Quantification of epoxide metabolite specific n-terminal globin adducts: a biomarker of internal dosimetry of 1,3-butadiene exposure and metabolism

Butadiene (BD) carcinogenicity in rodents shows gender, species and concentration dependency, making the extrapolation of animal results to humans complex. BD is a multispecies multisite carcinogen, with mice being a much more sensitive species than rats. This is considered to be related to the metabolism of BD to its epoxy metabolites, 1,2-epoxy-3-butene (EB), 1,2;3,4-diepoxybutane (DEB) and 1,2-epoxy-butanediol (EB-diol). The mutagenic potency of individual epoxides varies up to 200-fold, with DEB being the most mutagenic metabolite. For accurate risk assessment it is important to elucidate species differences in the internal formation of the individual epoxides in order to assign the relative risks associated with their different mutagenic potencies. N-terminal globin adducts have been widely used for measurements of the formation of BD derived epoxides. In this study, the formation of each epoxide was evaluated in globin samples from both genders of mice and rats exposed to BD by inhalation for 10 days. The numbers of globin adducts were then converted into EB dose equivalents. These were calculated on the basis of the combined internal dose (globin adducts) and the relative genotoxic potency of the respective epoxides inferred from the efficiency of inducing mutations at the Hprt locus. Then, the multiplicative cancer risk model was applied to quantitatively estimate tumor incidence by using the EB dose equivalent and long-term cancer bioassay data. Based on the EB equivalent, higher exposures formed lower amounts per ppm BD. This indicates that metabolism of BD to epoxides is most effective at low exposures. The EB equivalent for mice was about 40-fold higher than that of rats at similar exposures. No gender differences were noted in globin adducts of mice or rats at all exposures. As such, EB dose equivalents provide quantitative data on biomarkers of exposure that can be extended to a scientific basis for BD risk assessment

Formation of 1,2:3,4-Diepoxybutane-Specific Hemoglobin Adducts in 1,3-Butadiene Exposed Workers

1,3-Butadiene (BD) is an important industrial chemical that is classified as a human carcinogen. BD carcinogenicity has been attributed to its metabolism to several reactive epoxide metabolites and formation of the highly mutagenic 1,2:3,4-diepoxybutane (DEB) has been hypothesized to drive mutagenesis and carcinogenesis at exposures experienced in humans. We report herein the formation of DEB-specific N,N-(2,3-dihydroxy-1,4-butadiyl)valine (pyr-Val) in BD-exposed workers as a biomarker of DEB formation. pyr-Val was determined in BD monomer and polymer plant workers that had been previously analyzed for several other biomarkers of exposure and effect. pyr-Val was detected in 68 of 81 (84%) samples ranging from 0.08 to 0.86 pmol/g globin. Surprisingly, pyr-Val was observed in 19 of 23 administrative control subjects not known to be exposed to BD, suggesting exposure from environmental sources of BD. The mean ± SD amounts of pyr-Val were 0.11 ± 0.07, 0.16 ± 0.12, and 0.29 ± 0.20 pmol/g globin in the controls, monomer, and polymer workers, respectively, clearly demonstrating formation of DEB in humans. The amounts of pyr-Val found in this study suggest that humans are much less efficient in the formation of DEB than mice or rats at similar exposures. Formation of pyr-Val was more than 50-fold lower than has been associated with increased mutagenesis in rodents. The results further suggest that formation of DEB relative to other epoxides is significantly different in the highest exposed polymer workers compared with controls and BD monomer workers. Whether this is due to saturation of metabolic formation or increased GST-mediated detoxification could not be determined

Accurate quantitation of standard peptides used for quantitative proteomics

MS-based proteomics has become an indispensable tool in system biology generating a need for accurate and precise quantitation of peptide standards. The presented method utilizes ultra performance LC-MS/MS (UPLC-MS/MS) to accurately quantify peptide standards at concentrations of 0.1-10 microM. The ability for accurate quantitation of micro-molar concentrations has the advantages that quantitation can be performed routinely with high precision and the high sensitivity of the method minimizes the amounts required

1,3-Butadiene: Biomarkers and application to risk assessment

1,3-Butadiene (BD) is a known rodent and human carcinogen that is metabolized mainly by P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB) and 1,2-epoxy-3,4-butanediol (EB-diol). The individual epoxides vary up to 200-fold in their mutagenic potency, with DEB being the most mutagenic metabolite. It is important to understand the internal formation of the individual epoxides to assign the relative risk for each metabolite and to understand the molecular mechanisms responsible for major species differences in carcinogenicity. We have conducted extensive exposure-biomarker studies on mice, rats and humans. Using low exposures that range from current occupational levels to human exposures from tobacco smoke has provided evidence that mice are very different from humans, with mice forming ~200 times more DEB than humans at exposures of 0.1–1.5 ppm BD. While no gender differences have been noted in mice and rats for globin adducts or N-7 guanine adducts, female rats and mice had 2–3-fold higher Hprt mutations and DNA-DNA cross-links, suggesting a gender difference in DNA repair. Numerous molecular epidemiology studies have evaluated globin adducts and Hprt mutations, SCEs and chromosomal abnormalities. None of the blinded studies have shown evidence of human genotoxicity at current occupational exposures and studies of globin adducts have shown similar or lower formation of adducts in females than males. If one calculates the EB dose-equivalents for the three species, mice clearly differ from rats and humans, being ~44 and 174 times greater than rats and humans, respectively. These data provide a scientific basis for improved risk assessment of BD

Effect on Vitamin D status of Breastfeeding Infants after Vitamin D3 Supplementation during Breastfeeding Lactation: A double-blind randomized controlled trial

Background: Vitamin D deficiency in pregnancy increases several risks of breastfed mothers. To prevent these adverse events, vitamin D supplementation during pregnancy and lactation is recommended, but suggested dose ranges vary. Objective: To determine whether vitamin D3 1,800 IU/d supplementation in lactating mothers improves the vitamin D status of their breastfed infants. Materials and Methods: A randomized, placebo–controlled trial with Thai pregnant women was conducted. Lactating mothers (n=72) and their breastfed infants with insufficient maternal 25 hydroxyvitamin D (25(OH)D) levels in the third trimester were randomly assigned to two groups, one of which received 1,800 IU/d vitamin D supplementation and the other a placebo. Maternal serum 25(OH)D during lactation, cord blood, and 6-week breastfed infant serum were measured using LC-MS/MS. Results: Mean maternal age (±SD) was 27±5 years, and pre-gestational BMI was 22.29±5 kg/m2. Maternal serum 25(OH)D at baseline was 22.29±7.15 nmol/L. At 6 weeks, both maternal 25(OH)D and infant 25 (OH)D levels had increased significantly in the vitamin D supplement group of mothers and infants (68.30±15.40, 40.40±12.56 nmol/L) compared to those in placebo groups (55.15±13.57, 24.28±17.20 nmol/L) (p <0.001, p<0.001). The changes in infant 25(OH)D levels increased substantially in the vitamin D supplement group but decreased in placebo(17.49±16.27 ng/ml compared to -1.34±19.23 nmol/L in the placebo group, p<0.001). The change of maternal 25(OH)D were positively correlation to the change of 25(OH)D level in breastmilk mothers and infants by r=0.697, p<0.001 and r=0.379, p=0.003 respectively. Conclusions: Vitamin D3 supplementation to breastfed mother during lactation can increase serum 25(OH)D level in Thai breastfed mother and infants. Further work is needed to determine the optimum duration of vitamin D supplementation to normalized breastfed infants with 25(OH)D level >75 nmol/L

Exposure-Response of 1,2:3,4-Diepoxybutane–Specific N-Terminal Valine Adducts in Mice and Rats after Inhalation Exposure to 1,3-Butadiene

1,3-Butadiene (BD) is a known rodent and human carcinogen that is metabolized mainly by P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-epoxy-3,4-butanediol. The individual epoxides vary up to 200-fold in their mutagenic potency, with DEB being the most mutagenic metabolite. It is important to understand the internal formation of the individual epoxides to assign the relative risk for each metabolite and to understand the molecular mechanisms responsible for extensive species differences in carcinogenicity. This study presents a comprehensive exposure-response for the formation of the DEB-specific N,N-(2,3-dihydroxy-1,4-butadiyl)valine (pyr-Val) in mice and rats. Using nano-ultra high pressure liquid chromatography-tandem-mass spectrometry allowed analysis of pyr-Val in mice and rats exposed to BD as low as 0.1 and 0.5 ppm BD, respectively, and demonstrated significant differences in the amounts and exposure-response of pyr-Val formation. Mice formed 10- to 60-fold more pyr-Val compared to rats at similar exposures. The formation of pyr-Val increased with exposures, and the formation was most efficient with regard to formation per parts per million BD at low exposures. While formation at higher exposures appeared linear in mice, in rats formation saturated at exposures ≥ 200 ppm for 10 days. In rats, amounts of pyr-Val were lower after 20 days than after 10 days of exposure, suggesting that the lifespan of rat erythrocytes may be shortened following exposure to BD. This research supports the hypothesis that the lower susceptibility of rats to BD-induced carcinogenesis results from greatly reduced formation of DEB following exposure to BD

Targeted proteomics using selected reaction monitoring reveals the induction of specific terpene synthases in a multi-level study of methyl jasmonate-treated Norway spruce (Picea abies)

Induction of terpene synthase (TPS) gene expression and enzyme activity is known to occur in response to various chemical and biological stimuli in several species of spruce (genus Picea). However, high sequence identity between TPS family members has made it difficult to determine the induction patterns of individual TPS at the protein and transcript levels and whether specific TPS enzymes respond differentially to treatment. In the present study we used a multi-level approach to measure the induction and activity of TPS enzymes in protein extracts of Norway spruce (Picea abies) bark tissue following treatment with methyl jasmonate (MeJA). Measurements were made on the transcript, protein, enzyme activity and metabolite levels. Using a relatively new proteomics application, selective reaction monitoring (SRM), it was possible to differentiate and quantitatively measure the abundance of several known TPS proteins and three 1-deoxy-d-xylulose 5-phosphate synthase (DXS) isoforms in Norway spruce. Protein levels of individual TPS and DXS enzymes were differentially induced upon MeJA treatment and good correlation was generally observed between induction of transcripts, proteins, and enzyme activities. Most of the mono-and diterpenoid metabolites accumulated with similar temporal patterns of induction as part of the coordinated multi-compound chemical defense response. Protein and enzyme activity levels of the monoTPS (+)-3-carene synthase and the corresponding accumulation of (+)-3-carene was induced to a higher fold change than any other TPS or metabolite measured, indicating an important role in the induced terpenoid defense response in Norway spruce. © 2009 Blackwell Publishing Ltd

Ultrasensitive Quantification of Recombinant Proteins Using AAA-MS

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