Dietary Methionine and Total Sulfur Amino Acid Restriction in Healthy Adults.

OBJECTIVES: Dietary restriction of methionine (Met) and cysteine (Cys) delays the aging process and aging-related diseases, improves glucose and fat metabolism and reduces oxidative stress in numerous laboratory animal models. Little is known regarding the effects of sulfur amino acid restriction in humans. Thus, our objectives were to determine the impact of feeding diets restricted in Met alone (MetR) or in both Met and Cys (total sulfur amino acids, SAAR) to healthy adults on relevant biomarkers of cardiometabolic disease risk. DESIGN: A controlled feeding study. SETTING AND PARTICIPANTS: We included 20 healthy adults (11 females/9 males) assigned to MetR or SAAR diet groups consisting of three 4-wk feeding periods: Control period; low level restriction period (70% MetR or 50% SAAR); and high level restriction period (90% MetR or 65% SAAR) separated by 3-4-wk washout periods. RESULTS: No adverse effects were associated with either diet and level of restriction and compliance was high in all subjects. SAAR was associated with significant reductions in body weight and plasma levels of total cholesterol, LDL, uric acid, leptin, and insulin, BUN, and IGF-1, and increases in body temperature and plasma FGF-21 after 4 weeks (P<0.05). Fewer changes occurred with MetR including significant reductions in BUN, uric acid and 8-isoprostane and an increase in FGF-21 after 4 weeks (P<0.05). In the 65% SAAR group, plasma Met and Cys levels were significantly reduced by 15% and 13% respectively (P<0.05). CONCLUSION: These results suggest that many of the short-term beneficial effects of SAAR observed in animal models are translatable to humans and support further clinical development of this intervention.

Oral supplementation with liposomal glutathione elevates body stores of glutathione and markers of immune function.

BACKGROUND/OBJECTIVES: Glutathione (GSH) is the most abundant endogenous antioxidant and a critical regulator of oxidative stress. Maintenance of optimal tissues for GSH levels may be an important strategy for the prevention of oxidative stress-related diseases. We investigated if oral administration of liposomal GSH is effective at enhancing GSH levels in vivo. SUBJECTS/METHODS: A 1-month pilot clinical study of oral liposomal GSH administration at two doses (500 and 1000 mg of GSH per day) was conducted in healthy adults. GSH levels in whole blood, erythrocytes, plasma and peripheral blood mononuclear cells (PBMCs) were assessed in 12 subjects at the baseline and after 1, 2 and 4 weeks of GSH administration. RESULTS: GSH levels were elevated after 1 week with maximum increases of 40% in whole blood, 25% in erythrocytes, 28% in plasma and 100% in PBMCs occurring after 2 weeks (P<0.05). GSH increases were accompanied by reductions in oxidative stress biomarkers, including decreases of 35% in plasma 8-isoprostane and 20% in oxidized:reduced GSH ratios (P<0.05). Enhancements in immune function markers were observed with liposomal GSH administration including Natural killer (NK) cell cytotoxicity, which was elevated by up to 400% by 2 weeks (P<0.05), and lymphocyte proliferation, which was elevated by up to 60% after 2 weeks (P<0.05). Overall, there were no differences observed between dose groups, but statistical power was limited due to the small sample size in this study. CONCLUSIONS: Collectively, these preliminary findings support the effectiveness of daily liposomal GSH administration at elevating stores of GSH and impacting the immune function and levels of oxidative stress.

Stearoyl-CoA desaturase-1, a novel target of omega-3 fatty acids for reducing breast cancer risk in obese postmenopausal women.

BACKGROUND/OBJECTIVES: Conversion of saturated fatty acids to monounsaturated fatty acids by the enzyme stearoyl-Co-A-desaturase (SCD-1) is emerging as a major factor in promoting carcinogenesis including breast cancer. The aim of our study was to explore the regulation of SCD-1 by Raloxifene and omega-3 fatty acids in women at increased risk of breast cancer based on high breast density. SUBJECTS/METHODS: As a reflection of SCD-1 activity, we measured the ratios of palmitoleic acid (C16:1n7) to palmitic acid (C16:0) (SCD-16) and oleic acid (C18:1n9) to steric acid (C18:0) (SCD-18) in plasma samples of postmenopausal women enrolled in our clinical trial (NCT00723398) designed to test the effects of the antiestrogen, Raloxifene and/or the omega-3 preparation Lovaza, on breast density, a validated biomarker of breast cancer risk. RESULTS: We report that Lovaza but not Raloxifene-reduced SCD-16 and SCD-18 for the 2-year duration of the trial. Importantly, decreasing levels of SCD-16 and SCD-18 were associated with a progressive reduction in breast density but only in obese women (body mass index ⩾30). CONCLUSIONS: Body mass index-related factors play an important role in the reduction of breast density and hence breast cancer risk by omega-3 fatty acids. SCD-1 may be a useful biomarker in future clinical trials testing the benefit of nutritional interventions in reducing obesity-associated breast cancer risk.

Preliminary studies on the effect of moderate physical activity on blood levels of glutathione.

Molecular epidemiological approaches are being used to study how physical activity may protect against cancer. Prior epidemiological data suggest that physical activity protects against lung cancer; however, interpretation of these data is complicated by potential confounding by smoking. Glutathione (GSH) detoxifies cigarette smoke carcinogens and the paper tests whether physical activity levels are associated with blood GSH levels. Study subjects were enrolled in a chemoprevention trial testing whether antioxidant micronutrient supplementation reduces genetic damage from cigarette smoking. Physical activity data were collected by questionnaire from 178 subjects at 12 months of follow-up in the trial. Total GSH (tGSH), which is the sum of free and protein-bound GSH and glutathione disulfide levels, was measured using the 5,5'-dithiobis-(2-nitrobenzenoic acid) colormetric assay with red blood cell samples collected at the 12-month time point. In multivariate linear regression analyses that controlled for gender and cigarettes smoked per day, tGSH was positively associated with hours per week of moderate intensity activity (beta=0.005, p=0.02). Hours per week of vigorous intensity activity were unassociated with tGSH and the effect of moderate activity remained after control for vigorous activity. The results are consistent with prior research showing differential effects of moderate and vigorous activity and suggest a mechanism through which physical activity may influence lung cancer risk.

p53 mutations in head and neck squamous cell carcinomas from Sudanese snuff (toombak) users.

Toombak is a type of snuff used extensively in the Northern Sudan by a virtually non-smoking/nondrinking population. This Sudanese snuff contains inordinately high levels of the tobacco-specific nitrosamines (TSNAs) Nl-nitrosonornicotine (NNN) and (4-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These are considered to be major contributors to the induction of cancers of the aerodigestive tract in tobacco chewers, snuff dippers, and smokers. To determine if toombak use may be associated with the induction of mutations in the p53 tumor suppressor gene, we screened four head and neck squamous cell carcinomas (SCCs) obtained from three toombak-using patients and one non-toombak-using patient using polymerase chain reaction/single-stranded conformational polymorphism analysis and DNA sequencing. p53 mutations were found in tumors resected from two of three toombak-using patients, one at codon 282 (CGGarg-->TGGtrp) and the other in intron 6 (AT-->GC). No p53 mutations were observed in the tumor from the non-toombak-using patient. The observed mutations were similar in spectrum to those induced by nitrosamines in mouse lung tumors. No K-ras (codons 12 and 13) or H-ras (codon 12) mutations were found in any of the tumors. These results suggest that toombak components such as TSNAs may induce p53 mutations in head and neck SCCs and are likely contributors to the tobacco-induced carcinogenic load in humans.

A low incidence of p53 mutations in pre-malignant lesions of the oral cavity from non-tobacco users.

To determine the incidence of p53 mutations in pre-malignant lesions of the oral cavity from individuals without prior history of tobacco use, we have analyzed the conserved regions of the p53 gene (exons 5-9) in archival oral cavity lesion specimens obtained from patients with varied tobacco use histories, by polymerase chain reaction/single strand conformational polymorphism (PCR/SSCP) and DNA sequencing analysis. Twenty-six lesions were analyzed from 14 patients, with multiple lesions obtained from 8 patients. Six of these patients used tobacco, (3 being cigarette smokers, 1 ex-cigarette smoker, 1 moderate cigar smoker and 1 snuff chewer). The remaining 8 patients had no prior history of tobacco use. Thirteen of the pre-malignant lesions exhibited severe dysplasia, 9 exhibited moderate dysplasia and 4 exhibited mild dysplasia. Four of the 26 lesions exhibited p53 mutations, each being from a tobacco user. None of the 13 lesions from never-tobacco users exhibited p53 mutations. There was a significantly higher p53 mutation incidence in pre-malignant lesions from tobacco users (including ex-smokers) than in non-tobacco users as well as in cigarette smokers plus snuff chewers than in non-tobacco users. Two of the mutations were observed in lesions exhibiting severe dysplasia: 1 in a lesion exhibiting moderate dysplasia and 1 in a lesion exhibiting mild dysplasia. These data suggest that p53 mutation may be a very early event in oral cavity tumor progression and demonstrate that pre-malignant lesions obtained from non-tobacco users do not exhibit p53 mutations.

Sequence of the rat hypoxanthine guanine phosphoribosyl transferase (HPRT) transcriptional promoter region in wild-type and mutant rat liver epithelial cell lines.

The hypoxanthine guanine phosphoribosyltransferase (HPRT) gene is mutated by a variety of genotoxic agents in adult rat liver (ARL) epithelial cell lines. By polymerase chain reaction (PCR) amplification and DNA sequencing of rat ARL cell HPRT gene sequences with mouse- and rat-specific oligonucleotides, a large portion of the rat HPRT transcriptional promoter region was sequenced. This region exhibits approximately 60% homology with the corresponding mouse sequence, contains a similar G/C-rich region at its 3' end, and contains a similar series of 6-nucleotide (nt) GGGCGG repeats. To determine if this region is a target for mutation by different genotoxins, HPRT-deficient ARL mutants induced by 2-acetylaminofluorene (AAF), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), or 7,12-dimethyl-benz[a]anthracene (DMBA) were isolated and studied. A 1003-nt fragment of predominantly HPRT regulatory sequences was amplified by PCR using purified genomic DNA from 17 independent mutants and sequenced directly. None of the 17 mutants examined exhibited any alterations in the transcriptional regulatory region or the 5' untranslated region of HPRT exon 1 after direct sequencing analysis of PCR products. In addition, none of the 2-AAF-induced mutants exhibited differences in in vitro transcription rates as determined by nuclear run-on analysis. These data suggest that regulatory sequences of the HPRT gene are not a primary target for mutation by the genotoxins studied.

Identification and partial characterization of the c-jun oncogene in Xenopus laevis.

Sequencing analysis of a 275-nt cDNA fragment encoding the DNA-binding region at the carboxyl terminal of the Xenopus c-jun protein product shows that this region exhibits 77-79% nucleotide homology and 93% amino acid homology with the DNA-binding region of mouse and human c-jun, and contains an intact leucine zipper motif and a basic DNA-binding region. Xenopus c-jun mRNA is induced in quiescent Xenopus A6 kidney cells by 12-O-tetradecanoylphorbol-13-acetate, PDGF and cycloheximide. This 'immediate early' response suggests that Xenopus c-jun may be similar in both structure and function to that observed for its mammalian counterpart.

Effect of vitamin B6 on L-glutamate dehydrogenase activity in mice brain.

Investigations were carried out to obtain information on the effect of vitamin B6 level on L-glutamate dehydrogenase activity in mice brain. Subcutaneous and intracerebral injection of pyridoxal phosphate or pyridoxamine resulted in a significant enhancement of the L-glutamate dehydrogenase activity. In the case of pyridoxine, much larger doses and more prolonged time were necessary to exhibit the effect. The above effect of vitamin B6 was much more evidently observed in vitamin B6-deficient animals.