Dietary selenite and azadeoxycytidine treatments affect dimethylhydrazine-induced aberrant crypt formation in rat colon and DNA methylation in HT-29 cells

Several observations implicate a role for altered DNA methylation in cancer pathogenesis. The global level of DNA methylation is generally lower; however, DNA methyltransferase (Dnmt1) activity is usually higher in tumor cells than in normal cells. The purpose of this study was to investigate whether the Dnmt1 inhibitor, 5-aza-2'-deoxycytidine (aza-dC) would alter the effect of dietary selenium on the formation of aberrant crypts. Weanling rats (n = 60) were fed three concentrations of selenium (deficient, 0.1 and 2.0 mg/kg diet) in a Torula yeast-based diet. Half of the rats were injected weekly with aza-dC (1 mg/kg, subcutaneously) and half were injected with the vehicle control (PBS). After 3.5 wk of consuming the experimental diets, the rats were given two injections of dimethylhydrazine (DMH; 25 mg/kg, intraperitoneally). Rats fed the selenium-deficient diet and injected with PBS had significantly (P < 0.006) more aberrant crypts than rats fed 0.1 or 2.0 mg selenium/kg diet (244 +/- 21 vs. 165 +/- 9 and 132 +/- 14, respectively). In contrast, when rats were injected with aza-dC, there was a significant (P < 0.0001) reduction in aberrant crypt formation and dietary selenium had no effect (62 +/- 8 vs. 77 +/- 13 vs. 54 +/- 8, in rats fed 0, 0.1 and 2.0 mg selenium/kg diet, respectively). HT-29 cells cultured in the absence of selenium had significantly hypomethylated DNA but significantly more Dnmt1 protein expression than cells cultured in the presence of 1 or 2 micromol/L selenium. These results suggest that aza-dC treatment may protect selenium-deficient rats against carcinogen-induced aberrant crypt formation.     

Choline deficiency and methotrexate treatment induces marked but reversible changes in hepatic folate concentrations,serum homocysteine and DNA methylation rates in rats.

The study compared the effects of feeding rats a choline deficient (CD) diet or injecting low doses of methotrexate (MTX) on hepatic folate concentration and distribution, homocysteine (Hcy) concentration and DNA methylation.

Thirty rats were divided into three groups and were fed either a choline sufficient (CS) or deficient diet (CD), or injected with low doses of MTX (0.1 mg/kg/day) for 2 weeks. Half the animals of each group were sacrificed and the remaining CD and MTX animals were fed repletion diets without methotrexate administration for two additional weeks.

CD or MTX resulted in a significantly lower folate concentrations (25-50%) compared to the control group. Folate distribution in the treated animals was associated with elongation of the glutamate chains: higher proportion of hexa (from 14%, control, to 35%, choline, p < 0.05), hepta (from 5% to 16%, p < 0.05), and appearance of octaglutamyl folates. MTX administration resulted in a similar pattern of hepatic folate distribution. Two weeks following the MTX administration and the restoration of an adequate choline diet for 2 weeks restored the hepatic folate levels to the control animals.

Results are discussed based on the possibility that CD and MTX treatment appear to impair the capacity of tissues to incorporate folate in only 2 weeks and affect other biomarkers of one-carbon metabolism such as Hcy concentration and DNA methylation. This adverse picture was partially reversed in a relative short time by simply feeding an adequate CS diet and discontinuing MTX injections.     

Folate supplementation increases genomic DNA methylation in the liver of elder rats

The availability of folate is implicated as a determinant of DNA methylation, a functionally important feature of DNA. Nevertheless, when this phenomenon has been examined in the rodent model, the effect has not always been observed. Several reasons have been postulated for the inconsistency between studies: the rodent is less dependent on folate as a methyl source than man; juvenile animals, which most studies use, are more resistant to folate depletion than old animals; methods to measure genomic DNA methylation might not be sensitive enough to detect differences. We therefore examined the relationship between folate and genomic DNA methylation in an elder rat model with a newly developed method that can measure genomic DNA methylation sensitively and precisely. Thirty-nine 1-year-old rats were divided into three groups and fed a diet containing 0, 4.5 or 18 mumol folate/kg (folate-deplete, -replete and -supplemented groups, respectively). Rats were killed at 8 and 20 weeks. At both time points, mean liver folate concentrations increased incrementally between the folate-deplete, -replete and -supplemented rats (P for trend <0.001) and by 20 weeks hepatic DNA methylation also increased incrementally between the folate-deplete, -replete and -supplemented rats (P for trend=0.025). At both time points folate-supplemented rats had significantly increased levels of DNA methylation compared with folate-deplete rats (P<0.05). There was a strong correlation between hepatic folate concentration and genomic DNA methylation in the liver (r 0.48, P=0.004). In the liver of this animal model, dietary folate over a wide range of intakes modulates genomic DNA methylation.     

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.     

Hepatic content of S-adenosylmethionine, S-adenosylhomocysteine and glutathione in rats receiving treatments modulating methyl donor availability

Because of evidence linking methyl group deficiency and increased tumor formation in experimental animals, we explored other possible methods of producing a methyl group deficiency. Rats fed a low methionine diet lacking choline (MCD) were injected intraperitoneally daily for 3 wk with large doses of nicotinamide. Hepatic levels of lipids were elevated, S-adenosylmethionine (SAM) levels and the SAM:S-adenosylhomocysteine (SAH) ratio were decreased, and SAH level was not consistently changed. In livers of rats fed the MCD diet without folate (MCFD), lipids were also elevated and SAM reduced as compared to MCD-fed rats. In rats fed the MCD diet plus a methionine (Met) supplement (MCD + Met), hepatic SAM levels and the SAM:SAH ratio were higher and lipid levels lower than in MCD-fed rats, indicating that the MCD diet is marginally deficient in methyl donor groups. The injection of nicotinamide or the removal of folate from the MCD diet increased the severity of methyl donor deficiency, as shown by lower hepatic SAM levels and higher hepatic lipid levels. Hepatic glutathione levels were similar in MCD- and MCFD-fed rats and were lower than in rats fed the methionine-supplemented MCD diet or injected with nicotinamide.     

High-dose folic acid supplementation in rats: effects on gestation and the methionine cycle

There is new evidence that a good folate status may play a critical role in the prevention of neural-tube defects and in lowering elevated homocysteine concentrations. This adequate folate status may be achieved through folic acid dietary supplementation. Folate is a water-soluble vitamin with a low potential toxicity. However, the possible consequences of long-term high-dose folic acid supplementation are unknown, especially those related to the methionine cycle, where folate participates as a substrate. With the aim of evaluating such possible effects, four groups of Wistar rats were classified on the basis of physiological status (virgin v. pregnant) and the experimental diet administered (folic-acid-supplemented, 40 mg/kg diet v. control, 2 mg folic acid/kg diet). Animals were fed on the diets for 3 weeks. Results showed that gestation outcome was adequate in both groups regardless of the dietary supplementation. However, there were reductions (P < 0.001) in body weight and vertex-coccyx length in fetuses from supplemented dams v. control animals. Folic acid administration also induced a higher (P < 0.01) S-adenosylmethionine: S-adenosylhomocysteine value due to increased S-adenosylmethionine synthesis (P < 0.01). However, hepatic DNA methylation and serum methionine concentrations remained unchanged. Serum homocysteine levels were reduced in supplemented dams (P < 0.05). Finally, pregnancy caused lower serum folate, vitamin B6 and vitamin B12 levels (P < 0.05). Folic acid administration prevented the effect of pregnancy and raised folate levels in dams, but did not change levels of vitamins B12 and B6. These new findings are discussed on the basis of potential benefits and risks of dietary folic acid supplementation.     

Effects of parental folate deficiency on the folate content,global DNA methylation,and expressions of FRα, IGF-2 and IGF-1R in the postnatal rat liver

We examined the effect of parental folate deficiency on the folate content, global DNA methylation, folate receptor-alpha (FRα), insulin-like-growth factor-2 (IGF-2) and -1 receptor (IGF-1R) in the liver and plasma homocysteine in the postnatal rat. Male and female rats were randomly fed a folic acid-deficient (paternal folate-deficient, PD and maternal folate-deficient, MD), or folic acid-supplemented diet (paternal folate-supplemented, PS and maternal-folate-supplemented, MS) for four weeks. They were mated and grouped accordingly: PSxMS, PSxMD, PDxMS, and PDxMD. Pups were killed on day 21 of lactation. The hepatic folate content was markedly reduced in the PDxMD and PSxMD and PDxMS as compared with the PSxMS group. The hepatic global DNA methylation was decreased in the PDxMS and PSxMD groups as much as in the PDxMD group, and all the three groups were significantly lower as compared to the PSxMS group. There were no significant differences in the hepatic FRα, IGF-2 and IGF-1R expressions among the groups. Positive correlations were found between the hepatic folate content and global DNA methylation and protein expressions of FRα, IGF-2 and IGF-1R, whereas an inverse correlation was found between hepatic folate content and plasma homocysteine level in the 3-week-old rat pup. The results of this study show that both paternal and maternal folate deficiency at mating can influence the folate content and global DNA methylation in the postnatal rat liver.     

Vitamin B-12 deficiency induces anomalies of base substitution and methylation in the DNA of rat colonic epithelium

Derangements of one-carbon metabolism can directly affect the integrity of the genome by producing inappropriate uracil insertion into DNA and by altering patterns of DNA methylation. Vitamin B-12, a one-carbon nutrient, serves as a cofactor in the synthesis of precursors of biological methylation and in nucleotide synthesis. We therefore examined whether vitamin B-12 deficiency can induce these molecular anomalies in the colonic mucosa of rats. Weanling male Sprague-Dawley rats (n = 30) were divided into 2 groups and fed either a vitamin B-12-deficient diet or a similar diet containing adequate amounts of the vitamin. Rats from each group were killed at 6 and 10 wk. Uracil misincorporation into DNA was measured by GC/MS and genomic DNA methylation was measured by LC/MS. Plasma vitamin B-12 concentrations in deficient rats were below detectable limits at 6 and 10 wk; in control rats, concentrations were 0.46 +/- 0.07 and 0.42 +/- 0.10 nmol/L at those times. Although the colon total folate concentration did not differ between the groups, the proportion that was methylfolate was marginally greater in the deficient rats at 10 wk (P = 0.05) compared with control, consistent with the "methylfolate trap" that develops during vitamin B-12 deficiency. After 10 wk, the colonic DNA of the deficient rats displayed a 35% decrease in genomic methylation and a 105% increase in uracil incorporation (P < 0.05). This vitamin B-12-deficient diet, which was of insufficient severity to cause anemia or illness, created aberrations in both base substitution and methylation of colonic DNA, which might increase susceptibility to carcinogenesis.     

Plasma S-adenosylhomocysteine is a better biomarker of atherosclerosis than homocysteine in apolipoprotein E-deficient mice fed high dietary methionine

Homocysteine (Hcy) and S-adenosylhomocysteine (AdoHcy) are critical intermediates of methionine metabolism. To investigate which, if either, of these compounds is more closely related to atherosclerosis, we fed 5 groups of apolipoprotein E (apoE)-deficient mice different diets for 8 wk to induce changes in their plasma Hcy and AdoHcy concentrations. These included an AIN-93G control diet (C), this C diet supplemented with methionine (M), the M diet deficient in folates, vitamin B-6, and vitamin B-12 (M-V), this M diet supplemented with these B vitamins (M+V), and a C diet deficient in B vitamins (C-V). Compared with controls, mice fed the C-V diet had a moderate elevation in their plasma total Hcy (tHcy) levels; however, their plasma AdoHcy concentration and atherosclerotic lesion areas were not different. In contrast, the mice fed the M+V diet had larger atherosclerotic lesion areas and elevated plasma AdoHcy concentrations but their plasma tHcy concentration did not differ from that of the group C mice. The plasma AdoHcy concentration and aortic sinus lesion areas were positively correlated (r = 0.866; P < 0.001). We observed a negative correlation between the plasma AdoHcy concentration and both the DNA methyltransferase activity (r = -0.792; P < 0.001) and global DNA methylation status (r = -0.824; P < 0.001) in the aortic tissue. Hence, our study suggests that plasma AdoHcy is a better biomarker of atherosclerosis than Hcy and may accelerate the development of atherosclerotic lesions in apoE-deficient mice that have been fed a high methionine diet. The mechanisms underlying this effect may be related to the AdoHcy-mediated inhibition of DNA methylation in the aortic tissue.     

DNA stability and genomic methylation status in colonocyte isolated from methyl-donor-deficient rats

Summary Background: Epidemiological studies report an inverse relationship between intake of the B vitamine folic acid and colon cancer. Folate is important for DNA synthesis and repair. Moreover, the production of S-adenosylmethionine (SAM), essential for normal DNA methylation and gene expression, is dependent on folic acid. Folate deficiency may increase the risk of malignant transformation by perturbing these pathways. Aims of the study: The principal aim of this study was to determine the effects of folate deficiency on DNA stability and DNA methylation in rat colonocytes in vivo. As the metabolic pathways of folate and other dietary methyl donors are closely linked, the effects of methionine and choline deficiency were also evaluated. Methods: Male Hooded-Lister rats were fed a diet deficient in folic acid, or in methionine and choline, or in folate, methionne and choline for 10 weeks. DNA strand breakage and misincorporated uracil were determined in isolated colonocytes using alkaline single cell gel electrophoresis. Global DNA methylation was measured in colonic scrapings. Folate was measured in plasma, erythrocyte and liver samples. Results: Methyl donor deficiency induced DNA strand breakage in colonocytes isolated from all experimental groups. Uracil levels in colonocytes DNA remained unchanged compared with controls. DNA methylation was unaffected either by folate and/or methionine and choline depletion. Rats fed a folate-deficient diet had less folate in plasma, red blood cells and liver than controls. Conclusions: Folate and methyl deficiency in vivo primarily afects DNA stability in isolated colonocytes of rats, without affecting overall DNA methylation. Received: 16 February 2000, Accepted: 25 April 2000     

Model of nonalcoholic steatohepatitis

BACKGROUND: Obesity and diabetes are frequently associated with nonalcoholic steatohepatitis (NASH), but studies have been hampered by the absence of a suitable experimental model. OBJECTIVE: Our objective was to create a rat model of NASH. DESIGN: Sprague-Dawley rats were fed a high-fat, liquid diet (71% of energy from fat, 11% from carbohydrates, 18% from protein) or the standard Lieber-DeCarli diet (35% of energy from fat, 47% from carbohydrates, 18% from protein). The diets were given ad libitum or as two-thirds of the amount consumed ad libitum. RESULTS: Rats fed the high-fat diet ad libitum for 3 wk developed panlobular steatosis, whereas those fed the standard diet had few fat droplets. Accordingly, total lipid concentrations with the high-fat and standard diets were 129.9 +/- 9.1 ( +/- SEM) and 66.7 +/- 4.6 mg/g liver, respectively (P < 0.001). The high-fat diet caused abnormal mitochondria and mononuclear inflammation, which were accompanied by increased hepatic tumor necrosis factor alpha (TNF-alpha; P < 0.001), TNF-alpha messenger RNA (mRNA) (P < 0.001), collagen type 1, and alpha1(I) procollagen mRNA (P < 0.001). In addition, these rats had increased cytochrome P4502E1 (CYP2E1) mRNA (P < 0.001), which was accompanied by CYP2E1 induction (P < 0.001) and oxidative stress with increased 4-hydroxynonenal (P < 0.001). Plasma insulin was elevated, which reflected insulin resistance, a NASH pathogenic factor. Rats fed a restricted high-fat diet developed only mild steatosis with attenuated biochemical changes, whereas those given a restricted standard diet had normal livers. CONCLUSION: This rat model reproduces the key features of human NASH and provides a realistic experimental model for elucidating its treatment.     

Dietary folate and selenium affect dimethylhydrazine-induced aberrant crypt formation,global DNA methylation and one-carbon metabolism in rats

Several observations suggest a role for DNA methylation in cancer pathogenesis. Although both selenium and folate deficiency have been shown to cause global DNA hypomethylation and increased cancer susceptibility, the nutrients have different effects on one-carbon metabolism. Thus, the purpose of this study was to investigate the interactive effects of dietary selenium and folate. Weanling, Fischer-344 rats (n = 23/diet) were fed diets containing 0 or 2.0 mg selenium (as selenite)/kg and 0 or 2.0 mg folate/kg in a 2 x 2 factorial design. After 3 and 4 wk of a 12-wk experiment, 19 rats/diet were injected intraperitoneally with dimethylhydrazine (DMH, 25 mg/kg) and 4 rats/diet were administered saline. Selenium deficiency decreased (P < 0.05) colonic DNA methylation and the activities of liver DNA methyltransferase and betaine homocysteine methyltransferase and increased plasma glutathione concentrations. Folate deficiency increased (P < 0.05) the number of aberrant crypts per aberrant crypt foci, the concentration of colonic S-adenosylhomocysteine and the activity of liver cystathionine synthase. Selenium and folate interacted (P < 0.0001) to influence one-carbon metabolism and cancer susceptibility such that the number of aberrant crypts and the concentrations of plasma homocysteine and liver S-adenosylhomocysteine were the highest and the concentrations of plasma folate and liver S-adenosylmethionine and the activity of liver methionine synthase were the lowest in rats fed folate-deficient diets and supplemental selenium. These results suggest that selenium deprivation ameliorates some of the effects of folate deficiency, probably by shunting the buildup of homocysteine (as a result of folate deficiency) to glutathione.     

Folate status and age affect the accumulation of L-isoaspartyl residues in rat liver proteins

Formation of atypical L-isoaspartyl residues in proteins and peptides is a common, spontaneous and nonenzymatic modification of aspartyl and asparaginyl sites. The enzyme protein-L-isoaspartyl methyltransferase (PIMT) catalyzes the transfer of the methyl group of S-adenosyl-L-methionine (SAM) to these L-isoaspartyl sites, thereby allowing reisomerization and restoration of the original alpha peptide linkage. Because SAM is in part a product of folate metabolism, the present study was undertaken to determine the effects of folate deficiency on the presence of L-isoaspartyl residues in hepatic proteins. Young (weanling) and older (12 mo) Sprague-Dawley rats were fed a folate-sufficient (2 mg folate/kg diet) or folate-deficient (0 mg folate/kg diet) diet for 20 wk. Liver proteins were analyzed for L-isoaspartyl residues. This analysis was based on the PIMT-dependent incorporation of [(3)H]-methyl groups from [(3)H]-SAM and the subsequent (nonenzymatic) sublimation of these methyl groups into a nonaqueous scintillant. The amount of L-isoaspartyl residues in hepatic proteins was higher in younger folate-deficient than in folate-sufficient rats (deficient: 187 +/- 71, sufficient: 64 +/- 43 pmol/mg protein, P < 0.025). This difference, however, was not seen among the older groups of rats who instead exhibited a much larger accumulation of L-isoaspartyl residues in their hepatic proteins (deficient: 528 +/- 151, sufficient: 470 +/- 204 pmol/mg protein, P = 0.568). The importance of these observations is discussed.     

A rice bran oil diet increases LDL-receptor and HMG-CoA reductase mRNA expressions and insulin sensitivity in rats with streptozotocin/nicotinamide-induced type 2 diabetes

A rice bran oil (RBO) diet can reduce plasma lipids; this was attributed to the specific components, gamma-oryzanol and gamma-tocotrienol, which individually were shown to be hypocholesterolemic; however, the mechanism of their effects on diabetic hyperlipidemia and the development of diabetes is not known. Rats with streptozotocin/nicotinamide-induced type 2 diabetes were divided into control, RO10, and RO15 groups, and fed cholesterol-free diets containing 0, 10, and 15 g RBO with 0, 352, and 528 g gamma-oryzanol and 0, 6.0 and 9.0 mg gamma-tocotrienol/100 g diet for 4 wk. Diabetic rats fed the RBO diet had greater insulin sensitivity (P = 0.02) than rats fed the control diet. Diabetic rats fed the RBO diet also had lower plasma triglyceride (P = 0.003), LDL cholesterol (P = 0.028), and hepatic triglyceride concentrations (P = 0.04), as well as greater fecal neutral sterol and bile acid excretion than those fed the control diet. After 4 wk, there was an approximately 100% (P < 0.001) increase in the abundance of hepatic cholesterol 7alpha-hydroxylase, an 89% (P < 0.001) increase in the hepatic LDL-receptor, and a 50% (P < 0.001) increase in hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA in rats fed the RBO diet compared with those fed the control diet. These findings support the conclusion that a rice bran oil-containing diet can significantly suppress hyperlipidemic and hyperinsulinemic responses in diabetic rats. The high contents of gamma-oryzanol and gamma-tocotrienol in RBO can lead to increased fecal neutral sterol and bile acid excretion, via upregulation of cholesterol synthesis and catabolism.     

Vitamin A status and metabolism of benzo[a]pyrene in the rat

Male Sprague-Dawley rats were maintained on a vitamin A-deficient diet for a period of five weeks. At the end of that time, hepatic cytochrome P450 levels in vitamin A-deficient rats were 65% that of rats fed a complete diet. However, the hepatic rate of benzo[a]pyrene metabolism was significantly greater (2 times) in vitamin A-deficient rats compared with those fed a complete diet. The pattern of metabolites separable by thin-layer chromatography was similar in both groups of rats. Benzo[a]pyrene induced its own metabolism by a slightly greater amount in the vitamin-sufficient rats, but it was not to the level of the deficient group, although the levels of cytochrome P450 were still below those of the deficient rats. In discussing lung microsomes, benzo[a]pyrene pre-treatment of deficient rats resulted in slightly elevated levels of cytochrome P450 and a slightly greater rate of metabolism of benzo[a]pyrene compared with rats fed the complete diet.     

Folate deficiency, methionine metabolism, and alcoholic liver disease.

Methionine metabolism is regulated by folate, and both folate deficiency and abnormal hepatic methionine metabolism are recognized features of alcoholic liver disease (ALD). Previously, histological features of ALD were induced in castrated male micropigs fed diets containing ethanol at 40% of kilocalories for 12 months, whereas in male micropigs fed the same diets for 12 months abnormal methionine metabolism and hepatocellular apoptosis developed. Folate deficiency may promote the development of ALD by accentuating abnormal methionine metabolism. Intact male micropigs received eucaloric diets that were folate sufficient, folate deficient, or each containing 40% of kilocalories as ethanol for 14 weeks. Folate deficiency alone reduced hepatic folates by one half, and ethanol feeding alone reduced methionine synthase, S-adenosylmethionine (SAM), and glutathione (GSH) levels and elevated plasma malondialdehyde (MDA) levels. The combined regimen elevated plasma homocysteine, hepatic S-adenosylhomocysteine (SAH), urinary 8-hydroxy-2-deoxyguanosine (oxy(8)dG), an index of DNA oxidation, and serum aspartate aminotransferase (AST) levels. Terminal hepatic histopathologic characteristics included typical features of steatonecrosis and focal inflammation in pigs fed the combined diet, with no changes in the other groups. Hepatic SAM levels correlated with those of GSH, whereas urinary oxy(8)dG and plasma MDA levels correlated with the SAM:SAH ratio and to hepatic GSH. The results demonstrate the linkage of abnormal methionine metabolism to products of DNA and lipid oxidation and to liver injury. The finding of steatonecrosis and focal inflammation only in the combined diet group supports the suggestion that folate deficiency promotes and folate sufficiency protects against the early onset of methionine cycle-mediated ALD.     

Folate depletion and elevated plasma homocysteine promote oxidative stress in rat livers

This study was designed to determine whether nutritional folate depletion exerts hepatic oxidative stress in relation to elevated plasma homocysteine. To mimic various extents of folate depletion status in vivo, male Wistar rats were fed an amino acid-defined diet containing either 8 (control), 2, 0.5, or 0 mg folic acid/kg diet. After a 4-wk feeding period, the plasma and hepatic folate concentrations of the rats decreased significantly with each decrement of dietary folate. Folate depletion did not significantly affect two major liver antioxidants: reduced glutathione and alpha-tocopherol. Conversely, folate depletion decreased Cu-Zn superoxide dismutase and glutathione peroxidase activities, but had no effect on catalase activity in liver homogenates. Lipid peroxidation products, as measured by thiobarbituric acid-reactive substances, were significantly higher in livers of folate-depleted rats than in those of the controls. This occurrence of hepatic oxidative stress in folate-depleted rats was confirmed by demonstrating an increased susceptibility of livers of folate-depleted rats to lipid peroxidation induced by additional H2O2 or Fe(2+) treatments compared with the controls. Decreasing dietary folate intake resulted in graded increases in plasma homocysteine concentrations of folate-depleted rats. Elevated plasma homocysteine and decreased plasma and hepatic folate concentrations in folate-depleted rats were all strongly and significantly correlated with increased liver lipid peroxidation (/r/ > or = 0.58, P < 0.0003). These data demonstrate that folate depletion and elevated plasma homocysteine promote oxidative stress in rat livers.     

Vitamin a status and metabolism of benzo[α]pyrene in the rat

Male Sprague‐Dawley rats were maintained on a vitamin A‐deficient diet for a period of five weeks. At the end of that time, hepatic cytochrome P₄₅₀ levels in vitamin A‐deficient rats were 65% that of rats fed a complete diet. However, the hepatic rate of benzo[a]pyrene metabolism was significantly greater (2 times) in vitamin A‐deficient rats compared with those fed a complete diet. The pattern of metabolites separable by thin‐layer chromatography was similar in both groups of rats. Benzo[a]pyrene induced its own metabolism by a slightly greater amount in the vitamin‐sufficient rats, but it was not to the level of the deficient group, although the levels of cytochrome P₄₅₀ were still below those of the deficient rats. In discussing lung microsomes, benzo[a]pyrene pre‐treatment of deficient rats resulted in slightly elevated levels of cytochrome P₄₅₀ and a slightly greater rate of metabolism of benzo[a]pyrene compared with rats fed the complete diet.