Transcriptional and post-translational modifications of B-Raf in quinol-thioether induced tuberous sclerosis renal cell carcinoma.

Increased activity of B-Raf has been identified in approximately 7% of human cancers. Treatment of Eker rats (Tsc-2(EK/+) ), bearing a mutation in one allele of the tuberous sclerosis-2 (Tsc-2) gene, with the nephrocarcinogen 2,3,5-tris-(glutathion-S-yl) hydroquinone (TGHQ) results in loss of the wild-type allele of Tsc-2 in renal preneoplastic lesions and tumors. These tumors have increased protein expression of B-Raf, C-Raf (Raf-1), and increased expression and activity of ERK kinase. Similar changes are observed in Raf kinases following TGHQ-mediated transformation of primary renal epithelial cells derived from Tsc-2(EK/+) rats (QTRRE cells), cells that are also null for tuberin. Herein, we utilized LC-MS/MS to identify constitutive phosphorylation of S345 and S483 in both 100- and 95-kDa forms of B-Raf in QTRRE cells. Using microRotofor liquid-phase isoelectric focusing, we identified four fractions of B-Raf that contain different post-translational modification profiles in QTRRE cells. Amplification of the kinase domain of B-Raf from QTRRE cells, outer-stripe of the outer medulla of 8-month TGHQ- or vehicle-treated Tsc-2(+/+) and Tsc-2(EK/+) rats, as well as tumors excised from 8-month TGHQ-treated Tsc-2(EK/+) rats revealed three splice variants of B-Raf within the kinase domain. These splice variants differed by approximately 340, 544, and 600 bp; confirmed by sequencing. No point mutations within the kinase domain of B-Raf were identified. In addition, B-Raf/Raf-1/14-3-3 complex formation in the QTRRE cells was decreased by sorafenib, with concomitant selective decreases in p-ERK levels. Transcriptional and post-translational characterization of critical kinases, such as B-Raf, may contribute to the progression of tuberous sclerosis RCC. (246/250) © 2015 Wiley Periodicals, Inc.

Characterization of a new polymeric food contact coating with emphasis on the chemical analysis and safety assessment of non-intentionally added substances (NIAS).

Regulators have established safety requirements for food packaging raw materials and finished products, including by-products of polymer synthesis known as non-intentionally added substances (NIAS). However, there are no official guidance or regulations for best practices to evaluate the safety of NIAS. Here we described the process we followed to identify, characterize, and prioritize for safety assessment low molecular weight NIAS from an epoxy coating (V70) made with tetramethyl bisphenol F-based diglycidyl ether resin (TMBPF-DGE). We assembled a database of 15000 potential oligomers with masses up to 1000 Da and conducted extraction and migration testing of V70 coating. Acetonitrile extract contained higher number and concentration of substances compared to ethanolic-based food simulants. The extract contained 16 substances with matches in the database with estimated concentration of 18.27 µg/6 dm2; seven of these substances have potentially genotoxic oxirane functionality. TMBPF-DGE + hydroquinone (TMBPF-DGE + HQ) was most abundant (55% of total concentration) and was synthesized and prioritized for safety assessment. TMBPF-DGE + HQ exposure from can beverage was estimated at 5.2 µg/person/day, and it was not mutagenic or genotoxic in in vitro assays. The overall mixture of substances that migrated into ethanolic simulant was also negative in the mutagenicity bioassay. Our findings suggest that exposure to TMBPF-DGE + HQ from the V70 coating is exceedingly small and that the coating migrates are not genotoxic.

An expanded toxicological profile of tetramethyl bisphenol F (TMBPF), a precursor for a new food-contact metal packaging coating.

Tetramethyl bisphenol F (TMBPF) has been shown to impart unique physical properties critical for high performance of epoxy can coatings without the estrogenic activity concerns associated with other bisphenols. To further characterize the toxicological profile of TMBPF, additional endocrine-related endpoints including in vitro aromatase inhibition and steroidogenesis assays, and in vivo androgen agonism/antagonism were performed. Systemic toxicity was also assessed by a repeat dose 90-day dietary toxicity study followed by 28-day recovery period. TMBPF did not inhibit aromatase activity, and induced estradiol and testosterone at highest non-cytotoxic concentrations (10 µM) in the steroidogenesis assay. In the Hershberger assay, TMBPF showed no androgenic activity at any dose and equivocal anti-androgenic activity at the highest dose (1000 mg/kg-bw/d). In a 90-day dietary toxicity study with 28-day recovery period, observations including changes in clinical pathology, absolute and relative organ weights, and microscopic findings are discussed. In this current study, the no observed adverse effect level was considered to be 750 mg/kg-bw/d for female rats and 1000 mg/kg-bw/d for male rats with no biologically significant changes to endocrine endpoints at any dose level. Our findings provide further evidence that TMBPF is a low-toxicity substance with a toxicology profile distinct from some other bisphenols.