Sex differences in DNA damage produced by the carcinogen 2-acetylaminofluorene in cultured human hepatocytes compared to rat liver and cultured rat hepatocytes.

Male rats are more susceptible to the induction of liver cancer by the aromatic amine 2-acetylaminofluorene (AAF) than are females. To assess the basis for this difference and to determine whether sex differences in susceptibility to AAF are present in human liver cells, the DNA reactivity of AAF was measured in livers of male and female Sprague-Dawley (SD) rats and in cultured SD rat and human hepatocytes of both sexes. In livers of rats administered oral doses of AAF, the total levels of adducts measured by nucleotide postlabelling at up to 8 weeks were about twofold greater in males than in females. Similarly, the level of AAF-DNA adducts formed in cultured male rat hepatocytes dosed with AAF was about twofold greater than in female rat hepatocytes. Also, the level of DNA repair synthesis was about threefold greater in AAF-dosed cultured male rat hepatocytes compared with female, indicating that the greater adduct levels in males was not due to lesser repair. In contrast, in cultured human hepatocytes of both sexes, AAF produced similar levels of adducts and DNA repair synthesis, which were intermediate between those produced in male and female rat hepatocytes. Thus, the greater susceptibility of male rats to AAF hepatocarcinogenicity is due at least in part to greater bioactivation and formation of AAF-DNA adducts in hepatocytes. Moreover, the data from human hepatocytes suggest that human liver could be less susceptible than male rat liver to the carcinogenic effects of aromatic amine carcinogens of the AAF type.

Dietary methionine restriction inhibits prostatic intraepithelial neoplasia in TRAMP mice.

BACKGROUND: Prostate cancer (PCa) is a major aging-related disease for which little progress has been made in developing preventive strategies. Over the past several years, methionine restriction (MR), the feeding of a diet low in methionine (Met), has been identified as an intervention which significantly extends lifespan and reduces the onset of chronic diseases, including cancer, in laboratory animals. We, therefore, hypothesized that MR may be an effective strategy for inhibiting PCa. METHODS: Control (0.86% Met) or MR (0.12% Met) diets were fed to 5-week old TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, a well-characterized model for PCa. The mice were sacrificed at 16 weeks of age and prostate and other tissues were harvested for histological and biochemical analyses. RESULTS: As previously reported, MR was associated with a decrease in body weight which was not associated with lowered food intake. MR led to significant reductions in the development of Prostatic Intraepithelial Neoplasia (PIN) lesions, specifically in the anterior and dorsal lobes of the prostate where the incidence of high-grade PIN was reduced by ∼50% (P < 0.02). The reduction in PIN severity was associated with 46-64% reductions in cell proliferation rates (P < 0.02) and plasma IGF-1 levels (P < 0.0001), which might, in part, explain the effects on carcinogenesis. Additionally, no adverse consequences of MR on immune function were observed in the TRAMP mice. CONCLUSIONS: Overall, these findings indicate that MR is associated with a reduction in prostate cancer development in the TRAMP model and supports the continued development of MR as a potential PCa prevention strategy.

A methionine-restricted diet and endurance exercise decrease bone mass and extrinsic strength but increase intrinsic strength in growing male rats.

Dietary methionine restriction (MR) has been suggested to be comparable to endurance exercise with respect to its beneficial effects on health. To further investigate the effects of MR and endurance exercise on growing bone, 7-wk-old male Sprague-Dawley rats were fed different l-methionine (Met)-containing diets with or without endurance exercise intervention (Ex; 0.86% Met, 0.52% Met, 0.17% Met, 0.86% Met-Ex, 0.52% Met-Ex, and 0.17% Met-Ex groups). After an 8-wk intervention period, exercise-trained rats had a 9.2% lower body weight (BW) than did sedentary rats (P < 0.05). Additionally, 0.17% Met-fed rats had 32% lower BW when compared with rats fed the other 2 diets (P < 0.05). Serum osteocalcin was lower in the 0.17% Met-Ex group compared with the other 2 exercise groups and the 0.17% Met group (P < 0.05). Serum concentrations of C-terminal telopeptide of type 1 collagen were lower in exercise-trained and 0.17% Met-fed rats than in sedentary rats and rats fed the other 2 diets (P < 0.05 for both). Rats fed the 0.17% Met diet had lower trabecular bone volume, bone mineralization activities, and bone mineral content (BMC; e.g., total, cortical, and spongy BMC) and bone mineral density (BMD; e.g., total and spongy BMD) indices compared with rats fed the other 2 diets (P < 0.05). Exercise-trained rats also had lower bone mineralization activity, trabecular osteoclast density, total BMC, cortical BMC, and total BMD compared with sedentary rats (P < 0.05). In total BMD, only the 0.17% Met-Ex group had values lower than the other 2 exercise groups and the 0.17% Met group (P < 0.05). Compared with rats fed the other 2 diets and sedentary rats, the femora of 0.17% Met-fed and exercise-trained rats, respectively, had smaller size and/or lower extrinsic strength but enhanced intrinsic biomechanical properties (P < 0.05). The results indicate that MR and endurance exercise caused lower whole bone mass, size, and/or strength but might enhance intrinsic bone strength.

Mitochondrial complex I inhibition triggers NAD+-independent glucose oxidation via successive NADPH formation, "futile" fatty acid cycling, and FADH2 oxidation.

Inhibition of mitochondrial complex I (NADH dehydrogenase) is the primary mechanism of the antidiabetic drug metformin and various unrelated natural toxins. Complex I inhibition can also be induced by antidiabetic PPAR agonists, and it is elicited by methionine restriction, a nutritional intervention causing resistance to diabetes and obesity. Still, a comprehensible explanation to why complex I inhibition exerts antidiabetic properties and engenders metabolic inefficiency is missing. To evaluate this issue, we have systematically reanalyzed published transcriptomic datasets from MPP-treated neurons, metformin-treated hepatocytes, and methionine-restricted rats. We found that pathways leading to NADPH formation were widely induced, together with anabolic fatty acid biosynthesis, the latter appearing highly paradoxical in a state of mitochondrial impairment. However, concomitant induction of catabolic fatty acid oxidation indicated that complex I inhibition created a "futile" cycle of fatty acid synthesis and degradation, which was anatomically distributed between adipose tissue and liver in vivo. Cofactor balance analysis unveiled that such cycling would indeed be energetically futile (-3 ATP per acetyl-CoA), though it would not be redox-futile, as it would convert NADPH into respirable FADH2 without any net production of NADH. We conclude that inhibition of NADH dehydrogenase leads to a metabolic shift from glycolysis and the citric acid cycle (both generating NADH) towards the pentose phosphate pathway, whose product NADPH is translated 1:1 into FADH2 by fatty acid cycling. The diabetes-resistant phenotype following hepatic and intestinal complex I inhibition is attributed to FGF21- and GDF15-dependent fat hunger signaling, which remodels adipose tissue into a glucose-metabolizing organ.

Cysteine supplementation reverses methionine restriction effects on rat adiposity: significance of stearoyl-coenzyme A desaturase.

Stearoyl-CoA desaturase-1 (SCD1) is a key enzyme in fatty acid and energy metabolism, but little is known about its nutritional regulation. Dietary methionine restriction in rats decreases hepatic Scd1 mRNA and protein, increases energy expenditure, and decreases fat-pad mass/body-weight% (FM/BW%). In humans, plasma concentrations of the methionine product, cysteine, are associated with obesity. To determine which consequences of methionine-restriction are mediated by decreased cysteine availability, we monitored obesity-related variables in 4 dietary groups for 12 weeks: control-fed (CF), methionine-restricted (MR), MR supplemented with 0.5% l-cysteine (MR+Cys) and CF+Cys rats. MR lowered weight gain and FM/BW% despite higher food intake/weight than CF, and lowered serum cysteine. Hepatic Scd1 expression was decreased, with decreased serum SCD1 activity indices (calculated from serum fatty acid profile), decreased serum insulin, leptin and triglycerides, and higher adiponectin. Cysteine supplementation (MR+Cys) essentially reversed all these phenotypes and raised serum cysteine but not methionine to CF levels. Adding extra cysteine to control diet (CF+Cys) increased serum taurine but did not affect serum cysteine, lipids, proteins, or total weight gain. FM/BW% and serum leptin were modestly decreased. Our results indicate that anti-obesity effects of MR are caused by low cysteine and that dietary sulfur amino acid composition contributes to SCD1 regulation.

Inhibition by acetaminophen of neoplastic initiation elicited in rat liver by the DNA-reactive hepatocarcinogen N-acetyl-2-aminofluorene.

Acetaminophen, a monocyclic phenolic compound and analgesic, when fed at 8900 p.p.m. in the diet, was reported to inhibit the hepatocarcinogenicity in rats of the aromatic amine proximate carcinogen N-hydroxy-N-acetyl-2-aminofluorene. To elucidate the mechanism(s) of this anticarcinogenicity, the present study examined whether acetaminophen at lower doses has the ability to inhibit the initiating effects in the rat liver of the precursor hepatocarcinogen N-acetyl-2-aminofluorene. Male F344 rats were allocated to six groups, which were maintained under reverse light cycle conditions to assure acetaminophen ingestion at the time of N-acetyl-2-aminofluorene administration during the dark phase, which was imposed from 07.00 to 19.00 h. Group 1 served as vehicle control (0.5% carboxymethylcellulose) for N-acetyl-2-aminofluorene, which was administered intragastrically 3 days per week at 2.6 mg/kg for 8 weeks (group 4) to achieve initiation. Acetaminophen was given in the diet either alone at 2400 or 4800 p.p.m. for 9 weeks (groups 2 and 3), or with N-acetyl-2-aminofluorene (groups 5 and 6), starting 1 week before N-acetyl-2-aminofluorene administration. Acetaminophen blood levels were about 1 and 4 microg/ml at the two dietary concentrations. N-acetyl-2-aminofluorene induced hepatocellular preneoplastic lesions measured as hepatocellular altered foci expressing glutathione S-transferase-P, reflecting initiation. Induced foci were reduced with administration of both concentrations of acetaminophen. Acetaminophen by itself produced no DNA adducts nor did it alter the high formation of N-acetyl-2-aminofluorene-DNA adducts, about 200 in 10 nucleotides, measured by nucleotide postlabeling. Acetaminophen did not affect background liver cell proliferation, but significantly reduced N-acetyl-2-aminofluorene-induced increased proliferation measured by proliferating cell nuclear antigen immunostaining. Thus, acetaminophen effectively protected hepatocytes from the initiating effects of N-acetyl-2-aminofluorene, possibly through a cytoprotective effect resulting from slowing the rate of induced cell turnover.

Effects of methionine restriction and endurance exercise on bones of ovariectomized rats: a study of histomorphometry, densitometry, and biomechanical properties.

To investigate the effects of dietary methionine restriction (MetR) and endurance exercise on bone quality under a condition of estrogen deficiency, female Sprague-Dawley rats (36-wk-old) were assigned to a sham surgery group or one of five ovariectomized groups subjected to interventions of no treatment (Ovx), endurance exercise (Exe), methionine restriction (MetR), methionine restriction plus endurance exercise (MetR + Exe), and estrogen treatment (Est). Rats in the exercise groups were subjected to a treadmill running regimen. MetR and control diets contained 0.172 and 0.86% methionine, respectively. After the 12-wk intervention, all animals were killed, and serum and bone tissues were collected for analyses. Compared with estrogen treatment, MetR diet and endurance exercise showed better or equivalent efficiency in reducing body weight gain caused by ovariectomy (P < 0.05). Whereas only the Est group showed evidence for reduced bone turnover compared with the Ovx group, MetR diet and/or endurance exercise demonstrated efficiencies in downregulating serum insulin, leptin, triglyceride, and thiobarbituric acid reactive substances (P < 0.05). Both the Exe and MetR groups showed higher femoral cortical and total volumetric bone mineral density (vBMD), but only the Exe and Est groups preserved cancellous bone volume and/or vBMD of distal femora (P < 0.05) compared with the Ovx group. After being normalized to body mass, femora of the MetR and MetR + Exe groups had relatively higher bending strength and dimension values followed by the Sham, Exe, and Est groups (P < 0.05). In conclusion, both MetR diet and endurance exercise improved cortical bone properties, but only endurance exercise preserved cancellous bone under estrogen deficiency.

Inhibition of human topoisomerase IIalpha by fluoroquinolones and ultraviolet A irradiation.

Some fluoroquinolone antibiotics (FQs) become toxic and mutagenic upon exposure to ultraviolet radiation (UV). Topoisomerase inhibition has been proposed as one possible mechanism involved in this photochemical genotoxicity. To study this reaction, inhibition of the human topoisomerase IIalpha enzyme by four FQs varying in photochemical genotoxic potency (Bay y3118 [y3118] > Lomefloxacin [Lmx] > Ciprofloxacin [Cpx] > Moxifloxacin [Mox]) was measured in vitro in the presence of UVA irradiation. None of the FQs inhibited topoisomerase IIalpha in the absence of irradiation. In contrast, with irradiation at 365 nm, the potent photochemically genotoxic y3118 produced strong inhibition of the enzyme by 15% and Cpx caused a weak 5% inhibition, but the more photochemically genotoxic Lmx only showed a transient inhibitory effect at one concentration and one irradiation dose. The photostable Mox had no effect with irradiation. Topoisomerase IIalpha inhibition by y3118 only occurred when the FQ, DNA, and enzyme were simultaneously present in the UVA-irradiated reaction mixture and was abolished in the absence of ATP, indicating the possible formation of a ternary structure. The y3118 photochemical topoisomerase inhibition correlated with the increased irradiation-mediated binding of radiolabeled FQ to DNA:topoisomerase complexes and was irreversible, like that of the topoisomerase poison, etoposide, without irradiation. The inhibitory effect of photoactivated y3118 on topoisomerase IIalpha was also observed in the presence of the antioxidant TEMPO, indicating that reactive oxygen species were not involved in the inhibition. These observations demonstrate that some but not all photochemically genotoxic FQs inhibit human topoisomerase IIalpha, possibly by UV-induced affinity of FQs to DNA:topoisomerase complexes.

Metabolic adaptations to methionine restriction that benefit health and lifespan in rodents.

Restriction of dietary methionine by 80% slows the progression of aged-related diseases and prolongs lifespan in rodents. A salient feature of the methionine restriction phenotype is the significant reduction of adipose tissue mass, which is associated with improvement of insulin sensitivity. These beneficial effects of MR involve a host of metabolic adaptations leading to increased mitochondrial biogenesis and function, elevated energy expenditure, changes of lipid and carbohydrate homeostasis, and decreased oxidative damage and inflammation. This review summarizes observations from MR studies and provides insight about potential mediators of tissue-specific responses associated with MR's favorable metabolic effects that contribute to health and lifespan extension.

Methionine restriction prevents the progression of hepatic steatosis in leptin-deficient obese mice.

OBJECTIVE: This study investigated the effects of dietary methionine restriction (MR) on the progression of established hepatic steatosis in the leptin-deficient ob/ob mouse. MATERIAL/METHODS: Ten-week-old ob/ob mice were fed diets containing 0.86% (control-fed; CF) or 0.12% methionine (MR) for 14 weeks. At 14 weeks, liver and fat were excised and blood was collected for analysis. In another study, blood was collected to determine in vivo triglyceride (TG) and very-low-density lipoprotein (VLDL) secretion rates. Liver histology was conducted to determine the severity of steatosis. Hepatic TG, free fatty acid levels, and fatty acid oxidation (FAO) were also measured. Gene expression was analyzed by quantitative PCR. RESULTS: MR reversed the severity of steatosis in the ob/ob mouse. This was accompanied by reduced body weight despite similar weight-specific food intake. Compared with the CF group, hepatic TG levels were significantly reduced in response to MR, but adipose tissue weight was not decreased. MR reduced insulin and HOMA ratios but increased total and high-molecular-weight adiponectin levels. Scd1 gene expression was significantly downregulated, while Acadvl, Hadha, and Hadhb were upregulated in MR, corresponding with increased ß-hydroxybutyrate levels and a trend toward increased FAO. The VLDL secretion rate was also significantly increased in the MR mice, as were the mRNA levels of ApoB and Mttp. The expression of inflammatory markers, such as Tnf-α and Ccr2, was also downregulated by MR. CONCLUSIONS: Our data indicate that MR reverses steatosis in the ob/ob mouse liver by promoting FAO, increasing the export of lipids, and reducing obesity-related inflammatory responses.

Effect of taurine and N-acetylcysteine on methionine restriction-mediated adiposity resistance.

OBJECTIVES: Methionine-restricted (MR) rats, which are lean and insulin sensitive, have low serum total cysteine (tCys) and taurine and decreased hepatic expression and activity indices of stearoyl-coenzyme A desaturase-1 (SCD1). These effects are partly or completely reversed by cysteine supplementation. We investigated whether reversal of MR phenotypes can be achieved by other sulfur compounds, namely taurine or N-acetylcysteine (NAC). METHODS: MR and control-fed (CF) rats were supplemented with taurine (0.5%) or NAC (0.5%) for 12weeks. Adiposity, serum sulfur amino acids (SAA), Scd1 gene expression in liver and white adipose tissue, and SCD1 activity indices (calculated from serum fatty acid profile) were monitored. RESULTS: Taurine supplementation of MR rats did not restore weight gain or hepatic Scd1 expression or indices to CF levels, but further decreased adiposity. Taurine supplementation of CF rats did not affect adiposity, but lowered triglyceridemia. NAC supplementation in MR rats raised tCys and partly or completely reversed MR effects on weight, fat %, Scd1 expression in liver and white adipose tissue, and estimated SCD1 activity. In CF rats, NAC decreased body fat % and lowered SCD1-18 activity index (P<0.001). Serum triglycerides and leptin were over 40% lower in CF+NAC relative to CF rats (P≤0.003 for both). In all groups, change in tCys correlated with change in SCD1-16 index (partial r=0.60, P<0.001) independent of other SAA. CONCLUSION: The results rule out taurine as a mediator of increased adiposity produced by cysteine in MR, and show that NAC, similar to L-cysteine, blocks anti-obesity effects of MR. Our data show that dietary SAA can influence adiposity in part through mechanisms that converge on SCD1 function. This may have implications for understanding and preventing human obesity.

Methionine-restricted C57BL/6J mice are resistant to diet-induced obesity and insulin resistance but have low bone density.

Dietary methionine restriction (MR) extends lifespan, an effect associated with reduction of body weight gain, and improvement of insulin sensitivity in mice and rats as a result of metabolic adaptations in liver, adipose tissue and skeletal muscle. To test whether MR confers resistance to adiposity and insulin resistance, C57BL/6J mice were fed a high fat diet (HFD) containing either 0.86% methionine (control fed; CF) or 0.12% methionine (methionine-restricted; MR). MR mice on HFD had lower body weight gain despite increased food intake and absorption efficiency compared to their CF counterparts. MR mice on HFD were more glucose tolerant and insulin sensitive with reduced accumulation of hepatic triglycerides. In plasma, MR mice on HFD had higher levels of adiponectin and FGF21 while leptin and IGF-1 levels were reduced. Hepatic gene expression showed the downregulation of Scd1 while Pparg, Atgl, Cd36, Jak2 and Fgf21 were upregulated in MR mice on HFD. Restriction of growth rate in MR mice on HFD was also associated with lower bone mass and increased plasma levels of the collagen degradation marker C-terminal telopeptide of type 1 collagen (CTX-1). It is concluded that MR mice on HFD are metabolically healthy compared to CF mice on HFD but have decreased bone mass. These effects could be associated with the observed increase in FGF21 levels.

Genomic and metabolic responses to methionine-restricted and methionine-restricted, cysteine-supplemented diets in Fischer 344 rat inguinal adipose tissue, liver and quadriceps muscle.

BACKGROUND/AIMS: Methionine restriction (MR) is a dietary intervention that increases lifespan, reduces adiposity and improves insulin sensitivity. These effects are reversed by supplementation of the MR diet with cysteine (MRC). Genomic and metabolomic studies were conducted to identify potential mechanisms by which MR induces favorable metabolic effects, and that are reversed by cysteine supplementation. METHODS: Gene expression was examined by microarray analysis and TaqMan quantitative PCR. Levels of selected proteins were measured by Western blot and metabolic intermediates were analyzed by mass spectrometry. RESULTS: MR increased lipid metabolism in inguinal adipose tissue and quadriceps muscle while it decreased lipid synthesis in liver. In inguinal adipose tissue, MR not only caused the transcriptional upregulation of genes associated with fatty acid synthesis but also of Lpin1, Pc, Pck1 and Pdk1, genes that are associated with glyceroneogenesis. MR also upregulated lipolysis-associated genes in inguinal fat and led to increased oxidation in this tissue, as suggested by higher levels of methionine sulfoxide and 13-HODE + 9-HODE compared to control-fed (CF) rats. Moreover, MR caused a trend toward the downregulation of inflammation-associated genes in inguinal adipose tissue. MRC reversed most gene and metabolite changes induced by MR in inguinal adipose tissue, but drove the expression of Elovl6, Lpin1, Pc, and Pdk1 below CF levels. In liver, MR decreased levels of a number of long-chain fatty acids, glycerol and glycerol-3-phosphate corresponding with the gene expression data. Although MR increased the expression of genes associated with carbohydrate metabolism, levels of glycolytic intermediates were below CF levels. MR, however, stimulated gluconeogenesis and ketogenesis in liver tissue. As previously reported, sulfur amino acids derived from methionine were decreased in liver by MR, but homocysteine levels were elevated. Increased liver homocysteine levels by MR were associated with decreased cystathionine ß-synthase (CBS) protein levels and lowered vitamin B6 and 5-methyltetrahydrofolate (5MeTHF) content. Finally, MR upregulated fibroblast growth factor 21 (FGF21) gene and protein levels in both liver and adipose tissues. MRC reversed some of MR's effects in liver and upregulated the transcription of genes associated with inflammation and carcinogenesis such as Cxcl16, Cdh17, Mmp12, Mybl1, and Cav1 among others. In quadriceps muscle, MR upregulated lipid metabolism-associated genes and increased 3-hydroxybutyrate levels suggesting increased fatty acid oxidation as well as stimulation of gluconeogenesis and glycogenolysis in this tissue. CONCLUSION: Increased lipid metabolism in inguinal adipose tissue and quadriceps muscle, decreased triglyceride synthesis in liver and the downregulation of inflammation-associated genes are among the factors that could favor the lean phenotype and increased insulin sensitivity observed in MR rats.

Dietary methionine restriction increases fat oxidation in obese adults with metabolic syndrome.

OBJECTIVE: In preclinical reports, restriction of dietary methionine intake was shown to enhance metabolic flexibility, improve lipid profiles, and reduce fat deposition. The present report is the outcome of a "proof of concept" study to evaluate the efficacy of dietary methionine restriction (MR) in humans with metabolic syndrome. METHODS: Twenty-six obese subjects (six male and 20 female) meeting criteria for metabolic syndrome were randomized to a diet restricted to 2 mg methionine/kg body weight per day and were provided capsules containing either placebo (n = 12) or 33 mg methionine/kg body weight per day (n = 14). Energy expenditure, body composition, insulin sensitivity, and biomarkers of metabolic syndrome were measured before and after 16 wk on the respective diets. RESULTS: Insulin sensitivity and biomarkers of metabolic syndrome improved comparably in both dietary groups. Rates of energy expenditure were unaffected by the diets, but dietary MR produced a significant increase in fat oxidation (MR, 12.1 ± 6.0% increase; control, 8.1 ± 3.3% decrease) and reduction in intrahepatic lipid content (MR liver/spleen attenuation ratio, 8.1 ± 3.3% increase; control ratio, 2.2 ± 2.1% increase) that was independent of the comparable reduction in weight and adiposity that occurred in both groups. CONCLUSIONS: Sixteen weeks of dietary MR in subjects with metabolic syndrome produced a shift in fuel oxidation that was independent of the weight loss, decreased adiposity, and improved insulin sensitivity that was common to both diets.

Sulfur amino acids in methionine-restricted rats: hyperhomocysteinemia.

OBJECTIVE: Dietary methionine restriction in Fischer-344 rats favorably influences visceral fat mass, insulin sensitivity, metabolic parameters, and longevity. However, little is known about the effects of methionine restriction on serum methionine and its downstream sulfur amino acids. We investigated the serum sulfur amino acid profile of male Fischer-344 rats fed a methionine-restricted diet for 3 mo. METHODS AND RESULTS: Using tandem mass spectrometry, we observed marked reduction in serum concentrations of methionine, cystathionine, cysteine, and taurine in methionine-restricted rats compared with control (P<0.001) and a 2.5-fold elevation of homocysteine (P<0.001). CONCLUSION: This suggests that homocysteine trans-sulfuration may be inhibited by methionine restriction, and that some of the effects of methionine restriction may be mediated by changes in sulfur amino acids downstream of methionine.

Methionine restriction effects on mitochondrial biogenesis and aerobic capacity in white adipose tissue, liver, and skeletal muscle of F344 rats.

Methionine restriction increases life span in rats and mice and reduces age-related accretion of adipose tissue in Fischer 344 rats. Recent reports have shown that adipose tissue mitochondrial content and function are associated with adiposity; therefore, the expression of genes involved in mitochondrial biogenesis and oxidative capacity was examined in white adipose tissue, liver, and skeletal muscle from Fischer 344 rats fed control (0.86% methionine) or methionine-restricted (0.17% methionine) diets for 3 months. Methionine restriction induced transcriptional changes of peroxisome proliferator-activated receptors, peroxisome proliferator-activated receptor coactivators 1alpha and 1beta, and some of their known target genes in all of these tissues. In addition, tissue-specific responses were elicited at the protein level. In inguinal adipose tissue, methionine restriction increased protein levels of peroxisome proliferator-activated receptor and peroxisome proliferator-activated receptor coactivator target genes. It also induced mitochondrial DNA copy number, suggesting mitochondrial biogenesis and corresponding with the up-regulation of citrate synthase activity. In contrast, methionine restriction induced changes in mitochondrial glycerol-3-phosphate dehydrogenase activity and stearoyl-coenzyme A desaturase 1 protein levels only in liver and uncoupling protein 3 and cytochrome c oxidase subunit IV protein levels only in skeletal muscle. No increase in mitochondrial DNA copy number was observed in liver and skeletal muscle despite an increase in mitochondrial citrate synthase activity. The results indicate that adiposity resistance in methionine-restricted rats is associated with mitochondrial biogenesis in inguinal adipose tissue and increased mitochondrial aerobic capacity in liver and skeletal muscle.

Methionine restriction effects on 11 -HSD1 activity and lipogenic/lipolytic balance in F344 rat adipose tissue.

Methionine restriction (MR) limits age-related adiposity in Fischer 344 (F344) rats. To assess the mechanism of adiposity resistance, the effect of MR on adipose tissue (AT) 11beta-hydroxysteroid dehydrogenase-1 (11beta-HSD1) was examined. MR induced 11beta-HSD1 activity in all ATs, correlating with increased tissue corticosterone. However, an inverse relationship between 11beta-HSD1 activity and adipocyte size was observed. Because dietary restriction controls lipogenic and lipolytic rates, MR's effects on lipogenic and lipolytic enzymes were evaluated. MR increased adipose triglyceride lipase and acetyl-coenzyme A carboxylase (ACC) protein levels but induced ACC phosphorylation at serine residues that render the enzyme inactive, suggesting alterations of basal lipolysis and lipogenesis. In contrast, no changes in basal or phosphorylated hormone-sensitive lipase levels were observed. ACC-phosphorylated sites were specific for AMP-activated protein kinase (AMPK); therefore, AMPK activation was evaluated. Significant differences in AMPKalpha protein, phosphorylation, and activity levels were observed only in retroperitoneal fat from MR rats. No differences in protein kinase A phosphorylation and intracellular cAMP levels were detected. In vitro studies revealed increased lipid degradation and a trend toward increased lipid synthesis, suggesting the presence of a futile cycle. In conclusion, MR disrupts the lipogenic/lipolytic balance, contributing importantly to adiposity resistance in F344 rats.

In ovo carcinogenicity assay (IOCA): evaluation of mannitol, caprolactam and nitrosoproline.

The in ovo carcinogenicity assay (IOCA) was used to examine whether the noncarcinogens epsilon-caprolactam (CAP), D-mannitol (MAN) and nitrosoproline (NPRO) induce toxicity and subsequently morphological changes in embryonic turkey livers compared with the carcinogen diethylnitrosamine (DEN). Various doses of the test compounds were injected into fertilized turkey or quail eggs prior to incubation. Embryonic livers were collected 3-4 days before hatching and processed for histology. The positive control DEN induced hepatocyte altered foci (HAF) and karyomegalic hepatocytes, whereas histological analysis of livers from embryos exposed to CAP, MAN and NPRO did not show such histological changes. The effects of the tested compounds on liver were further examined in hepatocytes cultured from exposed turkey and quail embryos. As observed in ovo, megalocytes as well as karyomegalic hepatocytes were present in hepatocyte cultures established from DEN-exposed turkey embryos, but not from embryos exposed to CAP, MAN or NPRO. It is concluded that CAP, MAN and NPRO do not induce histological changes in embryonic liver of the type produced by the carcinogen DEN, correlating with findings for these compounds in rodent studies.

Embryonic turkey liver: activities of biotransformation enzymes and activation of DNA-reactive carcinogens.

Avian embryos are a potential alternative model for chemical toxicity and carcinogenicity research. Because the toxic and carcinogenic effects of some chemicals depend on bioactivation, activities of biotransformation enzymes and formation of DNA adducts in embryonic turkey liver were examined. Biochemical analyses of 22-day in ovo turkey liver post-mitochondrial fractions revealed activities of the biotransformation enzymes 7-ethoxycoumarin de-ethylase (ECOD), 7-ethoxyresorufin de-ethylase (EROD), aldrin epoxidase (ALD), epoxide hydrolase (EH), glutathione S-transferase (GST), and UDP-glucuronyltransferase (GLUT). Following the administration of phenobarbital (24 mg/egg) on day 21, enzyme activities of ECOD, EROD, ALD, EH and GLUT, but not of GST, were increased by two-fold or higher levels by day 22. In contrast, acute administration of 3-methylcholanthrene (5 mg/egg) induced only ECOD and EROD activities. Bioactivation of structurally diverse pro-carcinogens was also examined using (32)P-postlabeling for DNA adducts. In ovo exposure of turkey embryos on day 20 of gestation to 2-acetylaminofluorene (AAF), 4,4'-methylenebis(2-chloroaniline) (MOCA), benzo[a]pyrene (BaP), and 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx) resulted in the formation of DNA adducts in livers collected by day 21. Some of the DNA adducts had (32)P-postlabeling chromatographic migration patterns similar to DNA adducts found in livers from Fischer F344 rats exposed to the same pro-carcinogens. We conclude that 21-day embryonic turkey liver is capable of chemical biotransformation and activation of genotoxic carcinogens to form DNA adducts. Thus, turkey embryos could be utilized to investigate potential chemical toxicity and carcinogenicity.