Lack of Evidence for Contact Sensitization by Pfiesteria Extract

Members of the estuarine dinoflagellate genus Pfiesteria are reported to have been responsible for massive fish kills between 1991 and 1998, primarily in the estuaries of North Carolina and Maryland. The classic signs of Pfiesteria poisoning in fish are skin ulcerations and altered, erratic swimming patterns (Burkholder et al. 1992; Glasgow et al. 1995). The etiology of the skin ulcers has yet to be determined. Several theories have been proposed, including a) that Pfiesteria complex organisms produce a potent exotoxin that destroys the integrity of the skin directly (Glasgow et al. 1995) or induces necrolysis followed by opportunistic microbial infection (Burkholder 1999; Noga et al. 1996); and b) that the dinoflagellate directly attaches to and feeds on fish epidermis (Drgon et al. 2005; Vogelbein et al. 2002). Another hypothesis is that stress associated with environmental factors such as a Pfiesteria bloom causes severe physiologic dysfunction that can induce the acute ulceration response (Udomkusonsri and Noga 2005) and indirectly lead to pathogenic ulceration by water molds. Other environmental parameters may also induce the initial lesion that subsequently becomes infected by water molds (Kiryu et al. 2002; Noga et al. 1996; Udomkusonsri and Noga 2005). The Centers for Disease Control and Prevention (CDC) includes Pfiesteria-related illness in a multiple system syndrome known as possible estuary-associated syndrome (PEAS) (Samet et al. 2001). Under the PEAS criteria, definitive exposure to estuarine water bearing pathogenic Pfiesteria must be established in concert with the development of specific symptoms. Although there has been no indication of toxicity from consumption of seafood from Pfiesteria-contaminated waters (Golub et al. 1998), intense occupational exposure to active fish-killing Pfiesteria species in a laboratory environment or during a large fish kill event has been reported to induce headache, disruptions in cognitive function, and skin lesions (Burkholder 1999; Glasgow et al. 1995; Grattan et al. 1998). Initial anecdotal reports suggested that people who had extensive environmental exposure to Pfiesteria-contaminated water or aerosols during occupational or recreational aquatic activities might exhibit flulike symptoms, malaise, and respiratory and dermal irritation and inflammation (Grattan et al. 1998; Lowitt and Kauffman 1998; Shoemaker 1997, 1998). The irritation usually resolved spontaneously or by washing with fresh water; several investigators suggested that the lesions might be due to an allergic or toxic reaction (Grattan et al. 1998; Lowitt and Kauffman 1998). In these exposed individuals, immune, liver and renal function profiles, blood chemistries, and blood counts were normal (Grattan et al. 1998). These initial reports of human exposure have been criticized because of the limited number of exposed individuals, the lack of preexposure data and proper controls, the inability to definitively measure toxin exposure, the lack of an environmental screening test for the toxin, the transient nature of the organism, and confounding medical and lifestyle factors of many of those evaluated (Haselow et al. 2001; Moe et al. 2001; Samet et al. 2001). More recently, the CDC-sponsored, 4-year, cross-sectional studies in Maryland, Virginia, and North Carolina found that routine occupational exposure to estuarine environments containing Pfiesteria did not pose any significant neuropsychologic health risk (Moe et al. 2001; Morris et al. 2006). Evaluation of the health and cognitive function of study participants in both the preliminary studies and in the CDC studies attributed the majority of cases to chronic irritation and trauma associated with fishing and crabbing work, estuarine microbes, allergens such as ragweed or pollen, intense sun and water exposures, and preexisting medical and neuropsychologic conditions (Burke and Tester 2002; Collier and Burke 2002; Haselow et al. 2001; Lowitt and Kauffman 1998). In two Maryland studies a small number of skin lesions (seven biopsies from six patients and eight biopsies from four patients) were not ascribed to a specific, alternate diagnosis. Those lesions were biopsied and exhibited either nonspecific chronic inflammatory responses or an infiltrate containing eosinophils and lymphocytes (Grattan et al. 1998; Lowitt and Kauffman 1998). The nondiagnostic lesions and dermal irritation that included erythema and an itching, burning sensation experienced by many people in multiple studies were deemed significant in reviews by scientists not associated with the Maryland studies (Burke and Tester 2002; Collier and Burke 2002). The exact cause of Pfiesteria toxicity is still unknown, although numerous efforts have been made to isolate the toxin and characterize the chemical structure and mechanism of action (Drgon et al. 2005; Moeller et al. 2001). Cell extracts showed very weak activity in a panel of bioassays relative to extracts from Pfiesteria-laden aquarium water, indicating that the putative toxin(s) is preferentially released from the cells (Moeller et al. 2001). Available data suggest both a hydrophilic, neurotoxic component and a hydrophobic, dermonecrotic component (Samet et al. 2001), although the relative toxicities of the components have been contested (Moeller et al. 2001). A series of studies conducted at Duke University in Durham, North Carolina, indicated that the neurotoxic component might induce subtle, specific deficits in the acquisition phase of learning and habituation to the environment in Sprague-Dawley rats but does not affect other neurologic, hematologic, or histopathologic parameters (Duncan et al. 2005; Levin et al. 1997, 2003; Rezvani et al. 2001). The toxic effects diminished with storage time of the water, suggesting that the putative toxin degrades quickly (Levin et al. 1997), possibly because of oxidative degradation (Moeller et al. 2001). Earlier studies have associated intense exposure to Pfiesteria with a variety of ailments (Burkholder 1999; Glasgow et al. 1995; Grattan et al. 1998; Lowitt and Kaufman 1998; Shoemaker 1997, 1998). The eosinophilic infiltration of dermal lesions, inflammation, and burning sensation reported in people exposed to Pfiesteria-contaminated waters suggest potential allergic or irritant dermatitis. Both allergic contact dermatitis and contact irritant dermatitis are mediated by keratinocyte-derived cytokines (Corsini and Galli 2000). The goal of our present investigation was to use physical responses and cytokine profiles to assess the potential of a C18-bound fraction of Pfiesteria extract (CPE) as an allergen and contact sensitizer and to distinguish a possible local irritancy response from dermal sensitization.