Modulation of DNA modification (I-compound) levels in rat liver and kidney by dietary carbohydrate, protein, fat, vitamin, and mineral content.

I-compounds are DNA modifications detected by 32P-postlabeling that increase with age in rodents without known carcinogen exposure. Diet type (natural ingredient versus purified) greatly influences patterns and levels of I-compounds. To test the hypothesis that I-compound formation is affected, also, by dietary macro- and micronutrients, effects of carbohydrate, protein, fat, vitamin, and mineral content on rat liver and kidney I-compounds were determined. Female Sprague-Dawley rats were fed basic or modified AIN-76A purified diets for 3-6 months. High protein (HP) diet (50%, w/w) increased I-compound levels in liver but not kidney. High carbohydrate (HC) diet (78%) produced a significant increase in the polar as well as total I-compound levels in both tissues. High fat diets (20%) elicited significantly lower levels of liver I-compounds than HC, HP, and basic diets. There were few significant differences between high polyunsaturated (safflower oil) and saturated fat (lard) diet groups. No qualitative differences in I-compound profiles were observed in either tissue. In rats fed basic diet supplemented with vitamins and/or minerals, increased vitamin content reduced the levels of polar I-compounds in liver. No extra diet-induced adducts were observed; all effects were of a quantitative nature. These data provide direct evidence that nutrients significantly influence I-compound levels and support the hypothesis that normal metabolism of nutrients leads to the production of small amounts of DNA-reactive electrophiles. These observations suggest a novel mechanism where nutrient composition of the diet may play a role in development of neoplasia and other adverse health effects.     

Age-dependent covalent DNA alterations (I-compounds) in rodent tissues: species, tissue and sex specificities

I-compounds are non-polar covalent DNA modifications of as yet undetermined structure that tend to accumulate in an age-dependent manner in tissues of untreated animals. They are detectable by 32P-postlabeling assay because of their adduct-like properties and chromatographically resemble DNA nucleotides containing bulky/hydrophobic moieties. To determine which factors may be involved in their formation, I-compounds were examined by 32P-postlabeling in liver and kidney DNA of female and male Sprague-Dawley rats and Syrian hamsters of different ages (1, 4 and 10 months and 1, 2.5 and 9.5 months, respectively). The following results were obtained: (i) Every tissue DNA studied contained characteristic I-compounds. (ii) Patterns and amounts of I-compounds were reproducible among animals of the same kind. (iii) There were pronounced organ and species differences. (iv) I-compound patterns were sex-dependent. (v) I-compound levels increased with age in all tissues studied, except in male hamster kidney, a target organ of estrogen-induced carcinogenesis. The highest levels were observed in liver and kidney of 10-month-old female rats. (vi) The rise of I-compound levels was less steep during the later part of the observation period for female but not male animals. (vii) Gonadectomy decreased I-compound levels in female hamster kidney DNA, while causing a slight increase in male animals later in life. These I-compounds were identical to previously reported DNA modifications that increased in male hamster kidneys after prolonged estrogen treatment. Points, iv, vi and vii strongly implicated sex hormones in I-compound formation. The qualitative effects of species, tissue differentiation, gender and sex hormones on these DNA modifications support the hypothesis that I-compounds are formed by the binding of endogenous electrophiles to DNA. As persistent DNA alterations, they are likely to affect DNA replication and to play a role in spontaneous and chemically induced carcinogenesis and in aging.     

Effects of different diets and dietary restriction on perinatal endogenous DNA adducts. Time dependence of oxidative and presumptive nonoxidative lesions

Type II I-compounds (indigenous DNA adducts) denote a class of bulky oxidative DNA lesions that are detectable by 32P-postlabeling and represent useful biomarkers of DNA damage induced by oxidative stress. Their levels are increased in tissue DNA under pro-oxidant conditions, for example, as previously shown, in newborn rat organs. Here we have investigated whether the maternal diet affects perinatal type II I-compound levels. Pregnant F344 rats were fed Purina-5001 natural-ingredient or AIN-93G purified diet from day 11 of gestation. Type II I-compounds were measured in liver DNA at three different developmental stages, i.e., fetus, and 24 h and 9 days postnatally. Higher adduct levels were detected in the Purina-5001 group at each stage. In a second experiment, pregnant F344 rats were subjected to dietary restriction (DR) (by 40%; Purina-5001) from day 12 of gestation. At 24 h postpartum hepatic type II I-compound levels were decreased compared to parallel ad libitum (AL) fed controls. As an unrelated observation, fetal lung, but not liver, kidney, and skin DNA contained a different pattern of nonpolar, apparently nonoxidative adducts, which were not diet-dependent. These spots were not detectable 24 h after birth and were observed at much reduced levels and only in a few samples at 9 days. The main results show for the first time that the maternal nutrition modulated levels of oxidative lesions in fetal and neonatal DNA, but the underlying mechanisms (e.g., differences in metal or caloric content of the diets) still need to be determined. The dietary effects were apparently transmitted through both placenta and the mother's milk.     

Bulky endogenous DNA modifications (I-compounds) -possible structural origins and functional implications

I-compounds are bulky covalent DNA modifications which increase with age in tissues of unexposed laboratory animals and are derived from endogenous DNA-reactive intermediates of nutrient and oxygen metabolism. They have been classified into 2 major groups, i.e., type I and type II. Profiles and levels of type I I-compounds show considerable variation depending on species, strain, tissue, and gender, but are also affected by diet and chemical and hormonal exposures, indicating their formation to be determined by genetic and environmental factors. For example, sex hormones, dietary oat lipids, and isoprenoids affect their profiles and/or levels in tissue DNA. A gradual depletion of many type I I-compounds occurs during carcinogenesis, as many carcinogens/tumor promoters significantly reduce their levels, and neoplasms display very low levels, apparently independent of growth rate, indicating a loss of the ability to form these modified nucleotides. Conversely, dietary restriction, the most effective method to retard carcinogenesis and aging, significantly elevates type I I-compound levels, as compared to age-matched ad libitum-fed animals. Levels of many liver and kidney I-compounds exhibit genotype- and diet-dependent positive linear correlations with median life span. Formation of high levels of oat-related type I I-compounds has been associated with reduced formation of carcinogen-induced preneoplastic hepatic foci. These results suggest that such DNA modifications may not represent DNA lesions but may rather be functionally important. This view is supported by circadian rhythms displayed by some I-compounds. Thus, certain type I I-compounds may play a protective role against carcinogenesis and age-associated degenerative processes. Type II I-compounds, on the other hand, represent DNA damage and include several bulky lesions, which are enhanced by pro-oxidant carcinogens such as ferric nitrilotri- acetate (Fe-NTA) in target organ (kidney) DNA of rodents and are identical to products generated by oxidizing DNA or oligonucleotides under Fenton reaction conditions in vitro. Some of these products appear to be base-base or base-sugar intrastrand crosslinks. Notably, Fe-NTA reduces the levels of type I I-compounds in renal DNA. Type II I-compound levels are increased in tissue DNA of normal newborn rats. The formation of oxidative DNA lesions in neonates is most likely caused by oxidative stress associated with the sudden increase of partial oxygen pressure in arterial blood and tissues at birth. In view of the rapid cell replication at this developmental stage, endogenous oxidative DNA lesions sustained early in life may contribute to the development of cancer and degenerative diseases later in life.     

Induction of rat liver DNA alterations by chronic administration of peroxisome proliferators as detected by 32P-postlabeling

The mechanisms of the hepatocarcinogenicity of non-mutagenic peroxisome proliferators, i.e. compounds used as hypolipidemic drugs and industrial plasticizers, are not sufficiently understood. To gain more information on the mechanism of their action, the chronic effects of two structurally diverse peroxisome proliferators on rat-liver DNA were investigated by the 32P-postlabeling assay. Male F-344 rats (1.5 month old) were fed ciprofibrate (0.025%) in the diet for 2, 5, 8, and 16 months or Wy-14643 (0.1%) for 18 months. Liver DNA from individual treated animals (3-4 per group) and age-matched controls was analyzed by the nuclease P1/bisphosphate version of the 32P-postlabeling assay. Three distinct types of exposure-related DNA alterations were observed: (i) A significant reduction of the age-dependent accumulation of I-compounds (putative indigenous DNA modifications) (type 1), (ii) adduct-like DNA derivatives induced by the treatments (type 2), and (iii) as yet structurally uncharacterized radiolabeled material occupying substantial areas of DNA adduct maps and accumulating in an exposure time-dependent manner (type 3). DNA from liver tumors generated by these agents displayed only traces of I-compounds, lacked all but one adduct-like derivatives, and had no type 3 alterations. Thus, in contrast to the non-mutagenicity of peroxisome proliferators in short-term assays, chronic administration of these compounds led to DNA alterations that were detectable by 32P-postlabeling assay.     

Effects of dietary transition metals on oxidative DNA lesions in neonatal rats

Bulky endogenous oxidative lesions (type II I-compounds) reflect DNA damage associated with oxidative stress. As shown by 32P-postlabeling, their levels are enhanced by pro-oxidant genotoxins and also shortly after normal birth in several rat tissues as a function of time and the maternal diet. In order to elucidate which dietary components contribute to postnatal DNA damage, we have focused, herein, on the possible role of transition metals (iron, copper, and nickel). Pregnant Fischer 344 (F344) rats were fed AIN-93G purified diet containing different amounts of iron, copper, and nickel, or Purina-5001 natural-ingredient diet (which contains relatively high concentrations of these metals). Type II I-compounds were estimated by nuclease P1-enhanced 32P-postlabeling in liver and lung DNA of fetuses and at 24h and day 9 post-partum. Increased postnatal oxidative damage was detected in liver but not lung DNA of neonates exposed to higher amounts of dietary transition metals. There were significant positive linear correlations between maternal transition metal intake and neonatal, but not fetal and maternal type II I-compound levels. The results show that transition metals in the maternal diet affect perinatal oxidative DNA damage, presumably via a Fenton-type reaction. They also provide evidence for optimal levels in the maternal diet of transition metals, which on one hand, are essential for life, but on the other, can cause potentially deleterious DNA alterations in the offspring.     

Age-related DNA modifications (I-compounds): Modulation by physiological and pathological processes

I-compounds are covalent DNA modifications that can be detected and measured by ³²P-postlabeling assay because of their DNA-adduct like properties. They accumulate in an age-dependent, highly reproducible manner in tissue DNA of untreated animals in the absence of exogenous carcinogens and, therefore, appear to arise via the interaction of DNA with endogenous reactants formed in the course of normal metabolism. Chromatographically, they exhibit a wide range of polarities, indicative of structural diversity. In addition to age-dependent increases, I-compound profiles exhibit prominent species-, sex-, tissue- and diet-dependent qualitative and quantitative differences. Natural-ingredient (chow) diets produce qualitative differences as well as substantially higher I-compound levels in rat liver and kidney, when compared with purified diets. Modified purified diets containing high carbohydrate, protein, or fat concentrations further modulate I-compound profiles. During liver regeneration, I-compounds behave like DNA adducts rather than m⁵C in that their levels are not quickly restored. Treatment of rats with the hepatocarcinogens 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), CCl₄, and peroxisome proliferators as well as with a choline-devoid hepatocarcinogenic diet depressed the age-related increases of I-compound levels in liver, the target organ. Additional ³²P-labeled derivatives were observed only with the peroxisome proliferators and presumably represent DNA adducts of exogenous origin. No I-compounds were detected in a series of Morris hepatomas with different degrees of differentiation. Thus, loss of I-compounds may be associated with altered gene expression/dedifferentiation. On the other hand, the age-dependent accumulation of I-compounds and their adduct-like character suggest potential relations to aging-associated dysdifferentiation and initiation of cancer. Structural complexity indicates different biological roles of I-compounds.     

Circadian rhythm of covalent modifications in liver DNA.

32P-postlabeling analysis recently revealed that in addition to 5-methylcytosine, mammalian DNA contains covalently modified nucleotides of unknown structures and functions termed I-compounds whose levels increase with age. I-compound levels, in addition, depend on species, strain, sex, tissue, and diet and are generally lowered by carcinogen exposure. As shown here, levels of several non-polar I-compounds in liver DNA of untreated male C3H mice were elevated 2 to 8.5 times at 1800 h and 2400 h as compared to 0600 h and 1200 h, while polar I-compounds and persistent carcinogen-DNA adducts induced by safrole were unaffected by time of day. In liver DNA of male F-344 rats 4 non-polar I-compounds and 4 polar I-compounds showed significant circadian rhythm at 2000 h compared to 0800 h. This novel circadian variation of DNA structure implies mechanisms precisely regulating I-compound levels in vivo and may conceivably be linked to diurnal differences of DNA synthesis and gene expression.     

Acute and long-term effects of carbon tetrachloride on DNA modifications (I-compounds) in male mouse liver

I-compounds are recently discovered species and tissue dependent covalent DNA modifications which are detectable by the 32P-postlabeling assay for DNA adducts and tend to increase with the animal's age. The effects of the hepatocarcinogen carbon tetrachloride (CCl4) on hepatic I-compounds were studied in 10-12-month-old male ICR mice using the 32P-postlabeling assay. CCl4 was dissolved in corn oil (20%, v/v) and intraperitoneally (i.p.) injected in doses of 0.75 ml/kg (0.375 ml/100 g body weight, 20% CCl4 in corn oil) while control mice received corn oil only (0.375 ml/100 g body wt). Twenty-four h after a single injection of CCl4, the intensity of non-polar I-spots in liver DNA was significantly increased as compared with corn oil treated controls, while the level of one polar I-compound was reduced at 24 h. DNA synthesis (as indicated by [3H]thymidine incorporation) was not significantly affected at 24 h after a single dose of CCl4. To study the long-term effects of CCl4, five groups of mice were given two consecutive weekly injections of 0.75 ml/kg CCl4 (as above) and were sacrificed 1, 4, 8, 12 and 22 weeks after the second treatment. In these groups the total liver I-compound levels were reduced to 17.3-49.0% compared with corresponding controls. The maximum decline was observed at 4 weeks (17.3% of control). Comparison of thymidine incorporation showed no significant increase between control and treated liver DNAs at 1, 4 and 8 weeks after CCl4, suggesting that the decrease in I-compound levels was probably not a secondary effect of increased DNA synthesis during postnecrotic proliferation. Even though there was a trend of recovery between 8 and 22 weeks, I-compound levels still remained significantly lower at 22 weeks (49.0%). Since I-compounds appear to be normal DNA modifications, the results suggest that persistent reduction of I-compound levels contributes to the hepatocarcinogenic effect of CCl4.     

Hepatic lipids of intact or thyroidectomized rats after administration of tetracycline or ethanol]

The purpose of this work was to study the quantitative modifications of the hepatic lipids in adult thyroidectomized rats after administration of tetracycline or ethanol (acute dose or prolonged ingestion). 1. - Thyroidectomy did not inhibit the accumulation of fat in the liver of fed euthyroid or hypothyroid rats after intraperitoneal infusion of tetracycline (320 mg/body weight in 2 injections at an interval of 16 h, the diet containing 6% of lipids). 2. - Sixteen hours after the oral administration of a single large dose of ethanol (5 g/kg body weight), there were only found some small modifications of the lipid composition of the liver in fasting euthyroid or thyroidectomized rats, receiving a diet with 6% of lipids before the experiment; on the contrary, when the diet contained 19% of lipids, a fatty liver occurred in the intact rat, but not in the thyroidectomized rat. 3. - The prolonged ethanol intake (in a 20% solution in water) for 5 months with a diet containing 19% of lipids did not induce a fatty liver in intact rats but produced a decrease of hepatic non-phosphorus lipid and an increase of the cholesterol amounts. After the administration of L-thyroxin (10 mug/100 g body weight per day) to these alcoholic thyroidectomized rats during 2 weeks, it was found an increase of the hepatic non phosphorus lipids till an higher amount than in the euthyroid rats. 4. - The hepatic phospholipid amounts were relatively constant in the different experiments. These results accounting for this differential effects were discussed.     

Chronologic studies of the effects of a hypoproteinic diet followed by an equilibrated diet on delta-6 and delta-5 desaturations of linoleic acid in liver microsomes in the rat

A low protein diet affects amounts of linoleic and arachidonic acids in hepatic microsomal phospholipids of growing rats. Are the changes related to modifications in microsomal delta 6- and delta 5- linoleic acid desaturase activities? Two groups of Wistar rats weighing 80 +/- 5 g at the beginning of the experiment were used: Control group (T) was fed on a 16% gluten + 4% casein diet for 53 days; Experimental group (E) was fed on a 4% gluten + 1% casein diet for 26 days (MP) then Control diet for 27 days (RE). After 2, 14 and 26 days of MP and 2, 15 and 27 days of RE, rats of each group were sacrificed. Protein and water contents of liver, quantitative fatty acid, composition of total lipids in liver and hepatic microsomes were determined. delta 6- and delta 5- linoleic acid desaturase activities were estimated from incubation of liver microsomes with [1-14C] C 18: 2 n-6 or [2(14)C] C 20: 3 n-6 respectively. The low protein diet stops practically ponderal growth. The fatty-acid compositions of microsomal total lipids of E rats were affected in comparison with values of T rats. These modifications persist after 27 days of RE. The C 20: 4 n-6/C 18: 2 n-6 ratio in microsomal total lipids was slightly different between T and E rats but increased strongly during refeeding. Same modifications take place in the fatty-acid composition of hepatic total lipids. After two days of MP, delta 6- and delta 5- desaturase activities were depressed, phenomenon that not persist in the course of MP. These enzyme activities increase to higher values than those of the T after two days of RE.     

A comparison of the lipid-lowering and intestinal morphological effects of cholestyramine, chitosan, and oat gum in rats

Cholestyramine, chitosan, and oat gum are lipid-lowering compounds. Cholestyramine use in humans may contribute to colonic adenocarcinoma; chitosan and oat gum are being studied in the rat to determine their potential for human use. To compare these compounds, we fed three groups of 10 male Sprague-Dawley rats one of the substances at 5% of diet with 1% cholesterol and 0.2% cholic acid; two other groups were fed cellulose with and without 1% cholesterol and 0.2% cholic acid. All groups had similar food intake and weight gains. Cholesterol feeding increased total liver lipids almost 3-fold and liver cholesterol concentration almost 10-fold. Cholestyramine, oat gum, and chitosan all significantly lowered liver cholesterol with cholestyramine feeding yielding levels identical to the noncholesterol-fed basal group. Chitosan and oat gum lowered liver cholesterol moderately. Cholestyramine and chitosan both significantly lowered serum cholesterol compared to the cellulose group. Oat gum was less effective. Hemoglobin and serum iron were similar in all groups except the oat gum group, which had decreased serum iron. Histological examination of small and large bowel with morphometry revealed statistically significant increases in both proximal and distal small bowel and distal large bowel mucosal thickness in the cholestyramine-fed group. No changes were noted in the proximal large bowel. Neither chitosan nor oat gum produced mucosal change other than an increase in the distal small bowel with the oat gum diet. Chitosan may have lipid-lowering effects similar to those of cholestyramine without the deleterious changes in intestinal mucosa.     

A 32P-postlabeling assay for the oxidative DNA lesion 8,5'-cyclo-2'-deoxyadenosine in mammalian tissues: evidence that four type II I-compounds are dinucleotides containing the lesion in the 3' nucleotide.

8,5'-Cyclopurine-2'-deoxynucleotides, which are strong blocks to mammalian DNA and RNA polymerases, represent a novel class of oxidative DNA lesion in that they are specifically repaired by nucleotide excision repair but not by base excision repair or direct enzymatic reversion. Previous studies using thin layer chromatography of (32)P-postlabeled DNA digests have detected several bulky oxidative lesions of unknown structure, called I-compounds, in DNA from normal mammalian organs. We investigated whether any of these type II I-compounds contained 8,5'-cyclo-2'-deoxyadenosine (cA). Two previously detected type II I-compounds were found to be dinucleotides of the sequence pAp-cAp and pCp-cAp. Furthermore, a modification of the technique resulted in detection of two additional I-compounds, pTp-cAp and pGp-cAp. Each I-compound isolated from neonatal rat liver DNA matched authentic (32)P-labeled cA-containing chromatographic standards under nine different chromatographic conditions. Their levels increased significantly after normal birth. The (32)P-postlabeling technique used here is capable of detecting 1-5 lesions/diploid mammalian cell. Thus, it should now be possible to detect changes of cA levels resulting from low level ionizing radiation and other conditions associated with oxidative stress, and to assess cA levels in tissues from patients with the genetic disease xeroderma pigmentosum who are unable to carry out nucleotide excision repair.     

Lipid Metabolism in the Fatty Liver of Rats Fed a Threonine-deficient Diet

Feeding of a threonine-deficient diet to rats weighing approximately 53 g or 99 g caused a significant rise in liver lipids compared to the control diet containing 7% amino acid mixture. Whereas, when rats weighing approximately 155 g were fed either the control diet or the threonine-deficient diet, liver lipid content was essentially the same for both groups. Therefore, in the present paper, young rats were used to clarify the mechanism of liver lipid accumulation in threonine-deficiency. The increase in dietary fat content of the threonine-deficient diet did not prevent the lipid accumulation in rat liver. The rates of in vivo incorporation from radioactive acetate into liver lipids, body lipids and respiratory CO₂ of rats fed either the control diet or the threonine-deficient diet were measured. The threonine-deficient group tended to be lower in total activity of both the liver lipids and body lipids than those of the control group. Thus, these results suggest that the development of this type of fatty liver might not be due to the stimulation of lipid synthesis in the liver. In the serum of rats fed the threonine-deficient diet, the protein content of β-lipoproteins was significantly lower and free fatty acid level tended to be lower than the values of the control animals, respectively. From these results, decreased trasport of lipids from the liver may thus be considered a potential major factor responsible for the excessive lipid accumulation in the liver of rats fed the threonine-deficient diet.     

Aspects in oat breeding: nutrition quality,nakedness and disease resistance,challenges and perspectives

Traditionally, the oat crop (Avena sativa) has been neglected in a number of respects, cultivated in cropping areas not optimal for wheat, barley or maize. In recent years the interest in oats has increased, particularly because of its dietary benefits and therapeutic potential for human health. The uniqueness and advantages of naked oats over other popular cereals, due to its potentially valuable nutritional composition, have been well studied and reported, opening new market “niches” for oats. Despite the well‐documented benefits, the status of the oat crop is still fragile, due to many reasons. The area cultivated for the oat crop is much less compared with other cereals, and therefore commercial efforts in oat breeding are less. Oat groat yield is lower than other cereals such as wheat and the nutritious uniqueness has not been reflected in agreeable market prices. The same price still exists for both naked and conventional/covered oats in the world grain market. The absence of visible market competitiveness, and some of the oat biological drawbacks, including low grain yield, keeps the oat crop as a lower profitability minor crop. This review is intended to analyse and summarise main achievements and challenges in oat genetics, agronomy and phytopathology to find possible ways of oat improvement and future perspectives for oat breeding.     

Age-related effects of methionine-enriched diet on plasma homocysteine concentration and methylation of hepatic DNA in rats

In the present study we determined the age-related effect of methionine-enriched diet, a model of hyperhomocysteinemia, on the level of plasma homocysteine and hepatic global DNA methylation in rats. Feeding methionine diet to middle-aged rats for only 14 days resulted in a significant increase in plasma homocysteine level and DNA hypomethylation. In contrast, feeding the methionine-containing diet for 2 weeks to juvenile or post-pubertal animals did not alter the level of plasma homocysteine or hepatic DNA methylation. Supplementation of the methionine-enriched diet with vitamins B6, B12 and folic acid prevented both hepatic DNA hypomethylation and an increase of plasma homocysteine concentration in the middle-aged rats. These findings indicate that the elevated level of plasma homocysteine may be indicative of much broader and deeper alterations in intracellular methylation dysfunction, and suggest that dietary enrichment with B-vitamins is essential for the metabolism of homocysteine, especially in adult animals.     

Bulky DNA-adduct formation induced by Ni(II) in vitro and in vivo as assayed by 32P-postlabeling

Various small oxidation products (e.g. 8-hydroxydeoxyguanosine) can be induced in DNA by nickel compounds. In this study, the ³²P-postlabeling assay was applied to determine whether Ni(II) compounds are able to induce bulky DNA-adduct formation in vitro and in vivo. In vitro studies detected two major and several minor adducts in DNA incubated with NiCl₂ and H₂O₂ at 37°C for 1 h. Formation of the two major adducts increased with incubation time (0–24 h) and NiCl₂ concentration (0–800 μM). Adduct levels were greatly reduced by hydroxyl free-radical scavengers, i.e. 0.4 M sodium formate or 0.05 M p-nitrosodimethylaniline, and by a singlet oxygen scavenger, 0.05 M sodium azide. The in vitro effects of NiCl₂ on DNA were significantly enhanced by (1) addition of 3 mM ascorbic acid, (2) replacement of H₂O with D₂O in the reaction, and (3) prior denaturation of DNA. Adduct formation presumably involved a Fenton-type reaction, in which DNA crosslinks may arise by reaction with hydroxyl free radicals and singlet oxygen.For in vivo studies, male 6–8 wk old B6C3F1 mice were used. In untreated mice, several I-compounds (putative indigenous DNA modifications that increase with age) were detected in liver, kidney, and lung. Two of these spots (1 and 2) were chromatographically identical to the two major spots induced by Ni(II) in vitro. The intensities of spots 1 and 2 in kidney and of some other spots in liver and lung were increased 1 and 2 h after i.p. injection with a single dose of 170 μmoles/kg NiAc₂. The effects of NiAc₂ were reduced or undetectable in the three tissues 24 h after treatment. These observations indicate the capacity of Ni(II) to induce and modulate bulky DNA modifications both in vitro and in vivo.     

The effect of methylene blue on the hepatocellular redox state and liver lipid content during chronic ethanol feeding in the rat.

Feeding of ethanol in a liquid diet to male Wistar rats caused decreases in the hepatic cytosolic and mitochondrial [NAD+]/[NADH] ratios. This redox-state change was attenuated after 16 days of feeding ethanol as 36% of the total energy intake. Supplementation of the ethanol-containing liquid diet with Methylene Blue largely prevented the ethanol-induced redox state changes, but did not significantly decrease the severity of the hepatic lipid accumulation that resulted from ethanol ingestion. Methylene Blue did not affect body-weight gain, ethanol intake or serum ethanol concentrations in ethanol-fed rats, nor did the compound influence the hepatic redox state or liver lipid content of appropriate pair-fed control animals. These findings suggest that the altered hepatic redox state that results from ethanol oxidation is not primarily responsible for the production of fatty liver after long-term ethanol feeding in the rat.     

Dietary prevention of cataracts in the pink-eyed RCS rat

The American Institute of Nutrition purified ingredient diet (AIN-76) prevented occurrence of mature cataracts associated with hereditary retinal degeneration in pink-eyed, tan-hooded Royal College of Surgeons (RCS) rats. Rats fed a natural ingredient open formula NIH diet or a closed formula commercial diet had a cataract incidence of 27-29% by 3 to 12 months of age. In contrast, only 1 of 50 rats fed the AIN diet developed a mature cataract in one year. When the NIH diet and the commercial diet were pelleted with 25% of ground sunflower kernels, rats fed these diets had a delay in onset and a reduced incidence of mature cataracts to 18% and 5%, respectively. No mature cataracts occurred in rats fed the AIN diet supplemented with 25% sunflower kernels. All diets were fed to the parental generation as well as the progeny (experimental group). The rats were reared at a low level of illumination (1-3 footcandles inside the cage) to minimize effects of light. Prevention of mature cataracts by the AIN purified diet suggests that diets permitting cataracts to occur may have a constituent at a concentration innocuous for normal rats but beyond the homeostatic control of the RCS rat. Posterior subcapsular cataracts of RCS rats are a model for cataracts associated with human hereditary retinal degenerations, such as retinitis pigmentosa and gyrate atrophy. Manipulation of dietary ingredients allowed by the use of the AIN diet may permit identification of nutrients, nutrient interactions of toxic factors involved in cataractogenesis and its prevention.     

Effects of Feeding a Histidine-excess Diet and Subsequent Starvation on Liver and Muscle Glycogen,and on Serum Glucose

The effects of feeding with a histidine-excess diet and subsequent starvation on liver and muscle glycogen, and on serum glucose were investigated in young and adult rats.

Feeding with a histidine-excess diet resulted in the accumulation of liver glycogen in both young and adult rats. The hepatic glycogen continued to decrease during starvation, and the liver became almost totally depleted of glycogen after starvation for 48 hr. Glycogen in the liver of young rats starved for 24 hr after previous feeding with a histidine-excess diet was significantly higher than that of young rats starved for 24 hr after previous feeding with a basal diet.

Muscle glycogen after feeding and subsequent starvation was not affected by the types of diets fed previously, muscle glycogen during starvation showing a slight decrease in young rats and a slight increase in adult rats.

Feeding with a histidine-excess diet caused a significant decrease of serum glucose in young rats, but not in adult rats. Serum glucose in young rats was markedly reduced by starvation after previous feeding with a basal diet, but not after previous feeding with a histidine-excess diet. In adult rats, there were no changes in serum glucose between rats starved after feeding with either a basal diet or a histidine-excess diet, and serum glucose was decreased slightly by starvation after feeding with the test diets.

The overall results indicate that the maintenance of serum glucose in young rate even during starvation after previous feeding with a histidine-excess diet might be partially concerned with the export of glucose from the accumulated glycogen in the liver due to the diet.