Omega-3 Fatty Acids Responsive Proteins and Reduction in Breast Density in Obese Postmenopausal Women.

We reported that breast density (BD) was inversely correlated with the plasma level of DHA in postmenopausal obese, but not in nonobese, women given Lovaza (n-3FA). To identify protein biomarkers for the possible differential effect of n-3FA on BD between obese and nonobese women, an iTRAQ method was performed to analyze plasma from obese and lean women at each time point (baseline, 12 and 24-months, n = 10 per group); 173 proteins with >95% confidence (Unuses Score >1.3 and local false discovery rate estimation <5%) were identified. Comparative analysis between various groups identified several differentially expressed proteins (hemopexin precursor, vitamin D binding protein isoform 1 precursor [VDBP], fibronectin isoform 10 precursor [FN], and α-2 macroglobulin precursor [A2M]). Western blot analysis was performed to verify the differential expression of proteins in the iTRAQ study, and those found to be altered in a tumor protective fashion by an n-3FA rich diet in our previous preclinical study; gelsolin, VDBP, and FN were altered by n-3FA in a manner consistent with reduction in inflammation in obese women. To test the impact of our findings on breast cancer risk reduction by n-3FA, a posthoc analysis revealed that n-3FA administration reduced BD selectively in obese postmenopausal women.

Elevated hepatic iron activates NF-E2-related factor 2-regulated pathway in a dietary iron overload mouse model.

Hepatic iron overload has been associated classically with the genetic disorder hereditary hemochromatosis. More recently, it has become apparent that mild-to-moderate degrees of elevated hepatic iron stores observed in other liver diseases also have clinical relevance. The goal was to use a mouse model of dietary hepatic iron overload and isobaric tag for relative and absolute quantitation proteomics to identify, at a global level, differentially expressed proteins in livers from mice fed a control or 3,5,5-trimethyl-hexanoyl-ferrocene (TMHF) supplemented diet for 4 weeks. The expression of 74 proteins was altered by ≥ ±1.5-fold, showing that the effects of iron on the liver proteome were extensive. The top canonical pathway altered by TMHF treatment was the NF-E2-related factor 2 (NRF2-)-mediated oxidative stress response. Because of the long-standing association of elevated hepatic iron with oxidative stress, the remainder of the study was focused on NRF2. TMHF treatment upregulated 25 phase I/II and antioxidant proteins previously categorized as NRF2 target gene products. Immunoblot analyses showed that TMHF treatment increased the levels of glutathione S-transferase (GST) M1, GSTM4, glutamate-cysteine ligase (GCL) catalytic subunit, GCL modifier subunit, glutathione synthetase, glutathione reductase, heme oxygenase 1, epoxide hydrolase 1, and NAD(P)H dehydrogenase quinone 1. Immunofluorescence, carried out to determine the cellular localization of NRF2, showed that NRF2 was detected in the nucleus of hepatocytes from TMHF-treated mice and not from control mice. We conclude that elevated hepatic iron in a mouse model activates NRF2, a key regulator of the cellular response to oxidative stress.

4-(Methylnitrosamino)-I-(3-pyridyl)-1-butanone enhances the expression of apolipoprotein A-I and Clara cell 17-kDa protein in the lung proteomes of rats fed a corn oil diet but not a fish oil diet.

The nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most potent lung carcinogens in rodents. Several epidemiologic studies indicated that the development of lung cancer in smokers is influenced by the type and amount of dietary polyunsaturated fatty acids. A high corn oil diet has been shown to increase lung tumor volume and to decrease tumor latency in rats treated with NNK. In this study, we investigated the effects of dietary polyunsaturated fatty acids in the form of corn oil or fish oil on lung proteomes in F344 rats treated with or without NNK. The fish oil diet contained 17% fish oil and 3% corn oil, and the corn oil diet contained 20% corn oil. Rats were sacrificed after 3 months, and lungs were excised. Whole lung tissue proteins were separated by two-dimensional liquid chromatography, and differentially expressed proteins were identified by trypsin digestion and tandem mass spectrometry. Apolipoprotein A-I and Clara cell 17-kDa protein were overexpressed in the lungs of rats fed corn oil diet, compared with fish oil diet. NNK further enhanced their expression in rats fed corn oil diet; this effect was not observed in animals fed fish oil diet. The results suggest that the elevated levels of apolipoprotein A-I and Clara cell 17-kDa protein may be involved in the development of NNK-induced lung cancer in rats fed a high corn oil diet. Therefore, we propose that both proteins may serve as potential biomarkers in future molecular epidemiologic and clinical chemoprevention intervention studies.

Spermidine/spermine-N1-acetyltransferase-2 (SSAT2) acetylates thialysine and is not involved in polyamine metabolism.

Spermidine/spermine-N1-acetyltransferase (SSAT1) is a short-lived polyamine catabolic enzyme inducible by polyamines and polyamine analogues. Induction of SSAT1 plays an important role in polyamine homoeostasis, since the N1-acetylated polyamines can be excreted or oxidized by acetylpolyamine oxidase. We have purified a recombinant human acetyltransferase (SSAT2) that shares 45% identity and 61% homology with human SSAT1, but is only distally related to other known members of the GNAT (GCN5-related N-acetyltransferase) family. Like SSAT1, SSAT2 is widely expressed, but did not turn over rapidly, and levels were unaffected by treatments with polyamine analogues. Despite similarity in sequence to SSAT1, polyamines were found to be poor substrates of purified SSAT2, having K(m) values in the low millimolar range and kcat values of <0.01 s(-1). The kcat/K(m) values for spermine and spermidine for SSAT2 were <0.0003% those of SSAT1. Expression of SSAT2 in NIH-3T3 cells was not detrimental to growth, and did not reduce polyamine content or increase acetylpolyamines. These results indicate that SSAT2 is not a polyamine catabolic enzyme, and that polyamines are unlikely to be its natural intracellular substrates. A promising candidate for the physiological substrate of SSAT2 is thialysine [S-(2-aminoethyl)-L-cysteine], which is acetylated predominantly at the epsilon-amino group with K(m) and kcat values of 290 muM and 5.2 s(-1). Thialysine is a naturally occurring modified amino acid that can undergo metabolism to form cyclic ketimine derivatives found in the brain and as urinary metabolites, which can undergo further reaction to form antioxidants. SSAT2 should be renamed 'thialysine N(epsilon)-acetyltransferase', and may regulate this pathway.