Frederick W. Benz

Metallothionein Disulfides Are Present in Metallothionein-overexpressing Transgenic Mouse Heart and Increase under Conditions of Oxidative Stress

Metallothionein (MT) releases zinc under oxidative stress conditions in cultured cells. The change in the MT molecule after zinc release in vivo is unknown although in vitro studies have identified MT disulfide bond formation. The present study was undertaken to test the hypothesis that MT disulfide bond formation occurs in vivo. A cardiac-specific MT-overexpressing transgenic mouse model was used. Mice were administered saline as a control or doxorubicin (20 mg/kg), which is an effective anticancer drug but with severe cardiac toxicity at least partially because of the generation of reactive oxygen species. A differential alkylation of cysteine residues in MT of the heart extracts was performed. Free and metal-bound cysteines were first trapped by N-ethylmaleimide and the disulfide bonds were reduced by dithiothreitol followed by alkylation with radiolabeled iodoacetamide. Analyses of the differentially alkylated MTs in the heart extract by high preformance liquid chromatography, SDS-PAGE, Western blot, and mass spectrometry revealed that disulfide bonds were present in MT in vivo under both physiological and oxidative stress conditions. More disulfide bonds were found in MT under the oxidative stress conditions. The MT disulfide bonds were likely intramolecular and both α- and β-domains were involved in the disulfide bond formation, although the α-domain appeared to be more easily oxidized than the β-domain. The results suggest that under physiological conditions, the formation of MT disulfide bonds is involved in the regulation of zinc homeostasis. Additional zinc release from MT under oxidative stress conditions is accompanied by more MT disulfide bond formation.

Acute acrylonitrile toxicity: studies on the mechanism of the antidotal effect of D- and L-cysteine and their N-acetyl derivatives in the rat.

Thiol-containing antidotes for acute acrylonitrile (AN) toxicity may exert their action by chemically reacting with AN, by replacing critical sulfhydryl groups cyanoethylated by AN, and by detoxifying cyanide produced from AN metabolism. We have evaluated the ability of the optical isomers of cysteine and N-acetylcysteine to act as antidotes against AN toxicity in order to assess the relative importance of each of these three antidotal mechanisms. The toxicity of AN was determined in male Sprague-Dawley rats and compared to the toxicity determined after treatment with 2 mmol/kg of thiol antidote by computing a protective index (median lethal dose with antidote/median lethal dose without antidote). The protective indices of L-cysteine, D-cysteine, N-acetyl-L-cysteine, and N-acetyl-D-cysteine were 2.03, 1.97, 1.76, and 1.25, respectively. Measurements of urinary mercapturates, derived from the non-oxidative pathway of AN metabolism, indicated that none of the antidotes was able to significantly increase the excretion of these metabolites. Blood cyanide generated from the oxidative metabolism of AN and butyronitrile was also determined. All of the antidotes, except N-acetyl-D-cysteine, lowered blood cyanide levels. A comparison of these results with the predicted relative abilities of the enantiomers to participate in each of the three antidotal mechanisms leads to the conclusion that, under these experimental conditions, the best correlation exists with the cyanide detoxification mechanism.

Acrylonitrile irreversibly inactivates glyceraldehyde-3-phosphate dehydrogenase by alkylating the catalytically active cysteine 149.

Acrylonitrile (AN) is a vinyl monomer used in the production of synthetic fibers, rubber and plastics. AN is acutely toxic but its mechanism of toxicity remains to be established. AN is metabolized to cyanide in vivo but cyanide production alone cannot explain acute AN toxicity. Previous work in our laboratory has shown that AN can alkylate highly reactive cysteine residues in proteins. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a critical enzyme involved in glycolysis, has a catalytically active cysteine 149 in its active site. We report that AN irreversibly inhibits GAPDH with second-order rate constants, at pH 7.4, of 3.7 and 9.2 M(-1) s(-1) at 25 and 37 degrees C, respectively. A combination of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) and electrospray ionization-mass spectrometry-mass spectrometry (ESI-MS-MS) was used to show that AN inactivates GAPDH by covalently binding to cysteine 149 in the active site of the enzyme. Inactivation of GAPDH by AN would be expected to impair glycolytic ATP production and when coupled with the inhibition of mitochondrial ATP synthesis by the AN metabolite cyanide would result in metabolic arrest. The brain can withstand metabolic arrest for only a few minutes thus these combined actions may account for the acute toxicity of AN in vivo.

Structural Insights into the Pharmacophore of Vinca Domain Inhibitors of Microtubules.

Antibody-drug conjugates (ADCs) have achieved great success in cancer therapy in recent years. Some peptidyl microtubule inhibitors consisting of natural and unnatural amino acids, such as monomethyl auristatin E (MMAE) and F (MMAF), are extremely cytotoxic and have been used as a payload in ADCs. However, their precise molecular interaction with tubulin and microtubules remains unclear. We determined the crystal structures of tubulin in complex with three ultra-potent peptidyl microtubule inhibitors [MMAE, taltobulin (HTI- 286), and tubulysin M] at 2.5 Å. Our data showed that the three peptides bound to the vinca domain and shared a common and key pharmacophore containing two consecutive hydrophobic groups (Val, Ile-like side chain). These groups protruded in opposite directions into hydrophobic pockets on the tubulin β and α subunits. Nitrogen and oxygen atoms from the same backbone formed hydrogen bonds with Asn329 from the α subunit and Asp179 from the β subunit in a direction normal to the surface formed by the aforementioned hydrophobic groups. In addition, our crystal structure data indicated that tubulysin M bound to the β subunit alone, providing a structural explanation for its higher affinity. We also compared the conformations of two representative structurally different vinca domain compounds, ustiloxin D and vinblastine, with those of the aforementioned peptidyl ligands, and found that they shared a similar pharmacophore. Our findings lay a foundation for the rational design of novel vinca domain ligands and may facilitate the development of microtubule inhibitors with high specificity, affinity, and efficiency as payloads for ADCs in cancer therapy.

Regular ArticleDose Dependence of Covalent Binding of Acrylonitrile to Tissue Protein and Globin in Rats

The dose dependence of acrylonitrile (AN) covalent binding to tissue protein, following a single acute exposure over a 100-fold range in dose, was measured. Covalent binding was a linear function of AN dose in the lower dose range (0.02–0.95 mmol AN/kg). The slopes of the dose–response curves indicated that tissues varied by nearly 10-fold in their reactivity with AN. The relative order of covalent binding was as follows: blood ⪢ kidney = liver > forestomach = brain > glandular stomach ⪢ muscle. Similar dose–response behavior was observed for globin total covalent binding and for globinN-(2-cyanoethyl)valine (CEValine) adduct formation. The latter adduct was found to represent only 0.2% of the total AN adduction to globin. Regression of tissue protein binding versus globin total covalent binding or globin CEValine adduct indicated that both globin biomarkers could be used as surrogates to estimate the amount of AN bound to tissue protein. At higher AN doses, above approximately 1 mmol/kg, a sharp break in the covalent binding dose–response curve was observed. This knot value is explained by the nearly complete depletion of liver glutathione and the resultant termination of AN detoxification. The toxicity of AN is known to increase sharply above this dose. The data suggest that a comparison of specific tissue proteins labeled by AN above and below this threshold dose may provide some insight into the mechanism of AN-induced toxicity.

The acute lethality of acrylonitrile is not due to brain metabolic arrest.

Acrylonitrile (AN) is an organic compound produced in large quantities by the chemical industry and is acutely toxic. One mechanism proposed to explain the toxicity of AN is metabolism by P450 into cyanide (CN). Although blood and brain levels of CN in rats following an LD90 dose of AN are consistent with acute toxicity, blocking CN formation with P450 inhibitors does not prevent lethality. Another mechanism implicated in toxicity is covalent binding of AN to cysteine residues in tissue proteins. Previous work in our laboratory has shown that AN can irreversibly inactivate the catalytically active cysteine-149 in glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Inactivation of GAPDH by AN would be expected to impair glycolytic ATP production and, when coupled to the inhibition of mitochondrial ATP synthesis by the AN metabolite CN, would result in metabolic arrest, particularly in brain. In this study we have measured the high energy metabolites phosphocreatine (PCr), ATP, ADP and AMP by HPLC and compared their levels in the brains of rats treated with an LD90 dose of AN, when respiration ceased, vs. controls. Two methods of rapid brain freezing in liquid nitrogen were used: funnel freezing (FF) and head immersion (HI). AN administration resulted in large decreases in PCr of 74% (FF) and 80% (HI) but relatively minor decreases in ATP of 5% (FF) and 21% (HI) and Energy Charge of 6% (FF) and 10% (HI). Thus, although substantial depletion of PCr was observed, possibly due to inhibition of creatine kinase by AN, we found no evidence that brain ATP is depleted when respiration ceases in AN-intoxicated rats.