Effect of arylhydroxylamine metabolites of sulfamethoxazole and dapsone on stress signal expression in human keratinocytes.

The initiation of an immune response to small molecules is believed to require the release of stress/danger signals that activate resident dendritic cells, presumably secondary to the formation of reactive metabolites. We hypothesized that exposure to arylhydroxylamine metabolites of dapsone and sulfamethoxazole lead to the expression/release of numerous stress signals in the skin. To test this hypothesis, we examined the effect of these metabolites on the expression of selected heat shock proteins, uric acid, cytokines, adhesion molecules, and costimulatory molecules in normal human epidermal keratinocytes (NHEKs). NHEKs showed a time-dependent up-regulation of heat shock protein 70 and translocation of heat shock protein 27 when exposed to the arylhydroxylamine metabolites. In addition, the secretion of several proinflammatory cytokines was increased upon incubation of these cells with metabolite. In contrast, the uric acid concentration was not altered. Moreover, intercellular adhesion molecule-1, CD80, and CD86 expressions did not change when NHEKs were exposed to these reactive metabolites. Our data suggest that NHEKs selectively up-regulate certain danger signals when exposed to arylhydroxylamine metabolites. These signals may subsequently activate dendritic cells and initiate an immune response within skin.

Enzyme-mediated protein haptenation of dapsone and sulfamethoxazole in human keratinocytes: I. Expression and role of cytochromes P450.

Cutaneous drug reactions (CDRs) are among the most common adverse drug reactions and are responsible for numerous minor to life-threatening complications. Several arylamine drugs, such as sulfamethoxazole (SMX) and dapsone (DDS), undergo bioactivation, resulting in adduction to cellular proteins. These adducted proteins may initiate the immune response that ultimately results in a CDR. Recent studies have demonstrated that normal human epidermal keratinocytes (NHEKs) can bioactivate these drugs, resulting in protein haptenation. We sought to identify the enzyme(s) responsible for this bioactivation in NHEKs. Using immunofluorescence confocal microscopy and an adduct-specific enzyme-linked immunosorbent assay (ELISA), we found that N-acetylation of the primary amine of SMX and DDS markedly reduced the level of protein haptenation in NHEKs. Detection of mRNA and/or protein confirmed the presence of CYP3A4, CYP3A5, and CYP2E1 in NHEKs. In contrast, although a faint band suggestive of CYP2C9 protein was detected in one NHEK sample, a CYP2C9 message was not detectable. We also examined the ability of chemical inhibitors of cytochromes P450 (aminobenzotriazole and 1-dichloroethylene) and cyclooxygenase (indomethacin) to reduce protein haptenation when NHEKs were incubated with SMX or DDS by either confocal microscopy or ELISA. These inhibitors did not significantly attenuate protein adduction with either SMX or DDS, indicating that cytochromes P450 and cyclooxygenase do not play important roles in the bioactivation of these xenobiotics in NHEKs and thus suggesting the importance of other enzymes in these cells.

Immune response to xenobiotics in the skin: from contact sensitivity to drug allergy.

Skin is the most frequent target of adverse drug reactions. These cutaneous drug reactions (CDRs) show varied clinical manifestations ranging from mildly discomforting rashes to life-threatening Stevens-Johnson syndrome or toxic epidermal necrolysis. Most CDRs appear to be immune mediated, although the mechanism by which they are initiated remains unclear. In this review, current knowledge of the mechanisms by which xenobiotics provoke immune responses in the skin after epicutaneous administration and how similar reactions may occur after systemic routes are summarised. This review also discusses a variety of genetic or environmental factors that may determine the susceptibility of individuals towards immune responses in skin following drug exposure.

Enhanced xenobiotic-induced hepatotoxicity and Kupffer cell activation by restraint-induced stress.

We tested the hypothesis that environmental stress is a predisposing factor for liver injury by examining the effect of acute restraint on liver injury provoked by carbon tetrachloride (CCl4) and allyl alcohol. Mice were immobilized using Plexiglas restraint cages, producing a form of psychogenic stress, whereas other animals were allowed to roam free. Serum alanine aminotransferase levels were elevated significantly in restrained animals after administration of varying doses of CCl4 or allyl alcohol that did not produce liver injury in unrestrained animals. This enhanced liver injury after CCl4 was seen in both male and female mice. The duration of acute restraint was found to be important because a period of 2.5 h of restraint enhanced hepatotoxicity, whereas shorter periods of restraint did not significantly increase liver injury. Serum corticosterone concentrations increased, whereas hepatic glutathione content decreased during and after acute restraint. In addition, delay in administration of CCl4 until 5 h after completion of restraint also produced an elevated level of liver injury compared with that seen in free roaming animals. Immunohistochemical examination of the livers showed significantly enhanced Kupffer cell activation in restrained mice compared with that of free roaming mice. These observations suggest that induction of psychogenic stress may increase the susceptibility to liver injury observed with classic hepatotoxicants and may represent an important predisposing factor to liver injury after xenobiotic exposure. The underlying mechanism seems to be increased macrophage activation in the liver, which may subsequently sensitize hepatocytes to xenobiotics and thus enhance hepatotoxicity.

Effect of pro-inflammatory cytokines on the toxicity of the arylhydroxylamine metabolites of sulphamethoxazole and dapsone in normal human keratinocytes.

Sulphonamides, such as sulphamethoxazole (SMX) and the related sulphone dapsone (DDS), show a higher incidence of cutaneous drug reactions (CDRs) in patients with the acquired immunodeficiency syndrome (AIDS) compared with human immunodeficiency virus (HIV) negative patients. During HIV infection, pro-inflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) are increased. We hypothesized that this increase in pro-inflammatory cytokines may increase the toxicity of the arylhydroxylamine metabolites of SMX (S-NOH) and DDS (D-NOH) in keratinocytes through a reduction in glutathione (GSH) content. We evaluated the effect of TNF-alpha on GSH levels in normal human epidermal keratinocytes (NHEK) and found a significant decrease in GSH after 24h. Pre-treatment with TNF-alpha also resulted in an increase in the recovery of D-NOH, but failed to alter drug-protein covalent adduct formation in NHEK. We also evaluated the effect of TNF-alpha, IL-1 beta, interferon-gamma (IFN-gamma), lipopolysaccharide (LPS) and conditioned media (obtained from monocytes stimulated with LPS) on the cytotoxicity of pre-formed arylhydroxylamine metabolites in NHEK. Priming cells with cytokines did not significantly alter the cytotoxicity of the metabolites. The effect of pre-treatment with TNF-alpha on reactive oxygen species (ROS) generation in NHEK was also determined. While ROS formation in NHEK was increased in the presence of D-NOH, TNF-alpha did not alter the level of ROS generation. Our data suggest that the level of GSH reduction induced by pro-inflammatory cytokines does not predispose NHEK to cellular toxicity from either S-NOH or D-NOH.