Mohamed Faisal

Aquatic Pollution-Induced Immunotoxicity in Wildlife Species

The potential for chemicals to adversely affect human immunologic health has traditionally been evaluated in rodents, under laboratory conditions. These laboratory studies have generated valuable hazard identification and immunotoxicologic mechanism data; however, genetically diverse populations exposed in the wild may better reflect both human exposure conditions and may provide insight into potential immunotoxic effects in humans. In addition, comparative studies of species occupying reference and impacted sites provide important information on the effects of environmental pollution on the immunologic health of wildlife populations. In this symposium overview, Peter Hodson describes physiological changes in fish collected above or below the outflows of paper mills discharging effluent from the bleaching process (BKME). Effects attributable to BKME were identified, as were physiological changes attributable to other environmental factors. In this context, he discussed the problems of identifying true cause and effect relationships in field studies. Mohamed Faisal described changes in immune function of fish collected from areas with high levels of polyaromatic hydrocarbon contamination. His studies identified a contaminant-related decreases in the ability of anterior kidney leukocytes to bind to and kill tumor cell line targets, as well as changes in lymphocyte proliferation in response to mitogens. Altered proliferative responses of fish from the contaminated site were partially reversed by maintaining fish in water from the reference site. Peter Ross described studies in which harbor seals were fed herring obtained from relatively clean (Atlantic Ocean) and contaminated (Baltic Sea) waters. Decreased natural killer cell activity and lymphoproliferative responses to T and B cell mitogens, as well as depressed antibody and delayed hypersensitivity responses to injected antigens, were identified in seals fed contaminated herring. In laboratory studies, it was determined that rats fed freeze-dried Baltic Sea herring had higher virus titers after challenge with rat cytomegalovirus (RCMV) than rats fed Atlantic Ocean herring; perinatal exposure of rats to oil extracted from Baltic herring also reduced the response to challenge with RCMV. Keith Grassman reported an association between exposure to polyhalogenated aryl hydrocarbons and decreased T cell immunity in the offspring of fish-eating birds (herring gulls and Capsian terns) at highly contaminated sites in the Great Lakes. The greatest suppression of skin test responses to phytohemagglutinin injection (an indicator of T cell immunity) was consistently found at sites with the highest contaminant concentrations. Judith Zelikoff addressed the applicability of immunotoxicity studies developed in laboratory-reared fish for detecting altered immune function in wild populations. She presented data from studies done in her laboratory with environmentally relevant concentrations of metals as examples. Although the necessity of proceeding with caution when extrapolating across species was emphasized, she concluded that published data, and results presented by the other Symposium participants, demonstrate that assays similar to those developed for use in laboratory rodents may be useful for detecting immune system defects in wildlife species directly exposed to toxicants present in the environment.

Alterations in cytometric characteristics of hemocytes from the American oyster Crassostrea virginica exposed to a polycyclic aromatic hydrocarbon (PAH) contaminated environment

The effects of residing in a polycyclic aromatic hydrocarbon (PAH) contaminated environment on the cytometric characteristics of hemocytes from the American oyster Crassostrea virginica (collected from the Rappahannock River, Virginia, USA in Spring 1991) were analyzed using a multichannel Coulter counter (10000 hemocytes oyster-1). The percentage and relative volume of small-sized hemocytes (>2.5 to 5.1 μm) was higher (P<0.01) in oysters from Hospital Point (HP, a PAH contaminated site) in the Elizabeth River (ER), Virginia, compared to oysters from the relatively unpolluted Rappahannock River (RR). On the contrary, a decrease (P<0.001) in the percentage and volume of larger hemocytes (>6.2 to 10 μm) was observed in the HP-oysters. Maintaining the RR-oysters at the HP site for 8 wk induced statistically significant increases in the number and relative volume contribution of the >2.5 to 5.1 μm hemocytes and sharp decreases in the occurrence and relative volume of >6.2 to 13 μm cells (P<0.01). By depurating the HP-oysters in the York River (the control site), the number of, and volume contributed by the small hemocytes (>2.5 to 5.1 μm) showed a significant decrease over time as compared to the baseline values. On the other hand, the relative number of >6.2 to 13 μm hemocytes increased sharply within 8 wk (P<0.001). This indicates that these changes are both inducible and reversible and, at least in part, due to exposure to the ER-sediment. Based on these studies, oyster hemocyte cytometric characteristics could be developed as a sensitive biomarker of exposure to PAH.

Evidence for the presence of protease inhibitors in eastern (Crassostrea virginica) and Pacific (Crassostrea gigas) oysters

The plasma of eastern (Crassostrea virginica) and Pacific (Crassostrea gigas) oysters were compared for levels of inhibitory activities against a variety of proteases. Representatives of the serine, cysteine, metallo and aspartic protease mechanistic classes were analyzed, including extracellular proteases produced by two oyster-associated pathogens; Perkinsus marinus and Vibrio vulnificus. In comparison to C. virginica, C. gigas plasma exhibited significantly higher specific inhibition levels (ng protease inhibited/μg plasma protein) for papain (P<0.001), pepsin (P<0.001), P. marinus protease (P<0.001), trypsin (P=0.015), and V. vulnificus protease (P<0.001). Plasma of C. gigas did not inhibit the metalloprotease thermolysin. Instead, a significant increase in substrate hydrolysis was seen in wells containing plasma and thermolysin in comparison to wells containing thermolysin only. A similar trend was noted for thermolysin with the eastern oyster samples. These studies indicate the presence of protease inhibitors in the plasma of Crassostrea spp., which may have an impact upon host defense mechanisms, in addition to other physiological roles.

Effects of polycyclic aromatic hydrocarbons on the lymphocyte mitogenic responses in spot, Leiostomus xanthurus

Abstract Several polycyclic aromatic hydrocarbons (PAH) modulate leukocyte activity in vertebrates 1 . PAH require metabolic activation to ultimate carcinogenic forms, 2 but it is unknown whether PAH metabolism is required to exert immunocyte modulation in fish. The Southern Branch of Elizabeth River, Virginia, is heavily contaminated with PAH, and fish collected from this system show high incidence of diseases and neoplasms. 3,4 Because T lymphocytes play an important role in host-defense mechanisms in vertebrates, we designed several experiments to examine the effects of PAH on T cell activity in vivo and in vitro . Our results show that PAH suppress the proliferative responses of the lymphocytes and that this modulation may involve cytochrome P450-mediated PAH metabolism.

Discrimination between two Perkinsus spp. isolated from the softshell clam, Mya arenaria , by sequence analysis of two internal transcribed spacer regions and the 5·8S ribosomal RNA gene

The internal transcribed spacer (ITS-1 and ITS-2) regions and the 5.8S ribosomal RNA gene of 2 Perkinsus spp. (G117 and H49) originating from the softshell clam, Mya arenaria, of the Chesapeake Bay were cloned and sequenced to obtain evidence for their genetic divergence. A high level of heterogeneity in both regions, probably resulting from deletions, insertions, and base substitutions, was evident from alignments of the sequences of the 2 isolates with published sequences of other Perkinsus spp. The isolate G117 and other Perkinsus spp. were highly divergent (13-26% and 19-20% sequence divergence in ITS-1 and ITS-2, respectively). These regions in the isolate H49 and Perkinsus marinus were similar (99.07% and 99% for ITS-1 and ITS-2, respectively). Evidence obtained from a phylogenetic analysis using the aligned sequences suggests that G117 and H49 belong to 2 distinct species of Perkinsus. The isolate G117 possibly belongs to an as yet undescribed species of Perkinsus, and H49 belongs to the species P. marinus. The conclusions drawn from the genetic analysis of H49 and G117 are supported by previously reported morphological characteristics (McLaughlin & Faisal, 1998b). Isolates H49 and G117 originated from the same molluscan species demonstrating that at least 2 different species of Perkinsus can co-exist in 1 host.

Effects of laboratory exposure to sediments contaminated with polycyclic aromatic hydrocarbons on the hemocytes of the American oyster Crassostrea virginica

Abstract We have previously shown that hemocytes of American oysters collected from polycyclic aromatic hydrocarbon (PAH)-contaminated areas in the Elizabeth River, Virginia, have a lower ability to phagocytose yeast particles and a decreased expression of concanavalin A-binding sites as compared to hemocytes of oysters collected from cleaner environments. In the present study, we exposed healthy oysters to PAH-contaminated sediments of the Elizabeth River under laboratory conditions for up to 11 weeks. The data obtained indicate that exposure to the contaminated sediments induced a significant suppression in the number of Con A-binding sites as early as four weeks post exposure, but had no significant effects on the phagocytosis. On the other hand, depuration of the Elizabeth River oysters was accompanied by a steady increase in the number of hemocytes expressing Con A and phagocytosis was slightly enhanced. The sum of our data indicates that phenotypic changes are probably more sensitive to PAH exposure than functional parameters.

Interactive effects of cadmium and benzo[a]pyrene on metallothionein induction in mummichog (Fundulus heteroclitus)

Previous experiments demonstrated that exposure of mummichog to cadmium (Cd) in combination with benzo[a]pyrene (BaP) caused a higher mortality than would be expected from simple additive effects. Experiments are described here that investigated whether BaP exposure inhibits the induction of metallothionein (MT), a major detoxifying protein for Cd, or if reactive BaP metabolites compete with Cd for binding sites on MT. Fish were injected with or without BaP (18 mg/kg) in combination with a low (1 mg/kg) or high (3.2 mg/kg) dose of Cd, and in one treatment BP was dosed 4 days after Cd. The results showed a rapid induction of MT to 1.5 mg/g wet weight liver, 1 day after injecting the low Cd dose. Simultaneous BaP exposure significantly delayed the induction of MT, for both low and high Cd doses, and BaP temporarily lowered the induced MT concentration when dosed 4 days after induction by Cd. To test if binding of BaP metabolites to MT reduces the detoxification potential for Cd, microsomes of CYP1A-induced fish were incubated with MT and radiolabeled BaP. Active metabolism of BaP was observed by high-performance liquid chromatography analysis, but no association of BaP metabolites with MT was found. Neither could this be demonstrated in vivo, in liver MT isolated from mummichog dosed with 3H-BaP and Cd. These results suggest that increased toxicity of Cd in combination with BaP exposure is likely to be caused by inhibited MT synthesis, rather than by interference of BaP metabolites with Cd binding on MT.

Interaction of cadmium and benzo[a]pyrene in mummichog (Fundulus heteroclitus): Effects on acute mortality

Abstract Mummichog (Fundulus heteroclitus), a common estuarine fish species, were exposed to cadmium (Cd) and benzo[a]pyrene (BP) to study the effects of single and combined exposure on acute mortality. Experiments were performed to establish acute, 14 day LD50 values for Cd and BP after intraperitoneal injection. Combinations of 5.6–56 mgkg−1 BP and 0.1–10mgkg−1 Cd were injected concomitantly to investigate interactive effects on mortality. The calculated LD50 value for BP was 35.7 mgkg−1 (95% confidence interval (c.i.): 29.3–43.6), the LD50 value for Cd was 6.5 mgkg−1 (95% c.i.: 4.4–9.5). Based on the LD50 values, toxic units were calculated for the combined dosages of Cd and BP. The results showed that when Cd was dosed at sublethal levels together with lethal BP doses, mortality was lower than expected. When BP was dosed at sublethal levels with sublethal Cd doses, mortality was higher than expected. These results indicate that, depending on the concentrations of the compounds, both synergistic and antagonistic effects can occur when mummichog are exposed to a combination of Cd and BP. Possible mechanisms for these interactive effects may include the inhibition of P450 by Cd and the inactivation of metallothioneins by reactive BP metabolites.

Characterization of Two Perkinsus spp. from the Softshell Clam, Mya arenaria Using the Small Subunit Ribosomal RNA Gene

Sequence analysis and riboprinting of the small subunit ribosomal RNA genes were used to characterize two morphologically different Perkinsus species isolates from the gill (G117) and the hemolymph (H49) of the softshell clam, Mya arenaria. Sequence data of the polymerase chain reaction amplified ribosomal RNA loci of G117 and H49 indicated that these genes are 1803 and 1806 base‐pair long, respectively. A sequence similarity of > 98.9% was calculated among ribosomal RNA sequences of the two isolates of this study and the published sequences of Perkinsus marinus from the American eastern oyster, Crassostrea virginica, and Perkinsus sp. from the blood cockle of the Australian mollusc, Anadara trapezia. From a phylogenetic tree obtained from Jukes‐Cantor distances of the aligned ribosomal RNA gene sequences of 13 eukaryotic taxa using the Neighbor‐Joining method, we showed that G117 and H49 clustered within the genus Perkinsus. Guided by the sequence data of Perkinsus marinus (accession # X75762) and Perkinsus sp. (accession # L07375), restriction endonucleases were selected for restriction fragment analysis of polymerase chain reaction products of the small subunit ribosomal RNA genes (riboprinting). Riboprinting was used to distinguish the four members of the genus Perkinsus from each other.

A comparison of diagnostic assays for detection of Perkinsus spp. in the softshell clam Mya arenaria

Perkinsus spp. infections are increasing in prevalence and intensity in softshell clams Mya arenaria, a commercially and ecologically important bivalve of the Chesapeake Bay. In the present study, the gills, labial palps, rectal tissue, and hemolymph from softshell clams were compared for their suitability in thioglycolate diagnosis of Perkinsus spp. infection. Gill and palp tissues were more sensitive than either rectal tissue or hemolymph for detecting light infections. Rectal tissues became effective measures of parasite burdens as infections increased in intensity. Hemolymph samples were positive only in advanced infections. Further examination of paraffin-embedded tissue sections from lightly infected clams resulted in false negatives when compared with gill or palp thioglycolate assays. Hence, the use of both rectal and gill thioglycolate assays are recommended for routine diagnosis of softshell clam Perkinsus spp.