In this issue tabolites of the arsenosugar were dimethylarsonate (DMA, 40-46%), thio-dimethylarsenoacetate (1519%), thio-dimethylarsenoethanol (5-9%), and an unknown thio-metabolite (38%). In contrast, among the low excretors, the only arsenic-containing compounds in the urine were the unmetabolized arsenosugar, thio-arsenosugar, and DMA. Analysis of plasma samplesobtainedfromthree of the volunteers revealed similar patterns of metabolites as those found in urine.
Seafood Arsenic Risk The toxicity of inorganic arsenic is well recognized, and human exposure, primarily throughcontaminateddrinking water, is a significant health concern. Less clear, however, is the health risk of exposure to arsenic in the form of organic arsenicals in seafood.Amongthesecompounds are the arsenosugars, which are formed in algae from naturally occurring arsenic in seawater and accumulate in mollusks and shellfish that feed on algae. Previous attempts to determine the fate of seafoodborne arsenosugars in humans have led to conflicting results, leading Raml et al. (p 1534) to study the metabolism of a pure synthetic arsenosugar in six human volunteers. Urine samples collected for 4 days following administration of the arsenosugar were analyzed by HLPC coupled with arsenic-selective inductively coupled plasma mass spectrometry. The results revealed a striking dichotomy in urinary excretion of arsenic, in that four of the subjects excreted 85-95% of the ingested dose within 90 h, while the other two excreted only about 4 and 15% of the dose.
Mass spectral analysis of the unknown metabolite revealed both thio- and oxospecies, with the thio-species predominating. The data supported the presence of a Me2As(O) or Me2As(S) group coupled to a dihydroxybutyryl moiety; however, the exact structure of that moiety was not determined.
A repeat experiment 6 months later with one “high excretor” and one “low excretor” produced identical results, indicating that the distinct patterns of excretion reflect intrinsic differences in absorption/metabolism/excretion of the compound. Among the high excretors, the primary urinary me-
The finding of strikingly different patterns of arsenic excretion is consistent with prior findings, suggesting wide variations in the metabolism of food-borne arsenosugars. Raml et al. argue that the low excretor pattern could reflect a failure of gut absorption of the arsenosugar or a failure of metabolism to a form that is excreted in the urine. The basis for the difference remains to be determined;
Published online 09/21/2009 • DOI: 10.1021/tx900244f © 2009 American Chemical Society
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however, Raml et al. note that the identification of dimethylarsinate as the primary arsenosugar metabolite is of toxicologic significance since DMA is the primary toxic metabolite of inorganic arsenic.
Imines and Autoimmunity Idiosyncratic drug reactions (IDRs) are a significant problem plaguing clinicians and the pharmaceutical industry. Although IDRs affect relatively few patients, their unpredictability and uncertain mechanism make them difficult to prevent and treat. The accumulated clinical and experimental data suggest an immune mechanism for most IDRs. This is supported by studies of penicillamine-induced autoimmunity in Brown Norway (BN) rats, an animal model of IDRs. These studies suggest an important role for activated macrophages in the early stages of the IDR. Recent evidence also indicates that a key mechanism by which macrophages interact with T-lympocytes is the formation of an imine bond between aldehyde-containing proteins
on the macrophage membrane and amino groups on the surface of the lymphocyte. These findings led Li and Uetrecht (p 1526) to propose that formation of an imine between penicillamine and macrophage surface aldehydes plays a role in macrophage activation in penicillamine-induced autoimmunity.
Transcriptomic analysis of macrophages isolated from the spleens of BN rats 6 h after penicillamine administration revealed upregulation and downregulation of 324 and 273 genes, respectively. Of these, changes in 20 pro-inflammatory genes correlated with macrophage activation. mRNA profiling of NK cells isolated from the rats provided evidence of NK cell activation, which is likely a downstream effect of macrophage activation and release of cytokines such as IL-15. Incubation of RAW264.7 murine macrophage-like cells with a biotinylated aldehyde-reactive probe (N(aminooxyacetyl)-N′-(D-bi-
Special Features Oxidative stress leading to lipid peroxidation generates a number of electrophilic R,β-unsaturated aldehydes, including acrolein and 4-hydroxynonenal (HNE). Increasing evidence suggests that the reaction of these species with cellular nucleophiles is key to their toxicity. Now, Editoral Advisory Board member Dennis Petersen and his colleagues [LoPachin et al. (p 1499)] review what is known about the reaction of acrolein and HNE with nucleophiles in proteins and provide an argument for the importance of reactive thiolates as the most likely site of adduction that leads to protein dysfunction. Vol. 22,
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In this issue otinoyl) hydrazine) resulted in a concentration-dependent labeling of the cells, confirming the presence of reactive aldehydes on the plasma membrane. Treatment of RAW264.7 cells with penicillamine induced timeandconcentration-dependent secretion of tumor necrosis factor-R, interleukin(IL)-6, and IL-23. The aldehyde-reactive compounds hydralazine and isoniazid also induced secretion of IL-6 by RAW264.7 cells. Both of these compounds trigger autoimmune reactions in humans. Together, the findings confirmed the ability of aldehyde-reactive compounds to activate macrophages, supporting the hypothesis that this chemical interaction may play a role in the aberrant immune response induced by penicillamine. Butadiene Metabolic Trap Butadiene (BD) is an ubiquitous compound produced during incomplete combustion of organic materials, including tobacco, fossil fuels, and wood. It is also used industrially in the production of synthetic rubber and thermoplastic resins. BD has been classified as a class I human carcinogen by the International Agency for Research on Cancer. Its toxicity is the result of cytochrome P450dependent oxidation to reactive epoxides. The epoxides are subject to detoxification by glutathione conjugation or hydrolysis by epoxide hydrolases (EHs). The sequential actions of P450 and EH enzymes result in the formation of multiple metabolites, including ep-
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oxybutene (EB), diepoxybutane (DEB), butene-diol (BDdiol), epoxybutane-diol (EBdiol), and butane-tetraol (BDtetraol). DEB is the most toxic of the metabolites. Interspecies variability in BD toxicity has been attributed to differences in the relative rates of the activation and detoxication reactions.
Comparative studies of BD metabolism are hindered by the instability of the epoxides. Now, Motwani et al. (p 1509) demonstrate the use of the reduced form of vitamin B12, cob(I)alamin, as an epoxide trapping agent to investigate the kinetics of BD metabolism. The supernucleophile cob(I)alamin reacts ∼105 times faster than conventional nucleophiles and almost instantaneously traps the epoxides as alkylcob(I)alamins.
Motwani et al. first used the cob(I)alamin trapping method with identification of the alkylcob(I)alamins by LC-MS/MS to show that the rates of nonenzymatic hydrolysis of EB, DEB, and EBdiol (t1/2 ) 20, 33, and 82 h, respectively) were slow as compared to the rates of enzymatic formation and degradation. Next, they incubated EB, DEB, and BDdiol with rat S9 fraction and used cob(I)alamin to trap all epoxide-containing substrates and products. This resulted in the formation of al-
CHEMICAL RESEARCH IN TOXICOLOGY
kylcobalamins, which were subsequently analyzed by LC-MS/MS. Kinetic analysis provided estimates of the Michaelis-Menten constants for the key enzymatic conversions. The data revealed that the efficiency of conversion of EB to DEB was approximately equal to that of hydrolysis of DEB to EBdiol. The efficiency of oxidation of EB to DEB was lower than that of its hydrolysis to BDdiol. In addition, EBdiol was more efficiently formed by hydrolysis of DEB than by oxidation of BDdiol. Together, the data form the foundation for understanding BD metabolism in the rat. Similar studies using liver S9 fractions from other species should help to explain species variations in BD carcinogenicity. Inhibition of Angiogenesis Angiogenesis plays a central role in numerous pathophysiological processes such as wound healing, tumorigenesis, inflammation, and atherosclerosis. Evidence suggests a role for Cu2+ in angiogenesis, and Cu2+ chelators have been shown to inhibit the process. Because some Cu2+ chelators exhibit different biological effects when administered as the Cu2+ complex than when administered alone, Fan et al. (p 1517) investigated the effects of a series of nine salicylaldehyde pyrazole hydrazones (SPHs) and their Cu2+ complexes (Cu-SPHs) on cultured human umbilical vein endothelial cells (HUVECs). The SPHs and Cu-SPHs inhibited the growth of HUVECs, and in most cases,
the Cu-SPHs exhibited greater potency. Six of the nine Cu-SPHs, but not their corresponding SPHs, triggered apoptosis in HUVECs as determined by Hoechst staining. One compound, 15, was chosen for detailed study.
The ability of Cu-15 to trigger apoptosis was verified in a PARP cleavage assay. Interestingly, Cu-15, or a combination of 15 and Cu2+ added separately, inhibited growth and induced apoptosis in HUVECs, while 15 or Cu2+ added alone did not. These results suggested that the Cu-15 complex could be formed in the culture medium. Cu-15 complexes, but not 15 or Cu2+ alone, inhibited the ability of HUVECs to form microvascular structures. At higher concentrations, Cu-15 also inhibited HUVEC migration. Because prior evidence suggested a role for integrin β4 in triggering HUVEC apoptosis, Fan et al. studied the effects of Cu-15 complexes on integrin β4 levels. Cu-15 but not 15 or Cu2+ alone induced increased integrin β4 levels in HUVECs, and RNAi knockdown of integrin β4 inhibited the Cu-15-mediated apoptotic response. The results suggest that Cu-15 may be a valuable antiangiogenic compound and that its mechanism of action is, at least in part, mediated by integrin β4. TX900244F
Published online 09/21/2009 •
DOI: 10.1021/tx900244f $40.75 © 2009 American Chemical Society
