW); group 3, 220 microg caffeine + 16 microg Zn/g BW. Blood from groups 1 and 3 was collected at 3 min, 30 min, 1 h, 3 h, and 5 h to determine the pharmacokinetics of caffeine. All groups were killed at 5 h. Caffeine and Zn concentrations of the brain, kidney, heart, and liver of all groups were determined. The plasma-caffeine curve in group 3 showed a lower concentration at 3 min and a slower caffeine-elimination rate during the first 3 h. Brain and kidney caffeine levels remained constant in all groups, whereas caffeine levels were increased in the heart in group 2 and in the liver in group 3. Zn concentrations in the brain and kidney were lower in group 2 compared with groups 1 and 3 and higher in group 3 compared to groups 1 and 2. Zn concentration in the heart was the same among the three groups but was increased in the liver in group 3 compared to groups 1 and 2. Therefore, we concluded that caffeine combined with Zn affects caffeine pharmacokinetics. With caffeine intake, levels of Zn (16 microg/g BW) that are slightly higher than the daily requirements (12 microg/g BW) may prevent a reduction of Zn in tissue. In addition, caffeine's effects on Zn concentration among organs are different.

The general public widely consumes caffeine which is contained in various foods, beverages, and over-the-counter medications. The relationships between caffeine intake and bone fractures is controversial. Therefore, the aim of the current study was to determine what effects, if any, caffeine intake in early life exerts on mechanical properties and mineral contents of bone in growing ovariectomized rats. A total of 8 dams with pups were divided into two groups. Group 1 was fed a 20% protein diet. Group 2 was fed a 20% protein diet supplemented with caffeine (4 mg/100 g). The respective diets were fed to the dams during lactation and to the pups continuously after weaning on day 22 until the end of the experimental period. On day 32, offspring from both groups were ovariectomized. On day 52, the rats were sacrificed and the femora removed. The biomechanical properties of the femora were determined by three-point bend testing to failure at a rate of 2 mm/min, with continuous data sampling at 10 samples/s. The properties determined included the modulus of elasticity, yield load, yield stress, ultimate load, ultimate stress, and the second moment of area. The caffeine group exhibited a decrease in the various mechanical properties (ranging from approximately 7 to 20%), except for yield strain and moment of inertia. The decreases in maximum stress and elastic modulus values were significant. Calcium, magnesium, and phosphorous values for the caffeine group were significantly decreased. These results suggest that the bone in the caffeine group is weaker and less stiff, with greater deformation under applied loading. It could be concluded that caffeine intake during the early growing period affects the mechanical properties of bone., (Copyright 2002 S. Karger AG, Basel)

Caffeine consumption has been implicated in the development of cardiovascular disease. Therefore, in the present study, litters of rats were combined upon birth, and 8 pups were randomly assigned to each dam. Dams with pups were divided into 2 groups: group 1 received a 20% protein diet as a control, and group 2 received the 20% protein diet supplemented with caffeine (4 mg/100 g body weight). Pups from both groups were killed on days 11 and 15. Transmission electron microscopy revealed swollen, disrupted, degenerating mitochondria and intracellular edema in the hearts of rats in the caffeine groups when compared with those of the controls. Plasma Cu concentration was significantly decreased. These results indicate that early exposure to caffeine through maternal milk adversely affects cardiac mitochondria of rat pups and may be associated with decreased plasma Cu levels. It is unclear whether these results apply to the human infant. Interspecies extrapolation from rat to human must be made with caution.

Background: The general public widely consumes caffeine (1,3,7-trimethylxanthine), which is contained in various foods, beverages and over-the-counter medications. We have shown previously that caffeine intake could affect bone metabolism in vivo., Methods: Because prostaglandin E2 (PGE2) is shown to be elevated in the periodontally diseased site, the possible interaction between caffeine and PGE2 was investigated in the present study using UMR106-01 rat osteoblast-like cells in vitro., Results: Although neither 0.1 mM caffeine nor 0.1 microg/ml of PGE2 alone showed any inhibitory effects on cell proliferation, the combination of caffeine and PGE2 showed significant inhibition. However, in order to have inhibitory effects, both caffeine and PGE2 had to be present at least 72 or 96 hours in the medium. Addition of the endogenous PGE2 synthesis inhibitor, indomethacin, showed no effects on cell proliferation. Neither cAMP-inducing agent IBMX (0.01 mM and 0.1 mM) nor forskolin (0.001 mM) inhibited cell proliferation, but combined with PGE2 these agents strongly inhibited proliferation as was observed with the combination of caffeine and PGE2, suggesting possibly that the increase of intracellular cAMP concentration plays an important role in the inhibitory effects of cell proliferation., Conclusions: The present data for the first time demonstrate the possible implication of routine caffeine intake in the acceleration of pathological conditions of periodontitis. Thus, we propose that chronic caffeine intake is one of the possible risk factors in the advancement of pathology in the periodontitis patient. Further research in this area is warranted.

Caffeine is a substance which many people consume in their daily life. Caffeine's effects on bone are still controversial. Using ovariectomized rats, the present study was conducted to determine to what extent caffeine intake affects the mechanical properties, bone minerals and histology. Aged rats were divided into 2 groups after ovariectomy. Group 1 was fed a 20% protein diet as a control, and group 2 was fed a 20% protein diet supplemented with caffeine (2 mg/100 g body weight). The respective diets were fed to the rats of each group for 90 days. Rats were then killed by heart puncture, blood was collected, and femurs were removed. In 1 group of femurs paraffin cross-sections were made at the midshaft of each bone. Total width, cortical width, total cross-sectional bone area of the midshaft, and the number of osteocytes in randomly selected areas were measured. Another group of bones was subjected to three-point bending testing until failure. Bones were then pulverized and Ca, P, Mg, Zn, Sr, Si, hydroxyproline and hexosamine contents and crystallite size were measured. Various mechanical properties, except modulus of elasticity, in the caffeine group were consistently 7-23% lower than the noncaffeine controls. Yield strain in the caffeine group was significantly less than in the noncaffeine controls. Zinc, Sr, and crystallite size of bone showed a significant decrease in the caffeine group, whereas Si contents significantly increased. Our current results indicate that routine intake of caffeine in the elderly should be regarded with some caution.

The combined effects of caffeine and alcohol on mineral contents of fetal mandibles and femurs were studied. Pregnant rats were divided into four groups: group 1, control; group 2, caffeine; group 3, alcohol; and group 4, caffeine-plus-alcohol. Alcohol (1.0 g ethanol/kg body weight) was intubated twice daily, beginning at day 9 of gestation. Caffeine (2 mg/100 g body weight) was given as a dietary supplement. Groups 1 and 2 were intubated with isocaloric sucrose solution. At birth, randomly selected pups were killed and the mandible and femur were removed and dried. Ca, P, Mg, Zn and hydroxyproline in these bones were measured. Notwithstanding the dams' intake of caffeine and alcohol administered separately, the present results suggest that the combination of caffeine and alcohol exhibited the most detrimental effects.

1. The growth of cardiac cells derived from newborn rats whose dams were either malnourished or malnourished with caffeine during pregnancy was inhibited in culture over the period of 5 days as compared to that of the normally nourished cells. 2. Cells derived from malnourished rats with caffeine added to their diets showed a greater inhibition than those from the malnourished rats not given caffeine. 3. Both DNA and protein synthesis showed an inhibition due to caffeine in a dose-dependent manner using normally nourished cells. 4. In the presence of exogenous 2 mM caffeine, the degree of percent inhibition of DNA and protein synthesis of cells derived from rats malnourished with caffeine was less than that from the rats with malnutrition alone. 5. The present data indicated that malnutrition combined with caffeine during pregnancy exerted a greater negative effect on the nature of cell growth than malnutrition alone and these cells became less sensitive to exogenous caffeine.

Pregnant rat dams were divided into four groups on the 3rd day of gestation. Group 1 dams were fed a 20% protein diet as controls. Dams of group 2 were fed a 20% protein diet supplemented with zinc (0.6 g ZnCl2/kg diet). Group 3 dams were fed a 20% protein diet supplemented with caffeine (2 mg/100 g body weight) and dams of group 4 were fed a 20% protein diet supplemented with both caffeine and zinc. Fetuses were surgically delivered on day 22, and brains were removed and analyzed for alkaline phosphatase activity, protein, zinc, cholesterol and DNA concentrations. Fetal brain caffeine levels, as well as maternal and fetal plasma caffeine levels, were determined in caffeine-supplemented groups. The body weight of group 4 and brain weights of groups 3 and 4 were higher than those of groups 1 and 2. Alkaline phosphatase activity of group 3 was less than that of group 1. The brain zinc concentration of group 2 was higher than in the other groups, but that of group 4 was less than that of group 1. The present study indicated that the supplementation of caffeine to the maternal diet decreased zinc levels in the fetal brain, and the addition of extra zinc to this diet did not return the zinc level to that of the control level as we had expected. In addition, the supplementation of caffeine and zinc together increased the body weights of the fetuses compared to the controls, but the addition of only one of these substances had no effect, suggesting that the combination of caffeine and zinc may have unique effects on fetal growth.

Pregnant dams were fed a 20% protein diet with caffeine (2 mg/100 g b.wt.), starting on day 9 of gestation. At birth, each dam with 8 assigned pups was fed this diet until weaning, day 22. On day 22, female rats were caged and fed this diet until day 93. Starting on day 93, the caffeine-supplemented diet was replaced with a caffeine-free, 20% protein diet until day 388. Starting on day 31, each animal was placed in a photoactivity cage, and locomotive activity was measured until day 375. On day 388, the animals were killed, and their brains were removed and divided into 7 regions. The weight, DNA, protein and zinc contents, and alkaline phosphatase activity of each region were determined. Locomotive activity of the caffeine-fed group was higher than in the noncaffeine control group. Accumulative activity scores showed 3 subgroups (high, medium, and low) in both groups at day 93. The medium activity subgroup in the caffeine group was greater than the controls from day 72 to day 93. These differences reappeared 5 weeks after cessation of caffeine supplementation and continued until day 375. The differences in activity were minimum in the high and low subgroups. Chronic caffeine intake in early life permanently affected the medium activity subgroup. Furthermore, various regions of the brain were biochemically altered in spite of the feeding of a noncaffeine diet for almost 300 days after caffeine.

The purpose of the present study was to determine the levels of zinc in the hearts of growing post-weaning offspring, fetuses and their dams chronically fed caffeine. A further study was conducted to determine the distribution of Zn in subcellular heart fractions affected by acutely injecting caffeine into the veins of the adult rats. After delivery pups were raised on a 200 g protein/kg diet until day 22 of weaning. On day 22 randomly selected male offspring from each litter were divided into two groups. Group 1 was fed continuously on the same diet as a control, whereas in the experimental group offspring were fed on a 200 g protein/kg diet supplemented with caffeine (20 mg/kg). On day 49 the animals were killed and Zn, calcium and magnesium concentrations of the hearts were measured. In the second series of studies pregnant dams were randomly divided into two groups. Group 1 was fed on a 200 g protein/kg diet from day 3 of gestation, whereas in the experimental group dams were fed on the diet supplemented with caffeine. On day 22 of gestation the fetuses were surgically removed. The Zn, Ca and Mg concentrations of hearts of fetuses and dams were determined. In the third phase a caffeine solution was injected into the vein. After 45 min the hearts were removed and Zn levels in the subcellular fractions determined. The hearts of the growing offspring fed on a caffeine-supplemented diet consistently showed decreased Zn and Ca levels compared with the non-caffeine group.(ABSTRACT TRUNCATED AT 250 WORDS)

Pregnant dams were divided into four groups on day 10 of gestation. Dams of group 1 were fed an 20% protein diet as controls. Dams of groups 2, 3 and 4 were fed a 20% protein diet supplemented with 0.5 mg, 1 mg and 2 mg caffeine/100 g body weight of dams, respectively. Pups were delivered surgically on day 22, and their brains were rapidly removed and analyzed for DNA, protein, cholesterol, zinc and alkaline phosphatase activity. The dams' brains were analyzed for the same parameters as those of the pups. Plasma and brain caffeine levels were also determined in caffeine-supplemented groups. The pups' brains in group 2 were heavier than those in group 4. The DNA concentration of group 2 was higher than that of the other groups. The protein concentration of group 4 was higher than that of the other groups. The cholesterol concentration of group 3 and 4 was less than that of the controls. The zinc concentration of group 4 was less than that of group 2. Alkaline phosphatase activity was decreased in groups 3 and 4 compared with either controls or group 2. Dams showed no significant difference among the groups in the same biochemical parameters except for cholesterol concentration that was higher in groups 2, 3 and 4 than in the controls. Plasma and brain caffeine levels of the fetuses and plasma caffeine of the dams in group 4 were higher than those of either group 2 or 3. It is concluded that various amounts of maternal caffeine intake exert different effects on fetal brain growth. In contrast, the effect of caffeine on the dams' brain is relatively minor.

Time-pregnant rats were fed a regular laboratory diet until delivery. Litters delivered within an 8-hour period were combined and 8 randomly selected pups were assigned to each dam. Dams were then divided into two groups. Group I received a 20% protein diet. Group II was pair-fed to group I with a 20% protein diet containing caffeine (1 mg/100 g body weight) until day 22 postpartum. On weaning (day 22), only male pups were selected and fed the 20% protein or 20% protein + caffeine diet according to which diet their respective dams had been fed. On day 43, rats were killed and the brains were divided into 6 different areas. DNA, RNA, protein and cholesterol contents were measured. Caffeine supplementation resulted in a decrease in the total brain weight, and DNA and RNA content. DNA value of cortex-midbrain was smaller in the caffeine group whereas the values of medulla oblongata and striatum were greater in comparison with the controls. Caffeine's effects on RNA included an increase in the cerebellum, cortex-midbrain and hippocampus and a decrease in the medulla oblongata. Protein content of cerebellum was increased and of hippocampus decreased due to caffeine supplementation. Cholesterol content of the medulla oblongata and hippocampus was less in the caffeine group than in the control group. The present study shows that chronic caffeine intake during rapid periods of growth influences various parts of the brain in entirely different biochemical manners.

Pregnant dams were divided into two subgroups on day 10 of gestation. Half were fed a 20% protein diet and the other half an 8% protein diet. A second group also subdivided was pair-fed with rats of the first group. Their diet was supplemented with caffeine in amount calculated to provide daily doses of 2 mg/100 g body weight. On days 18, 20, and 22 randomly selected dams were injected with 3H-thymidine intraperitoneally and 1.5 h later their fetuses were delivered surgically in order to determine the rate of DNA synthesis along the gestation. The rest of the fetuses were delivered surgically on day 22. Pups' brains were rapidly removed and DNA, RNA, protein and 3H-thymidine uptake were studied. Average body weights of the fetuses in the caffeine-supplemented control group were smaller than those of the noncaffeine group. Effects of caffeine that were similar in both diet groups included a decrease in brain DNA content and concentration and an increase in brain protein content and concentration. However, the percent decrease and increase, respectively, was different depending on the nutritional status. DNA synthesis was not affected by malnutrition or caffeine supplementation on day 18 of gestation. Caffeine's effect on the rate of DNA synthesis was different on day 20 of gestation depending on nutritional status. Caffeine supplementation resulted in a decrease in DNA synthesis in both groups on day 22 of gestation. These data indicate that caffeine intake during pregnancy produces differential effects on fetal rat brain depending on dietary protein content.

At birth, dams with 8 randomly assigned pups were divided into three groups. Dams of group 1 were fed a control diet. Dams of groups 2 and 3 were fed the control diet supplemented with caffeine (1 mg and 2 mg/100 g body weight, respectively). Pups were killed at day 15 and their brains removed. After weighing, brains were analyzed for DNA, protein, cholesterol, zinc and alkaline phosphatase activity. Brain and plasma caffeine levels were also determined on groups 2 and 3. The dams were milked to measure caffeine levels. The brains from the dams were analyzed for the same parameters as the pups. Caffeine levels in group 3 were consistently higher than in group 2. In the pups, body and brain weights were heavier in group 3 than in the controls. Protein and cholesterol concentrations in group 2 were less than either controls or group 3. Alkaline phosphatase activity in group 2 was higher than either controls or group 3. In the dams, DNA concentration in groups 2 and 3 was less than the controls. Protein and cholesterol concentration in group 2 was less than group 3. It was concluded that low levels of caffeine in the maternal diet during lactation could affect various parameters in the newborn brain. These effects were different from those when the dietary caffeine level was doubled. In contrast, the effects of caffeine on brains of the dams were relatively minor.

Various levels of maternal caffeine ingestion during pregnancy were investigated to determine whether caffeine will affect the mineral contents of the growing bones of fetal rats. On day 8 of gestation, rat dams were fed with a 20% protein diet supplemented with 0.5 mg, 1 mg, or 2 mg caffeine/100 g of dams body weight as an experimental group and the same without caffeine as a control until day 22 of gestation. Fetuses were removed by cesarean section on day 22 and mandibular bones were removed to study the mineral contents of calcium, phosphorus, magnesium, and zinc. Although the mandible weighed more in the 0.5-mg caffeine group as compared to the controls, an additional increase of caffeine resulted in a decrease in weight. All calcium, magnesium, and zinc contents per bone decreased in the 1- and 2-mg caffeine groups as compared to either controls or 0.5-mg caffeine group, whereas phosphorus stayed relatively constant regardless of the different levels of caffeine intake. When data are expressed as per gram of bone tissue, most of the mineral contents among the groups disappeared suggesting that normal growth and development of the fetal bone are impaired as a results of maternal caffeine intake. Caffeine intake during gestation in the present study indicates that different levels of caffeine intake may exert not only different effects on mineral contents in bone development, but also affect the growth of the fetal bone.