Modulation by dietary vitamin E of I-compounds (putative indigenous DNA modifications) in rat liver and kidney

I(indigenous)-compounds are age-related, carcinogen adduct-like, putative indigenous DNA modifications detectable by 32P-postlabeling assay in untreated animals. To investigate the origins of these DNA derivatives, we examined the effects of dietary vitamin E, a natural antioxidant, on I-compounds of rat liver and kidney DNA. Weanling female Sprague-Dawley rats were fed Draper's diets containing 0, 100, 1000, or 10,000 mg/kg alpha-tocopheryl acetate for 6 mo. The DNA from four individual rats of each group was analyzed by a nuclease P1-enhanced version of the 32P-postlabeling assay for DNA adducts. The amount of vitamin E in the liver was measured by high performance liquid chromatography. Rats fed vitamin E-deficient diet (0 mg/kg) showed identical profiles and similar levels of I-compounds as those fed the 100 mg/kg diet. Most I-spots were significantly intensified and one tissue-specific extra spot was found in both liver and kidney DNA of rats fed the 1000 or 10,000 mg/kg vitamin E diet. However, one of the five major I-spots detected in the kidney was weaker in the 1000 and 10,000 mg/kg groups than in the 0 and 100 mg/kg groups. These results show that formation of most I-compounds was not affected by vitamin E-deficient diet, and that long-term feeding of diet containing high levels of vitamin E may cause metabolic alterations leading to an increased formation of DNA-reactive (potentially mutagenic or carcinogenic) electrophiles.     

High dietary lipids induce liver glucose-6-phosphatase expression in rainbow trout (Oncorhynchus mykiss)

To contribute to the understanding of the mechanisms involved in poor metabolic utilization of dietary carbohydrates by rainbow trout (Oncorhynchus mykiss), we explored in this study the effects of dietary lipids on the regulation of two hepatic key enzymes, i.e., glucokinase (GK, first enzyme of the glycolytic pathway) and glucose-6-phosphatase (G6Pase, last enzyme of the gluconeogenesis). Two groups of juvenile trout were pair-fed for 8 wk either a low (10%) or a high (25%) level of dietary lipids supplied as fish oil; the pair-feeding technique was adopted to vary fat intake while keeping the protein and carbohydrate intakes more or less constant. Fish fed the high level of dietary lipids had inefficient control of glycemia compared with fish fed the low level of lipids. Levels of dietary lipids did not affect GK activity even though there was a small increase of GK mRNA level at 3 h after feeding high levels of lipids. By contrast, the high level of dietary lipids significantly increased G6Pase mRNA expression at 3, 6 and 12 h and enzyme activity at 6 h after food consumption. Thus, these data suggest that poor dietary carbohydrate utilization in rainbow trout may be related at least in part to increased hepatic glucose production under conditions of high dietary fat intake.     

High dietary intake of sodium selenite induces oxidative DNA damage in rat liver

One mechanism for the cancer-chemopreventive effects of high selenium (Se) intake is hypothesized to be antioxidant protection of DNA. In this work we examine DNA oxidation in whole animals as a function of dietary Se intake and carcinogen administration. Weanling male Sprague-Dawley rats were fed a basal, Torula yeast-based, Se-deficient diet supplemented with 0, 0.15, or 2.0 ppm Se as sodium selenite for 10 wk. They were then injected with 0, 0.1, or 10 mg /kg body weight of the pro-oxidant carcinogen N-nitrosodiethylamine. High levels of carcinogen and high levels of selenite intake each increased concentration of 8-hydroxy-2'-deoxyguanosine in liver DNA. Se-dependent glutathione peroxidase I gene expression and enzyme activity were dramatically reduced by dietary Se deficiency but were unaffected by carcinogen administration. There were no significant main or interactive effects of Se or carcinogen on activity or gene expression of the DNA repair enzyme 8-oxoguanine glycosylase I. These results do not support the hypothesis that high Se intake may be cancer-preventive by inhibiting oxidative DNA damage. Rather than inhibiting oxidative DNA damage, these findings suggest that high dietary intake of inorganic Se may promote in vivo DNA oxidation.     

Effect of dietary lipids and vitamin E on in vitro lipid peroxidation in rat liver and kidney homogenates

Rats were fed for 5 wk 10% (wt/wt) menhaden oil (MO) or a 10% corn oil-lard (COL) mixture (1:1) in diets with a low vitamin E content (less than or equal to 5 mg/kg) or supplemented with d-alpha-tocopheryl succinate to a total of 30 or 150 mg per kg. Thiobarbituric acid-reactive substances (TBARS), conjugated dienes (CD), hexanal and total volatiles (TOV) were measured in tissue homogenates incubated at 37 degrees C for 1 h in the absence (uninduced) and presence of 15 microM ferrous sulfate (induced). The fatty acid composition of liver and kidney reflected that of dietary lipids. For uninduced peroxidation, there was in general a significant inverse correlation of TBARS, CD and TOV with the log of dietary vitamin E content for liver and kidney from rats fed either lipid. For induced peroxidation, the inverse correlation was significant for liver, but not for kidney, from rats fed either lipid. The correlation was generally higher for liver and kidney from rats fed COL than for tissues from rats fed MO. Vitamin E was thus a more effective antioxidant for liver than for kidney regardless of the dietary lipid, and for liver and kidney from rats fed COL than from rats fed MO. Dietary MO enhanced tissue susceptibility to both peroxidation systems. A simulation model developed to mimic the experiments showed good correlations between experimental data and simulated values.     

Localization of ultrastructural alterations induced in rat liver by dietary polybromobiphenyls (FireMaster BP-6)

In 1973 and 1974, widespread environmental pollution and animal losses in Michigan were caused by the accidental incorporation of polybrominated biphenyls (PBBs, FireMaster FF-1) in livestock feed. This study documents the deleterious changes in the liver during an early state of polybrominated biphenyl-induced toxicosis. Male weanling Fischer rats were fed diets containing either 0 or 100 ppm of polybromobiphenyls (Fire-Master BP-6) (PBBs) for 10 days. Dietary PBBs did not affect body weight or food consumption. At necropsy, the liver was removed and processed for light and electron microscopy. The livers of PBB-treated rats were enlarged and friable. Light microscopic changes included moderate enlargement of centrilobular and midzonal hepatocytes which exhibited a foamy cytoplasm. Periportal hepatocytes appeared normal except that most bile canaliculi were dilated. Ultrastructural alterations within centrilobular hepatocytes consisted of small and dense mitochondria, abundant smooth endoplasmic reticulum, deep stacks of rough endoplasmic reticulum which appeared disorganized with fewer attached ribosomes, numerous lipid droplets, and dilated bile canaliculi which often contained myelin-like figures. The ultrastructural changes within mid-zonal hepatocytes were similar to centrilobular hepatocytes except there was less proliferation of smooth and rough endoplasmic reticulum, larger and less dense mitochondria, and more numerous lipid droplets which varied greatly in size. Glycogen depletion was apparent in both centrilobular and midzonal hepatocytes as was numerous multi-vesicular bodies associated with the Golgi apparatus.     

Effect of dietary proteins on insulin-like growth factor-1 (IGF-1) messenger ribonucleic acid content in rat liver.

Effects of quantity and quality of dietary proteins on plasma immunoreactive insulin-like growth factor-1 (IGF-1) concentration, and content of IGF-1 mRNA in rat liver were investigated in rats. Plasma immunoreactive IGF-1 concentration in rats given a casein diet was higher than that in rats given a soya-bean-protein or protein-free diet. The IGF-1 mRNA content in liver was estimated by the Northern blot hybridization technique employing 32P-labelled rat IGF-1 complementary DNA (cDNA). At least four molecular species of IGF-1 mRNA of different molecular weight were found in rat liver. The sizes were 0.8-1.2, 2.0, 3.6-4.0 and 7.4 kb. Most of the mRNA species decreased in the livers of rats given a gluten diet (120 g gluten/kg diet) compared with rats given the casein diet. In particular, mRNA of 7.4 kb decreased markedly. When rats were fed on the protein-free diet, mRNA of all species decreased significantly. The estimated IGF-1 mRNA in the livers of rats fed on the gluten or protein-free diet was almost 0.4 of that of the rats given the casein diet. Feeding the soya-bean-protein diet did not result in a marked effect on the hepatic content of mRNA species of IGF-1. The results showed that liver IGF-1 mRNA content is sensitively regulated by quantity and nutritional quality of dietary proteins.     

Effect of dietary excess leucine on nicotinamide nucleotide level in rat liver.

1. Six groups of rats were freely fed diets containing casein at 5, 10 and 20% levels with and without nicotinic acid. After 2 weeks on these diets, hepatic nicotinamide nucleotide and free nicotinic acid concentrations were studied. 2. Hepatic nicotinamide nucleotide level was kept in the normal range in rats fed the 10 and 20% casein diets with and without nicotinic acid. 3. L-leucine supplemented at the 5% level to the 10 and 20% casein diets caused significant decrease in hepatic nicotinamide nucleotide level only in rats fed nicotinic acid devoid diet. 4. Hepatic nicotinamide nucleotide in rats fed the diet in which casein was replaced by zein increased significantly by adding nicotinic acid. This increase in the hepatic nicotinamide nucleotide caused by dietary supplemented nicotinic acid was not reduced by the addition of L-leucine. 5. The hepatic free nicotinic acid level did not change even in rats of which hepatic nicotinamide nucleotide was significantly reduced. 6. Urinary excretion of nicotinic acid and N-methylnicotinamide was increased significantly by adding nicotinic acid to the diet but was not affected by adding L-leucine at the 5% level. 7. From the above results, a possible mechanism of L-leucine action was discussed.     

Effect of dietary supplementation with carotenoids on xenobiotic metabolizing enzymes in the liver, lung, kidney and small intestine of the rat.

The effect of 16 d intake of 300 mg carotenoids/kg diet (beta-carotene (beta C), bixin (BX), lycopene (LY), lutein (LU), canthaxanthin (CX) or astaxanthin (AX) on xenobiotic metabolizing enzymes in the liver, lung, kidney and small intestine of male Wistar rats was assessed. A control group received the basal diet (AIN-76) without carotenoids and a positive control group for enzyme induction received 3-methylcholanthrene (3-MC) at 666 mg/kg diet. Cytochrome P450 activity was assessed using the substrates ethoxyresorufin for P450 1A1, methoxyresorufin for P450 1A2, pentoxyresorufin for P450 2B1/2 and benzyloxyresorufin for P450 types 1A1/2, 2B1/2 and 3A. Glutathione-S-transferase (EC 2.5.1.18) and reduced glutathione status were assessed. Carotenoid uptake by the tissues was also determined. 3-MC and the carotenoids BX, CX and AX led to significant increases compared with control in liver, lung and kidney ethoxyresorufin-O-deethylation. Methoxyresorufin-O-demethylation activity was significantly increased in liver and lung by BX, CX and AX but only CX and AX significantly increased activity in kidney. Pentoxyresorufin-O-depentylation and benzyloxyresorufin-O-dearylation increased in liver of 3-MC-, BX-, CX- and AX-treated rats, but to a much lesser degree than for the other two substrates. Benzyloxyresorufin-O-dearylation in lung was significantly decreased by all carotenoids. Activities of any of the measured enzymes in the small intestine were undetectable in all treatment groups except the 3-MC group. Glutathione status was unaffected by any of the treatments. This is the first study identifying the carotenoids BX, CX and AX as inducers of rat lung and kidney xenobiotic metabolizing enzymes.     

Age, dietary selenium and quantity of 7,12-dimethylbenz(a)anthracene influence the in vivo occurrence of rat mammary DNA adducts.

The present studies determined the impact of age, dietary selenium and 7,12-dimethylbenz(a)anthracene (DMBA) dosage on the occurrence of DMBA-DNA adducts in rat mammary tissue. Diets formulated to contain selenium, as sodium selenite, at 0.1 (control) or 2.0 mg/kg were fed for 2 wk before DMBA treatment. Food intake and weight gain were not influenced by selenium intake. Anti- and syn-dihydrodiol epoxide adducts reached maximum binding by 24 and 36 h, respectively, after treatment with DMBA. Consumption of the diet containing 2.0 mg Se/kg inhibited the appearance of both anti- and syn-adducts by approximately 50% compared with controls. The occurrence of DMBA-DNA adducts correlated with a dosage of DMBA from 5 to 50 mg/kg body wt (r greater than or equal to 0.95). The ability of supplemental selenite to lower DMBA binding to mammary cell DNA increased as the quantity of the carcinogen administered increased. DMBA-DNA binding was found to increase with the increasing age of the rat. Nevertheless, dietary selenium supplementation was effective in reducing DMBA binding to DNA in all ages examined. These data confirmed the ability of dietary selenium to inhibit the in vivo metabolism of DMBA under a variety of conditions.     

Acute elevation by short-term dietary restriction or food deprivation of type I I-compound levels in rat liver DNA

Type I I-compounds are bulky endogenous DNA modifications detectable by 32P postlabeling that exhibit age, species, tissue, genotype, gender, and diet dependence. Their formation appears unrelated to oxidative stress. In fact, several lines of indirect evidence suggest that many type I I-compounds may represent normal functional DNA modifications. For example, long-term dietary restriction (DR), which retards the development of age-related diseases including cancer and extends median and maximum life spans, unexpectedly elicits significant increases rather than decreases in the levels of many I-compounds in different rodent tissues. Positive linear correlations have been observed between such levels and median life spans of the animals. In the present work we have investigated 1) whether elevation of I-compound levels does not depend on chronic DR, i.e., occurs after a short period of DR or fasting, and 2) whether I-compound levels return to control values after the animals are returned to unrestricted feeding after food deprivation. Female Fischer 344 rats (approx 140 g each) were randomized into three groups. Group I was fed a natural ingredient (Purina 5001) diet ad libitum (AL) throughout the study, Group 2 was switched to 60% of the AL amount (40% DR) at 0 hour, and Group 3 was given no food for up to 72 hours and then returned to AL feeding until the end of the experiment. Liver DNA of individual rats (n = 4) was isolated for I-compound analysis at 24, 72, and 240 hours. Restricted and food-deprived rats showed elevated levels of hepatic I-compounds, with fasting eliciting the highest levels. These effects were seen as early as the 24-hour time point. Refeeding after 72 hours of food deprivation restored the levels to control values, measured at 240 hours. Our observations are discussed in relation to carcinogenesis and tumor promotion. The almost instantaneous changes of endogenous DNA modifications showed their exquisite sensitivity to nutritional factors and provided strong new evidence for precise regulation of their formation and removal.     

Postweaning dietary folate deficiency provided through childhood to puberty permanently increases genomic DNA methylation in adult rat liver

Folate plays an important role in the pathogenesis of several chronic diseases by its potential ability to modulate DNA methylation. We hypothesized that the postweaning period might be a highly susceptible period to dietary folate intervention for DNA methylation patterning. We determined the effects of timing and duration of dietary folate intervention provided during the postweaning period on genomic DNA methylation in adult rat liver. In study 1, weanling rats were randomized to receive an amino acid-defined diet containing 0 (deficient), 2 (control), or 8 (supplemented) mg folic acid/kg until 8 wk of age, after which all the rats were fed the control diet until 30 wk of age. In study 2, weanling rats were fed the control diet until 8 wk of age and then randomized to receive the diet containing 0, 2, or 8 mg folic acid/kg until 30 wk of age. In study 3, weanling rats were randomized to receive these diets until 30 wk of age. Dietary folate deficiency, but not supplementation, provided during the postweaning period through childhood to puberty significantly increased genomic DNA methylation by 34-48% (P < 0.04) in rat liver that persisted into adulthood following a return to the control diet at puberty. In contrast, dietary folate deficiency or supplementation continually imposed at weaning or at puberty did not significantly affect genomic DNA methylation in adult rat liver. Our data suggest that early folate nutrition during postnatal development plays an important role in epigenetic programming that can have a permanent effect in adulthood.     

Combined effects of high-fat diet and ethanol induce oxidative stress in rat liver

Individuals affected by liver steatosis seldom have symptoms of liver injury, but may be particularly vulnerable to oxidative insults. In this study, we evaluated liver redox alterations produced by acute ethanol administration to rats that were fed a high-fat diet (HFD). Adult male Wistar rats were fed HFD or standard diet (controls) for 1 month; a group of animals from each condition were gavaged with 35% (vol/vol) ethanol every 12 h for the last 3 days of the experiment. Total lipid content determined in liver showed lipid accumulation after HFD or HFD combined with ethanol. HFD alone induced a significant rise of seric alanine aminotransferase levels and a marked reduction of antioxidant enzyme activities (catalase, superoxide dismutase, glutathione transferase). Ethanol alone caused a significant rise of seric cholesterol levels and enhanced mitochondrial H₂O₂ production, but without apparent oxidative stress as evaluated by thiobarbituric acid-reactive substances (TBARS) assay. The combination of HFD and acute ethanol caused an increase of TBARS, indicating lipid peroxidation, most likely as a consequence of a decrease in antioxidant defenses induced by HFD and of an increase in reactive oxygen species production induced by ethanol. Principal component analysis, based on all the measured parameters, that is, serum liver function tests, antioxidant enzyme activities, mitochondrial H₂O₂ release, and TBARS, indicated that HFD and ethanol act as two independent factors. In conclusion, our results show that HFD or acute ethanol alone produce, at the most, mild liver injury, whereas their combination triggers oxidative stress, possibly inducing a progression toward liver disease. Hence, our data indicate that a diet too rich in fat is a serious risk factor for the occurrence of liver injury deriving from acute ethanol consumption.     

Effect of varying dietary fat levels on rat growth and oxidative DNA damage.

Dietary fat has previously been shown to have somewhat complicated relationships to levels of oxidative stress in rats. In this study, we examined the effects of five different dietary fat intakes on levels of oxidative DNA damage in rats. Animals fed diets containing 3%, 5%, 10%, or 15% corn oil had body weights that were similar after 20 weeks. Animals fed a 20% fat diet, however, had significantly higher mean body weight than any other group. Levels of 5-hydroxymethyl-2'-deoxyuridine, one marker of oxidative DNA damage, had different relationships to dietary fat in blood and mammary gland. In blood, levels increased with dietary fat levels, and the highest levels were observed with the 20% fat diet (65% higher levels than with the 3% fat diet). In mammary gland, a plateau-type effect was observed, with maximal levels of oxidative DNA damage being obtained using 10% fat (representing a 68% increase relative to the 3% fat diet). This could be a result of induction of compensatory mechanisms in response to a high-fat diet in mammary gland but not in the short-lived nucleated blood cells. Oxidative DNA damage levels in blood thus appear to be a marker of dietary fat intake. In mammary gland, however, levels of DNA damage are consistent with previously observed promotional effects of dietary fat on mammary gland tumorigenesis at lower levels of fat intake with little or no incremental promoting effects at higher levels of fat intake.     

Inhibition of liver, kidney, and erythrocyte delta-aminolevulinic acid dehydratase (porphobilinogen synthase) by gallium in the rat

Selective inhibition of enzymes in the heme biosynthesis pathway with concomitant urinary excretion of heme precursors serve as potentially important biological markers of chemical exposure and cell injury. Intratracheal administration of gallium arsenide particulate suspensions has been shown to result in inhibition of delta-aminolevulinic acid dehydratase (ALAD) in several tissues and increased excretion of the heme precursor aminolevulinic acid (ALA). This study was undertaken to evaluate in vivo the role of gallium alone in ALAD inhibition and increased urinary excretion of ALA. Male CD rats received a single ip injection of Ga2(SO4)3 at doses of 12.5, 25, 50, 100, and 200 mg Ga/kg. A dose-dependent inhibition of ALAD was observed 24 hr later in liver, kidney, and erythrocytes. After injection of 25 mg Ga/kg, maximal inhibition (42 to 49% of control) of ALAD occurred between 6 and 24 hr in liver and kidney with full recovery of activity at 96 hr. In erythrocytes, maximal inhibition (48% of control) occurred between 24 and 48 hr with recovery of activity at 96 hr. Mild to moderate renal proximal tubular necrosis in the pars recta was observed 24 hr after administration of 100 and 200 mg/kg, but no histopathologic changes were evident at lower doses. No consistent changes in urinary excretion of ALA were observed. Lineweaver-Burk analyses of renal and hepatic ALAD activities in the absence and presence of gallium indicated that the inhibition of ALAD by this element is noncompetitive (same Km, decreased Vmax). Gallium was shown to possess an inhibition constant (Ki) of approximately 3 microns for ALAD, similar to the Ki obtained for lead in other studies. Incubation of ALAD in vitro with gallium and lead, an active thiol group inhibitor, resulted in a greater inhibition of the enzyme. Further in vitro studies demonstrated the attenuation of gallium inhibition of hepatic and renal ALAD by zinc, suggesting that the mechanism of gallium action may involve competition for or displacement of zinc from the sulfhydryl group of the enzyme active site. Since ALAD inhibition occurred at doses at which no histopathologic changes were evident, the determination of ALAD activity in various tissues, including blood, may be of potential value as a biomarker of exposure/toxicity to metals such as gallium. The effect of chemical form and route of exposure of gallium and effects of other Group III metals on inhibition of ALAD and excretion of ALA is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental changes in the distribution of rat liver ribosomes in response to dietary manipulations.

Ribosomal profiles were prepared from liver postmitochondrial supernatant preparations of 2- to 21-day old and 60-day-old rats. Rats fed or starved for 3 or 15 to 16 hours were compared. Relative ribosomal distribution, calculated from planimetric analysis of the hepatic ribosomal profiles, was found to be approximately constant at all ages when fed animals were compared. In contrast, starvation for 15 hours resulted in profound changes in ribosomal distribution, i.e., a relative accumulation of oligosomes as compared with polysomes. This change in ribosomal distribution was most extensive in animals younger than 2 weeks of age, 60-day-old rats showing no effect. Refeeding of 6-day-old starved rats with diets containing 10% protein resulted in a prompt decrease in the planimetric yield of monsomes and disomes. Similarly, dietary protein was shown to be required to prevent accumulation of these two species. It is suggested that it is the response of the ribosomal cycle to starvation that changes with age rather than the maximum capacity for protein synthesis.