Free radical production from the interaction of 2-chloroethyl vesicants (mustard gas) with pyridine nucleotide-driven flavoprotein electron transport systems.

The biochemical sequelae to chloroethyl mustard exposure correspond very well to toxic processes initiated by free radicals. Additionally, mustard solutions contain spontaneously formed cyclic onium ions which produce carbon free radicals when reduced electrochemically. Therefore, we hypothesized that the onium ions of sulfur or nitrogen mustards might produce carbon free radicals upon being reduced enzymatically, and that these radicals might constitute a metabolic activation. We set out to document radical production using an in vitro metabolic system and electron paramagnetic resonance (EPR). Our system consisted of NADPH, one of several pyridine nucleotide-driven flavoprotein reductases, cytochrome c as a terminal electron acceptor, various sulfur or nitrogen mustards and the spin trap alpha-[4-pyridyl-1-oxide]-N-tert-butylnitrone in buffer. Reactions were started by adding the reductase to the other materials, vortexing and immediately transferring the mixture to a 10 mm EPR flat cell. Repeated scans on a Bruker ESP 300E EPR spectrometer produced a triplet of doublets with hyperfine splitting constants of a(N)=15.483 G and a(H)=2.512 G. The outcome supported our hypothesis that carbon-centered free radicals are produced when mustard-related onium ions are enzymatically reduced. The EPR results varied little with the chloroethyl compound used or with porcine or human cytochrome P450 reductase, the reductase domain of rat brain neuronal nitric oxide synthase or rat liver thioredoxin reductase. Our results offer new insight into the basis for mustard-induced vesication and the outcome of exposure to different mustards. The free radical model provides an explanation for similarities in the lesions arising from mustard exposure and energy-based lesions such as those from heat, ultraviolet and nuclear radiation as well as damage across tissue types such as skin, eyes or airway epithelium.

A conceptual framework for predicting the toxicity of reactive chemicals: modeling soft electrophilicity.

Although the literature is replete with QSAR models developed for many toxic effects caused by reversible chemical interactions, the development of QSARs for the toxic effects of reactive chemicals lacks a consistent approach. While limitations exit, an appropriate starting-point for modeling reactive toxicity is the applicability of the general rules of organic chemical reactions and the association of these reactions to cellular targets of importance in toxicology. The identification of plausible "molecular initiating events" based on covalent reactions with nucleophiles in proteins and DNA provides the unifying concept for a framework for reactive toxicity. This paper outlines the proposed framework for reactive toxicity. Empirical measures of the chemical reactivity of xenobiotics with a model nucleophile (thiol) are used to simulate the relative rates at which a reactive chemical is likely to bind irreversibly to cellular targets. These measures of intrinsic reactivity serve as correlates to a variety of toxic effects; what's more they appear to be more appropriate endpoints for QSAR modeling than the toxicity endpoints themselves.

Accelerated cytotoxicity mechanism screening using drug metabolising enzyme modulators.

By assessing how drug/new chemical entity (NCE) cytotoxicity is affected when their metabolic pathways are inhibited or activated, the metabolic pathways that activate versus detoxify drugs/NCEs can be identified. Reactive metabolites contributing to cytotoxicity can also be identified. In the following, the drug metabolizing enzyme inhibitors and activators used in vitro with freshly isolated rat hepatocytes for the accelerated cytotoxicity mechanism screening (ACMS) of drugs/NCEs (a technique used in our laboratory) are reviewed and, this technique is useful for determining in vivo rat hepatotoxicity mechanisms. The enzyme inhibitors/activators have been chosen on the basis of their selectivity, modulator effectiveness, and their lack of toxicity. The use of these inhibitors/activators with human hepatocytes or subcellular fractions for assessing human hepatotoxicity mechanisms is also reviewed.

Hydrogen peroxide supports hepatocyte P450 catalysed xenobiotic/drug metabolic activation to form cytotoxic reactive intermediates.

1. A H2O2 generating system markedly increased the cytotoxicity of catechols, hydroquinone, in isolated hepatocytes, but not in P450 inhibited hepatocytes. 2. H2O2 or NADPH supported microsomal catalysed GSH conjugate formation with catechols or hydroquinone. Cytochrome P450 inhibitors inhibited conjugate formation. However, superoxide dismutase inhibited NADPH, but did not affect H2O2 supported GSH conjugate formation. The conjugate formed with dihydrocaffeic acid was identified as a mono-GSH conjugate indicating that the o-quinone was the major metabolite formed. 3. Dopamine (a catecholamine) induced cytotoxicity was prevented by inhibitors of monoamine oxidase (MAO) or P450, but was markedly increased by hepatocyte catalase inhibition or NAD(P)H:quinone oxidoreductase inhibition. This suggests that H2O2 formed by the mitochondrial metabolism of monoamine oxidase then oxidised dopamine to cytotoxic o-quinone catalysed by P450. Dihydrocaffeic acid cytotoxicity was also increased by the monoamine oxidase substrate tyramine. 4. It is concluded that polyphenolics are oxidised by H2O2/P450 in hepatocytes to form quinone metabolites.

Superoxide radical scavenging and attenuation of hypoxia-reoxygenation injury by neurotransmitter ferric complexes in isolated rat hepatocytes.

Reactive oxygen species have been implicated in the pathogenesis of hypoxia-reoxygenation injury. Previously, we demonstrated that 2:1 catecholic iron complexes were more effective than uncomplexed catechols at (a) scavenging superoxide radicals generated enzymatically, and (b) protecting hepatocytes against hypoxia-reoxygenation injury [25]. Based on these findings, we sought to demonstrate similar effects using catecholamine neurotransmitters. Various catecholamine-iron complexes were shown to be more effective than uncomplexed catecholamines at scavenging superoxide radicals and could be used to protect cells from hypoxia-reoxygenation injury. alpha-Methyl-3, 4-dihydroxyphenylalanine (alpha-methylDOPA) complexed with ferric ion (2:1) showed the greatest superoxide scavenging potency amongst the catecholamine-iron complexes. The uncomplexed catecholamines were much less effective at scavenging superoxide radicals than the iron-catecholamine complexes. Dopamine was the most effective superoxide scavenger among the uncomplexed catecholamines. The superoxide scavenging effectiveness of the latter seemed to correlate with their reduction potentials, but not directly to their pK(a) values. Furthermore, dopamine:iron(III) complex protected isolated hepatocytes against hypoxia-reoxygenation injury at concentrations four-fold lower than that required for protection by dopamine alone.