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The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a component of tobacco smoke and is rapidly metabolized to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NN...

There are 660 pyrrolizidine alkaloids (PAs) and PA N-oxides present in the plants, with approximately half being possible carcinogens. We previously reported that a set of four PA-derived DNA adducts is formed in the liver of rats administered a series of hepatocarcinogenic PAs and a PA N-oxide. Based on our findings, we hypothesized that this set of DNA adducts is a common biological biomarker of PA-induced liver tumor formation. In this study, we determined that rat liver microsomal metabolism of five hepatocarcinogenic PAs (lasiocarpine, retrorsine, riddelliine, monocrotaline, and heliotrine) and their corresponding PA N-oxides produced the same set of DNA adducts. Among these compounds, lasiocarpine N-oxide, retrorsine N-oxide, monocrotaline N-oxide, and heliotrine N-oxide are for first time shown to be able to produce these DNA adducts. These results further support the role of these DNA adducts as potential common biomarkers of PA-induced liver tumor initiation.

Diet is an important variable in toxicology. There are mixed reports on the impact of soy components on energy utilization, fat deposition, and reproductive parameters. Three generations of CD-1 mice were fed irradiated natural ingredient diets with varying levels of soy (NIH-41, 5K96, or 5008/5001), purified irradiated AIN-93 diet, or the AIN-93 formulation modified with ethanol-washed soy protein concentrate (SPC) or SPC with isoflavones (SPC-IF). NIH-41 was the control for pairwise comparisons. Minimal differences were observed among natural ingredient diet groups. F0 males fed AIN-93, SPC, and SPC-IF diets had elevated glucose levels and lower insulin levels compared with the NIH-41 group. In both sexes of the F1 and F2 generations, the SPC and SPC-IF groups had lower body weight gains than the NIH-41 controls and the AIN-93 group had an increased percent body fat at postnatal day 21. AIN-93 F1 pups had higher baseline glucose than NIH-41 controls, but diet did not significantly affect breeding performance or responses to glucose or uterotrophic challenges. Reduced testes weight and sperm in the AIN-93 group may be related to low thiamine levels. Our observations underline the importance of careful selection, manufacturing procedures, and nutritional characterization of diets used in toxicological studies.

Consumption of soy infant formula represents a unique exposure scenario in which developing children ingest a mixture of endocrine-active isoflavones along with a substantial portion of daily nutrition. Genistein and daidzein were administered as glucoside conjugates to neonatal rhesus monkeys in a fortified commercial soy formula at 5, 35, and 70 days after birth. A single gavage dosing with 10 mg/kg bw genistein and 6 mg/kg bw daidzein was chosen to represent the upper range of typical daily consumption and to facilitate complete pharmacokinetic measurements for aglycone and total isoflavones and equol. Adult monkeys were also gavaged with the same formula solution at 2.8 and 1.6 mg/kg bw genistein and daidzein, respectively, and by IV injection with isoflavone aglycones (5.2 and 3.2 mg/kg bw, respectively) to determine absolute bioavailability. Significant differences in internal exposure were observed between neonatal and adult monkeys, with higher values for dose-adjusted AUC and Cmax of the active aglycone isoflavones in neonates. The magnitude and frequency of equol production by the gut microbiome were also significantly greater in adults. These findings are consistent with immaturity of metabolic and/or physiological systems in developing non-human primates that reduces total clearance of soy isoflavones from the body.

High levels of inorganic arsenic (iAs) exposure are associated with severe health effects. Less clear are effects of lower exposure levels on neurodevelopment. Relative to maternal intake, there is limited lactational transfer of arsenic in humans or rodents, yet there are few rodent studies which directly exposed preweaning animals. To more clearly determine iAs developmental neurotoxicity, 28 pregnant Sprague-Dawley rats were exposed to arsenate (AsV) via drinking water (0, 23.6, 47.7, 71.0 ppm) (n = 5–7/group) from gestational day (GD) 6 through GD 22 with targeted doses of 0, 2.33, 4.67, 7.00 mg/kg/day, respectively. Offspring were dosed by gavage daily with the same mg/kg AsV dose as intended for their dam from postnatal day (PND) 1 to 21. Gestational water intake was reduced at all AsV doses, but returned to control levels on lactational day (LD) 1 when control water was returned. Gestational body weight was reduced only at the highest dose on GD 22 and lactational body weight was unaffected. Food intake was unaffected. iAs exposure did not alter offspring body weight (PNDs 1–21) or age at fur development and bilateral ear opening. Incisor eruption, however, was significantly delayed in offspring of the 4.67 and 7.00 mg/kg groups. Further, all iAs groups were significantly delayed in bilateral eye opening. Righting reflex (PNDs 3–6) was unaffected, while slant board performance (PNDs 8–11) was significantly poorer at the highest dose. Brains of culled pups (PND 1) showed dose-dependent increases of iAs. There were no significant AsV-related effects on PND 21 brain regional concentrations of dopamine, DOPAC, HVA, 5-HT or 5-HIAA. These hazard identification results will guide the study designs of developmental iAs exposure at human-relevant levels essential for risk-assessment.

Bisphenol A (BPA) is a chemical used in the production of numerous consumer products resulting in potential daily human exposure to this chemical. The FDA previously evaluated the body of BPA toxicology data and determined that BPA is safe at current exposure levels. Although consistent with the assessment of some other regulatory agencies around the world, this determination of BPA safety continues to be debated in scientific and popular publications, resulting in conflicting messages to the public. Thus, the National Toxicology Program (NTP), National Institute of Environmental Health Sciences (NIEHS), and U.S. Food and Drug Administration (FDA) developed a consortium-based research program to link more effectively a variety of hypothesis-based research investigations and guideline-compliant safety testing with BPA. This collaboration is known as the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA). This paper provides a detailed description of the conduct of the study and a midterm update on progress of the CLARITY-BPA research program.

We report the data from the guideline-compliant two-year toxicology study conducted as part of the Consortium Linking Academic and Regulatory Insights on Bisphenol A Toxicity (CLARITY-BPA). BPA (0, 2.5, 25, 250, 2,500, and 25,000 μg/kg body weight (bw)/day) was administered daily by gavage in 0.3% carboxymethylcellulose vehicle to NCTR Sprague-Dawley rats from gestation day 6 through the start of parturition and then directly to pups from the day after birth until postnatal day 21 (stop-dose arm) or continuously until termination at one or two years. The stop-dose arm was included to assess the potential for any BPA effects that were due to developmental exposure. No BPA-related effects were evident in the in-life and non-histopathology data. Neoplastic and nonneoplastic lesions diagnosed in both females and males were common age-associated lesions that were variable across control and BPA-treated groups. The lack of consistent responses within the continuous- and stop-dose arms within and across tissues brought into question the plausible relationship of most of these lesions to BPA treatment. There was a possible relationship between the increased incidences of lesions in the female reproductive tract and the male pituitary and exposure to the 25,000 μg BPA/kg bw/day dose level.

Pharmaceutical products that are contaminated with Burkholderia cepacia complex (BCC) bacteria may pose serious consequences to vulnerable patients. Benzyldimethylalkylammonium chloride (BZK) cationic surfactants are extensively used in medical applications and have been implicated in the coselection of antimicrobial resistance. The ability of BCC to degrade BZK, tetradecyldimethylbenzylammonium chloride (C14BDMA-Cl), dodecyldimethylbenzylammonium chloride (C12BDMA-Cl), decyldimethylbenzylammonium chloride (C10BDMA-Cl), hexyldimethylbenzylammonium chloride, and benzyltrimethylammonium chloride was determined by incubation in 1/10-diluted tryptic soy broth (TSB) to determine if BCC bacteria have the ability to survive and inactivate these disinfectants. With BZK, C14BDMA-Cl, and C12BDMA-Cl, inhibition of the growth of 20 BCC strains was observed in disinfectant solutions that ranged from 64 to 256 µg/ml. The efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone increased the sensitivity of bacteria to 64 µg/ml BZK. The 20 BCC strains grew well in 1/10-diluted TSB medium with BZK, C12BDMA-Cl, and C10BDMA-Cl; they absorbed and degraded the compounds in 7 days. Formation of benzyldimethylamine and benzylmethylamine as the initial metabolites suggested that the cleavage of the C alkyl-N bond occurred as the first step of BZK degradation by BCC bacteria. Proteomic data confirmed the observed efflux activity and metabolic inactivation via biodegradation in terms of BZK resistance of BCC bacteria, which suggests that the two main resistance mechanisms are intrinsic and widespread., IMPORTANCE Benzyldimethylalkylammonium chloride is commonly used as an antiseptic in the United States. Several recent microbial outbreaks were linked to antiseptics that were found to contain strains of the Burkholderia cepacia complex. Burkholderia species survived in antiseptics, possibly because of the degradation of antiseptic molecules or regulation of relevant gene expression. In this study, we assessed the efflux pump and the potential of B. cepacia complex bacteria to degrade benzyldimethylalkylammonium chloride and improved our understanding of the resistance mechanisms, by using proteomic and metabolic information. To our knowledge, this is the first systematic report of the intrinsic mechanisms of B. cepacia complex strain resistance to benzyldimethylalkylammonium chloride, based on the metabolic and proteomic evidence for efflux pumps and the complete biodegradation of benzyldimethylalkylammonium chloride.

Effects of the dosing matrix and timing on the onset of renal crystal formation were evaluated in male and non-pregnant female rats (Fisher 344) exposed to both melamine (MEL) and cyanuric acid (CYA) for 28 days. Rats were fed ground feed containing 60 ppm MEL and 60 ppm CYA, (5 mg/kg bw/day equivalent), or exposed via oral gavage to carboxymethylcellulose containing 5 mg/kg bw MEL followed by 5 mg/kg bw CYA either consecutively (

Tricaine methanesulfonate, a sedative for temporarily immobilizing fish, has a 21-day withdrawal time. Benzocaine has been proposed as an alternative sedative because a withdrawal period may not be required. Since benzocaine is known to induce methemoglobinemia, the potential for orally administered benzocaine to induce methemoglobin was assessed in rats. Sprague-Dawley rats were given a single gavage administration of 64mg benzocaine hydrochloride per kg bw and then euthanized at intervals up to 120min. Plasma levels of benzocaine were relatively low at all times, whereas methemoglobin peaked at 24min. Additional rats were orally gavaged with 0-1024mg benzocaine hydrochloride per kg bw and euthanized after 24min. Plasma levels of benzocaine increased from 0.01μM at 2mg per kg bw to 2.9μM at 1024mg per kg bw. Methemoglobin levels did not differ from controls at doses up to 32mg per kg bw in females and 64mg per kg bw in males, whereupon the value increased to ∼80% at 1024mg per kg bw. These data were used to estimate the potential impact of benzocaine residues in fish and suggest that the consumption of fish treated with benzocaine hydrochloride will not cause methemoglobinemia in humans.

Antimicrobial residues found in municipal wastewater may increase selective pressure on microorganisms for development of resistance, but studies with mixed microbial cultures derived from wastewater have suggested that some bacteria are able to inactivate fluoroquinolones. Medium containing N -phenylpiperazine and inoculated with wastewater was used to enrich fluoroquinolone-modifying bacteria. One bacterial strain isolated from an enrichment culture was identified by 16S rRNA gene sequence analysis as a Microbacterium sp. similar to a plant growth-promoting bacterium, Microbacterium azadirachtae (99.70%), and a nematode pathogen, “ M. nematophilum ” (99.02%). During growth in medium with norfloxacin, this strain produced four metabolites, which were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) analyses as 8-hydroxynorfloxacin, 6-defluoro-6-hydroxynorfloxacin, desethylene norfloxacin, and N -acetylnorfloxacin. The production of the first three metabolites was enhanced by ascorbic acid and nitrate, but it was inhibited by phosphate, amino acids, mannitol, formate, and thiourea. In contrast, N -acetylnorfloxacin was most abundant in cultures supplemented with amino acids. This is the first report of defluorination and hydroxylation of a fluoroquinolone by an isolated bacterial strain. The results suggest that some bacteria may degrade fluoroquinolones in wastewater to metabolites with less antibacterial activity that could be subject to further degradation by other microorganisms.

Bisphenol A (BPA) is a high-production volume industrial chemical used in the manufacture of polycarbonate plastic products and epoxy resin-based food can liners. The presence of BPA in urine of >90% of Americans aged 6-60 is controversial because of the potential for endocrine disruption, particularly during perinatal development, as suggested by in vitro, experimental animal, and epidemiological studies. The current study used LC/MS/MS to measure serum pharmacokinetics of aglycone (active) and conjugated (inactive) BPA in adult and neonatal rhesus monkeys by oral (PND 5, 35, 70) and intravenous injection (PND 77) routes using d6-BPA to avoid sample contamination. The concentration-time profiles observed in adult monkeys following oral administration of 100 μg/kg bw were remarkably similar to those previously reported in human volunteers given a similar dose; moreover, minimal pharmacokinetic differences were observed between neonatal and adult monkeys for the receptor-active aglycone form of BPA. Circulating concentrations of BPA aglycone were quite low following oral administration (< 1% of total), which reflects the redundancy of active UDP-glucuronosyl transferase isoforms in both gut and liver. No age-related changes were seen in internal exposure metrics for aglycone BPA in monkeys, a result clearly different from developing rats where significant inverse age-related changes, based on immaturity of Phase II metabolism and renal excretion, were recently reported. These observations imply that any toxicological effect observed in rats from early postnatal exposures to BPA could over-predict those possible in primates of the same age, based on significantly higher internal exposures and overall immaturity at birth.

Soy-based infant formulas are widely used in the United States and some other countries. These formulas contain high levels of the estrogenic isoflavone genistein, leading to concern that neonatal genistein exposure could cause acute and/or long-term adverse effects on reproductive and other organs. However, previous work to assess genistein effects in rodent models has not typically replicated the route of delivery and/or serum genistein concentrations reported for soy formula-fed human infants. Our objective was to develop a mouse model that more closely mimics the oral genistein exposure and total serum genistein concentrations observed in soy formula-fed infants. Mouse pups were dosed orally with genistein in a soy formula-corn oil emulsion from Postnatal Day (PND) 1 to PND 5, then effects on reproductive and non-reproductive organs were assessed after dosing and during subsequent development. Neonatal treatment resulted in changes both at the completion of dosing (PND 5) and in adult animals. At PND 5, neonatal genistein treatment caused increased relative uterine weight and down-regulation of progesterone receptor in uterine epithelia. Estrogenic effects of genistein were also seen in the neonatal ovary and thymus, which had an increase in the incidence of multioocyte follicles (MOFs) and a decrease in thymic weight relative to body weight, respectively. The increased incidence of MOFs persisted into adulthood for neonatally treated genistein females, and estrous cycle abnormalities were seen at 6 mo of age despite normal fertility in these mice. The immediate and long-term effects in this neonatal animal model raise concerns that high serum concentrations of genistein are estrogenic and could potentially impact the development of human infants fed soy formula.

Genistein is an estrogenic soy isoflavone widely promoted for healthy aging, but its effects on cognitive function are not well-understood. We examined the cognitive effects of once daily oral genistein treatment at two doses (approximately 162 μg/kg/day low dose and a 323 μg/kg/day high dose) in ovariectomized young (7 month), middle-aged (16 month), and old (22 month) Long–Evans rats. Operant tasks including delayed spatial alternation (DSA), differential reinforcement of low rates of responding (DRL), and reversal learning that tap prefrontal cortical function were used to assess working memory, inhibitory control/timing, and strategy shifting, respectively. At the conclusion of cognitive testing, brains were collected and relative densities of D1 and D2 dopamine receptors and dopamine transporter (DAT) were measured in the prefrontal cortex. On the DSA task, the high dose old group performed worse than both the high dose young and middle-aged groups. On the DRL task, the high dose of genistein resulted in a marginally significant impairment in the ratio of reinforced to non-reinforced lever presses. This effect was present across age groups. Age effects were also found as old rats performed more poorly than the young and middle-aged rats on the DSA overall. In contrast, middle-aged and old rats made fewer lever presses on the DRL than did the young rats, a pattern of behavior associated with better performance on this task. Moreover, while DAT levels overall decreased with age, genistein treatment produced an increase in DAT expression in old rats relative to similarly aged control rats. D1 and D2 densities did not differ between genistein dose groups or by age. These results highlight the fact that aspects of executive function are differentially sensitive to both genistein exposure and aging and suggest that altered prefrontal dopamine function could potentially play a role in mediating these effects.

Background Developmental exposure to environmental estrogens is associated with adverse consequences later in life. Exposure to genistin (GIN), the glycosylated form of the phytoestrogen genistein (GEN) found in soy products, is of concern because approximately 20% of U.S. infants are fed soy formula. High circulating levels of GEN have been measured in the serum of these infants, indicating that GIN is readily absorbed, hydrolyzed, and circulated. Objectives We investigated whether orally administered GIN is estrogenic in neonatal mice and whether it causes adverse effects on the developing female reproductive tract. Methods Female CD-1 mice were treated on postnatal days 1–5 with oral GIN (6.25, 12.5, 25, or 37.5 mg/kg/day; GEN-equivalent doses), oral GEN (25, 37.5, or 75 mg/kg/day), or subcutaneous GEN (12.5, 20, or 25 mg/kg/day). Estrogenic activity was measured on day 5 by determining uterine wet weight gain and induction of the estrogen-responsive gene lactoferrin. Vaginal opening, estrous cyclicity, fertility, and morphologic alterations in the ovary/reproductive tract were examined. Results Oral GIN elicited an estrogenic response in the neonatal uterus, whereas the response to oral GEN was much weaker. Oral GIN altered ovarian differentiation (i.e., multioocyte follicles), delayed vaginal opening, caused abnormal estrous cycles, decreased fertility, and delayed parturition. Conclusions Our results support the idea that the dose of the physiologically active compound reaching the target tissue, rather than the administered dose or route, is most important in modeling chemical exposures. This is particularly true with young animals in which phase II metabolism capacity is underdeveloped relative to adults.

Genistein (GEN), a soy isoflavone, stimulates growth of estrogen-dependent human tumor cells (MCF-7) in a preclinical mouse model for postmenopausal breast cancer. Antiestrogens and aromatase inhibitors are frontline therapies for estrogen-dependent breast cancer. We have demonstrated that dietary GEN can negate the inhibitory effect of tamoxifen. In this study, we evaluated the interaction of dietary GEN (at 250-1000 p.p.m. in the American Institute of Nutrition 93 growth diet) and an aromatase inhibitor, letrozole (LET), on the growth of tumors in an aromatase-expressing breast cancer xenograft model (MCF-7Ca) in the presence and absence of the substrate androstenedione (AD). Dietary GEN (250 and 500 p.p.m.) or implanted AD stimulated MCF-7Ca tumor growth. Implanted LET inhibited AD-stimulated MCF-7Ca tumor growth. In the presence of AD and LET, dietary GEN (250, 500 and 1000 p.p.m.) reversed the inhibitory effect of LET in a dose-dependent manner. Uterine wet weight, plasma estradiol (E(2)) levels (enzyme-linked immunosorbent assay) and total plasma GEN and LET levels (liquid chromatography-electrospray/tandem mass spectrometry) were measured. Ki-67 (cellular proliferation), aromatase and pS2 protein expression in tumors were evaluated using immunohistochemical (IHC) analysis. In conclusion, dietary GEN increased the growth of MCF-7Ca tumors implanted in ovariectomized mice and could also negate the inhibitory effect of LET on MCF-7Ca tumor growth. These findings are significant because tumors, which express aromatase and synthesize estrogen, are good candidates for aromatase therapy dietary and GEN can reverse the inhibitory effect of LET on tumor growth and adversely impact breast cancer therapy. Caution is warranted for consumption of dietary GEN by postmenopausal women with estrogen-dependent breast cancer taking LET treatment.

Many different organic compounds may be converted by microbial biotransformation to high-value products for the chemical and pharmaceutical industries. This review summarizes the use of strains of Aspergillus niger, a well-known filamentous fungus used in numerous biotechnological processes, for biochemical transformations of organic compounds. The substrates transformed include monocyclic, bicyclic, and polycyclic aromatic hydrocarbons; azaarenes, epoxides, chlorinated hydrocarbons, and other aliphatic and aromatic compounds. The types of reactions performed by A. niger, although not unique to this species, are extremely diverse. They include hydroxylation, oxidation of various functional groups, reduction of double bonds, demethylation, sulfation, epoxide hydrolysis, dechlorination, ring cleavage, and conjugation. Some of the products may be useful as new investigational drugs or chemical intermediates.

Bisphenol A (BPA) is a high production volume industrial chemical to which there is widespread human oral exposure. Guideline studies used to set regulatory limits detected adverse effects only at doses well above human exposures and established a no-observed-adverse-effect level (NOAEL) of 5 mg/kg body weight (bw)/day. However, many reported animal studies link BPA to potentially adverse effects on multiple organ systems at doses below the NOAEL. The primary goals of the subchronic study reported here were to identify adverse effects induced by orally (gavage) administered BPA below the NOAEL, to characterize the dose response for such effects and to determine doses for a subsequent chronic study. Sprague Dawley rat dams were dosed daily from gestation day 6 until the start of labor, and their pups were directly dosed from day 1 after birth to termination. The primary focus was on seven equally spaced BPA doses (2.5–2700 μg/kg bw/day). Also included were a naïve control, two doses of ethinyl estradiol (EE2) to demonstrate the estrogen responsiveness of the animal model, and two high BPA doses (100,000 and 300,000 μg/kg bw/day) expected from guideline studies to produce adverse effects. Clear adverse effects of BPA, including depressed gestational and postnatal body weight gain, effects on the ovary (increased cystic follicles, depleted corpora lutea, and antral follicles), and serum hormones (increased serum estradiol and prolactin and decreased progesterone), were observed only at the two high doses of BPA. BPA-induced effects partially overlapped those induced by EE2, consistent with the known weak estrogenic activity of BPA.

Oxidant damage from reactive oxygen species (ROS) and reactive nitrogen species (RNS) is a major contributor to the cellular damage seen in numerous types of renal injury. Resveratrol (trans-3,4',5-trihydroxystilbene) is a phytoalexin found naturally in many common food sources. The anti-oxidant properties of resveratrol are of particular interest because of the fundamental role that oxidant damage plays in numerous forms of kidney injury. To examine whether resveratrol could block damage to the renal epithelial cell line, mIMCD-3, cells were exposed to the peroxynitrite donor 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride (SIN-1). Resveratrol produced a concentration-dependent inhibition of cytotoxicity induced by SIN-1. To examine the mechanism of protection, resveratrol was incubated with authentic peroxynitrite and found to block nitration of bovine serum albumin with an EC(50) value of 22.7 microM, in contrast to the known RNS scavenger, N-acetyl-l-cysteine, which inhibited nitration with an EC(50) value of 439 microM. These data suggested that resveratrol could provide functional protection by directly scavenging peroxynitrite. To examine whether resveratrol was a substrate for peroxynitrite oxidation, resveratrol was reacted with authentic peroxynitrite. Resveratrol nitration products and dimers were detected using liquid chromatograph with tandem electrospray mass spectrometry. Similar products were detected in the media of cells treated with SIN-1 and resveratrol. Taken collectively, the data suggest that resveratrol is able to provide functional protection of renal tubular cells, at least in part, by directly scavenging the RNS peroxynitrite. This property of resveratrol may contribute to the understanding of its anti-oxidant activities.

2-acetylbenzothiophene-S-oxide (2-ABT-S-oxide or M1) is a reactive metabolite of zileuton, a drug used in the treatment of asthma and is capable of conjugating with glutathione in vitro. Human serum albumin (HSA) is the most abundant protein in plasma and plays a critical role in detoxifying reactive oxygen species. The current research is focused on understanding the interaction between M1 and HSA. The stability studies revealed the half-life of M1 to be about 0.85 h in HSA, 1.82 h in human plasma, and 4.48 h in phosphate-buffered saline (PBS) as determined by first-order approximation. The alkylation rate constant k for HSA was 20 M(-1) min(-1). After quenching with acetonitrile, the half-life of M1 did not change significantly, indicating that M1 is covalently bound to HSA. LC-MS and LC-MS/MS analysis of human plasma revealed the M1 alkylated peptide P (m/z 870) formed by HSA conjugation and concomitant water elimination. The specific amino acid on HSA bound to M1 was identified as Cys-34. This alkylation is observed to be concentration- and incubation-time-dependent in human plasma. HSA oxidized by N, N'-diacetyl-L-cystine exhibits a compromised ability of HSA to react with M1. The alkylated HSA diminished the binding affinity for warfarin. Furthermore, the alkylation was found to be irreversible in the dialysis experiment. In addition, M1 decomposes to 2-ABT in the presence of HSA, presumably acting as an oxidant. The formation of 2-ABT in the incubation and the self-condensation of M1 in PBS indicate that the alkylation of Cys-34 is only one of a number of reactions that occur in the presence of HSA. Irreversible protein modification may potentially lead to a loss of its function. HSA irreversible alkylation represents a model for other proteins to be potentially toxic and thus may help explain zileuton hepatotoxicity.

A series of phosphorylated test peptides was studied by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS). The extensive ECD-induced fragmentation made identification of phosphorylation sites for these peptides straightforward. The site(s) of initial phosphorylation of a synthetic peptide with a sequence identical to that of the phosphorylation site domain (PSD) of the myristoylated alanine-rich C kinase (MARCKS) protein was then determined. Despite success in analyzing fragmentation of the smaller test peptides, a unique site on the PSD for the first step of phosphorylation could not be identified because the phosphorylation reaction produced a heterogeneous mixture of products. Some molecules were phosphorylated on the serine closest to the N-terminus, and others on one of the two serines closest to the C-terminus of the peptide. Although no definitive evidence for phosphorylation on either of the remaining two serines in the PSD was found, modification there could not be ruled out by the ECD fragmentation data.