The past several years have seen the accumulation of evidence demonstrating that tissue injury induced by diverse toxicants is due not only to their direct effects on target tissues but also indirectly to the actions of resident and infiltrating macrophages. These cells release an array of mediators with cytotoxic, pro- and anti-inflammatory, angiogenic, fibrogenic, and mitogenic activity, which function to fight infections, limit tissue injury, and promote wound healing. However, following exposure to toxicants, macrophages can become hyperresponsive, resulting in uncontrolled or dysregulated release of mediators that exacerbate acute tissue injury and/or promote the development of chronic diseases such as fibrosis and cancer. Evidence suggests that the diverse activity of macrophages is mediated by distinct subpopulations that develop in response to signals within their microenvironment. Understanding the precise roles of these different macrophage populations in the pathogenic response to toxicants is key to designing effective treatments for minimizing tissue damage and chronic disease and for facilitating wound repair.

Tissue injury induced by a diverse group of xenobiotics appears to involve both direct and indirect damage to target cells. Thus, while chemicals may act directly on target cells resulting in toxicity, they may also act indirectly by recruiting and activating resident and inflammatory tissue macrophages. Macrophages are potent secretory cells that release an array of mediators, including proinflammatory and cytotoxic cytokines and growth factors, bioactive lipids, hydrolytic enzymes, reactive oxygen intermediates, and nitric oxide--each of which has been implicated in the pathogenesis of tissue injury. The potential role of macrophages and their mediators in tissue injury has been extensively investigated in the lung and the liver. In both of these tissues, xenobiotics induce localized macrophage accumulation and mediator release. Furthermore, when macrophage functioning is blocked, pulmonary and hepatic injury-induced agents such as ozone, bleomycin, acetaminophen, carbon tetrachloride, and galactosamine are reduced. These data provide direct support for the hypothesis that macrophages and the mediators they release contribute to xenobiotic-induced tissue injury.

Macrophages function as control switches of the immune system, providing a balance between pro- and anti-inflammatory responses. To accomplish this, they develop into different subsets: classically (M1) or alternatively (M2) activated macrophages. Whereas M1 macrophages display a cytotoxic, proinflammatory phenotype, much like the soldiers of The Dark Side of The Force in the Star Wars movies; M2 macrophages, like Jedi fighters, suppress immune and inflammatory responses and participate in wound repair and angiogenesis. Critical to the actions of these divergent or polarized macrophage subpopulations is the regulated release of inflammatory mediators. When properly controlled, M1 macrophages effectively destroy invading pathogens, tumor cells and foreign materials. However, when M1 activation becomes excessive or uncontrolled, these cells can succumb to The Dark Side, releasing copious amounts of cytotoxic mediators that contribute to disease pathogenesis. The activity of M1 macrophages is countered by The Force of alternatively activated M2 macrophages which release anti-inflammatory cytokines, growth factors and mediators involved in extracellular matrix turnover and tissue repair. It is the balance in the production of mediators by these two cell types that ultimately determines the outcome of the tissue response to chemical toxicants.

The biological effects of monocyte chemoattractant protein (MCP) 1 are mediated by binding to C-C chemokine receptor (CCR) 2. In the present studies, we used CCR2 knockout (CCR2؊/؊) mice to examine the role of MCP-1 in acetaminophen-induced macrophage accumulation in the liver, expression of inflammatory cytokines, and hepatotoxicity. We found that hepatic expression of CCR2 and MCP-1 was increased 10-fold and 20-fold, respectively, 12 to 72 hours after administration of acetaminophen to wild-type mice. Expression of these proteins was localized in centrilobular regions of the liver. Whereas MCP-1 was expressed by both hepatocytes and macrophages, CCR2 was identified in inflammatory macrophages. F4/80 is a marker of mature macrophages expressed in large quantities by Kupffer cells. In wild-type mice, a 75% decrease in F4/80-positive macrophages was observed 24 to 48 hours after administration of acetaminophen. In contrast, expression of macrosialin (CD68), a marker of activated macrophages, increased 2-fold 24 to 72 hours after administration of acetaminophen and was associated with inflammatory cells. Although there was a decrease in the overall severity of inflammation and in the number of macrosialin-positive macrophages 72 hours after administration of acetaminophen in CCR2؊/؊ mice, the number of F4/80-positive cells did not change. Loss of CCR2 was also found to alter acetaminophen-induced expression of tumor necrosis factor ␣, monocyte chemoattractant protein 3, and KC/gro. However, the overall outcome of acetaminopheninduced hepatic injury was not affected. In conclusion, these data indicate that MCP-1 and CCR2 contribute to the recruitment of a subset of activated macrophages into the liver during acetaminophen-induced hepatotoxicity that may be important in resolution of tissue injury.

Sulfur mustard (SM), a chemical weapon first employed during World War I, targets the skin, eyes, and lung. It remains a significant military and civilian threat. The characteristic response of human skin to SM involves erythema of delayed onset, followed by edema with inflammatory cell infiltration, the appearance of large blisters in the affected area, and a prolonged healing period. Several in vivo and in vitro models have been established to understand the pathology and investigate the mechanism of action of this vesicating agent in the skin. SM is a bifunctional alkylating agent which reacts with many targets including lipids, proteins, and DNA, forming both intra- and intermolecular cross-links. Despite the relatively nonselective chemical reactivity of this agent, basal keratinocytes are more sensitive, and blistering involves detachment of these cells from their basement membrane adherence zones. The sequence and manner in which these cells die and detach is still unresolved. Much has been discovered over the past two decades with respect to the mechanisms of SM-induced cytotoxicity and the intracellular and extracellular targets of this vesicant. In this review, the effects of SM exposure on the skin are described, as well as potential mechanisms mediating its actions. Successful therapy for SM poisoning will depend on following new mechanistic leads to develop drugs that target one or more of its sites of action.

Acetaminophen is a mild analgesic and antipyretic agent known to cause centrilobular hepatic necrosis at toxic doses. Although this may be due to a direct interaction of reactive acetaminophen metabolites with hepatocyte proteins, recent studies have suggested that cytotoxic mediators produced by parenchymal and nonparenchymal cells also contribute to the pathophysiological process. Nitric oxide is a highly reactive oxidant produced in the liver in response to inflammatory mediators. In the present studies we evaluated the role of nitric oxide in the pathophysiology of acetaminophen-induced liver injury. Treatment of male Long Evans Hooded rats with acetaminophen (1 g/kg) resulted in damage to centrilobular regions of the liver and increases in serum transaminase levels, which were evident within 6 hours of treatment of the animals and reached a maximum at 24 hours. This was correlated with expression of inducible nitric oxide synthase (iNOS) protein in these regions. Hepatocytes isolated from both control and acetaminophen-treated rats were found to readily synthesize nitric oxide in response to inflammatory stimuli. Cells isolated from acetaminophen-treated rats produced more nitric oxide than cells from control animals. Production of nitric oxide by cells from both control and acetaminophen-treated rats was blocked by aminoguanidine, a relatively specific inhibitor of iNOS. Arginine uptake and metabolism studies revealed that the inhibitory effects of aminoguanidine were due predominantly to inhibition of iNOS enzyme activity. Pretreatment of rats with aminoguanidine was found to prevent acetaminophen-induced hepatic necrosis and increases in serum transaminase levels. This was associated with reduced nitric oxide production by hepatocytes. Inhibition of toxicity was not due to alterations in acetaminophen metabolism since aminoguanidine had no effect on hepatocyte cytochrome P4502E1 protein expression or N-acetyl-pbenzoquinone-imine formation. Taken together, these data demonstrate that nitric oxide is an important mediator of acetaminophen-induced hepatotoxicity. (HEPATOLOGY 1998; 26:748-754.)

Inhaled nitric oxide is a targeted pulmonary vasodilator that improves clinical outcomes for newborn patients with persistent pulmonary hypertension of the newborn, and may be effective in treating some premature patients with acute respiratory distress syndrome or lung disease of prematurity. Nitric oxide is now recognized as playing an important role in the regulation of diverse physiological processes. However, the pharmacological properties of inhaled nitric oxide are not easy to separate from its toxicological effects. For example, the intended effect of inhaled nitric oxide, vasodilation in the lung, is mediated, in part, by increased cellular cyclic GMP (cGMP). However, increased cGMP can also interfere with normal cellular proliferation. Nitric oxide has also been shown to cause DNA strand breaks and/or base alterations that are potentially mutagenic. Inhaled nitric oxide can rapidly react with oxygen in the lung to form nitrogen dioxide, which is a potent pulmonary irritant. Nitric oxide also reacts with superoxide anion to form peroxynitrite, a cytotoxic oxidant that can interfere with surfactant functioning. The overall effect of inhaled nitric oxide in potentiating or attenuating inflammation and oxidative damage in diseased lung is dependent on the dose administered. Furthermore, despite rapid inactivation by circulating hemoglobin, inhaled nitric oxide exerts effects outside the lung, including blocking platelet aggregation, causing methemoglobinemia, and possibly inducing extrapulmonary vasodilation. The toxicology of inhaled nitric oxide is not completely understood and must be considered in the design of protocols for its safe and effective clinical use.

Persistent inflammation and the generation of reactive oxygen and nitrogen species play pivotal roles in tissue injury during disease pathogenesis and as a reaction to toxicant exposures. The associated oxidative and nitrative stress promote diverse pathologic reactions including neurodegenerative disorders, atherosclerosis, chronic inflammation, cancer, and premature labor and stillbirth. These effects occur via sustained inflammation, cellular proliferation and cytotoxicity and via induction of a proangiogenic environment. For example, exposure to the ubiquitous air pollutant ozone leads to generation of reactive oxygen and nitrogen species in lung macrophages that play a key role in subsequent tissue damage. Similarly, studies indicate that genes involved in regulating oxidative stress are altered by anesthetic treatment resulting in brain injury, most notable during development. In addition to a role in tissue injury in the brain, inflammation, and oxidative stress are implicated in Parkinson's disease, a neurodegenerative disease characterized by the loss of dopamine neurons. Recent data suggest a mechanistic link between oxidative stress and elevated levels of 3,4-dihydroxyphenylacetaldehyde, a neurotoxin endogenous to dopamine neurons. These findings have significant implications for development of therapeutics and identification of novel biomarkers for Parkinson's disease pathogenesis. Oxidative and nitrative stress is also thought to play a role in creating the proinflammatory microenvironment associated with the aggressive phenotype of inflammatory breast cancer. An understanding of fundamental concepts of oxidative and nitrative stress can underpin a rational plan of treatment for diseases and toxicities associated with excessive production of reactive oxygen and nitrogen species.