Eugene Jackim

Detection of DNA damage in individual cells from marine organisms using the single cell gel assay

The single cell gel (SCG) or comet assay is a simple method by which DNA damage is expressed as relative nuclear ‘tail’ length of gel-embedded cells following alkaline electrophoresis. While potentially applicable to any cell type, laboratory experiments were conducted to examine the utility of the SCG method for the detection of genotoxicity in cells of marine fish and invertebrates. Selected cells included those from flounder (Pleuronectes americanus) and oysters (Crassostrea virginica). In vitro exposures were used to optimize parameters and evaluate sensitivity, reproducibility and dose-responsiveness of the SCG method. In vivo exposures were used to examine the effects of factors such as intra-animal variability on low level DNA damage detection. In one experimental series, individually identified oysters were repeatedly sampled to monitor DNA damage and recovery following in vivo exposure to genotoxicant-spiked sediment. Preliminary results suggest that the SCG may be a useful tool to monitor for genotoxic environmental exposures and investigate pollution-mediated health effects.

Protein synthesis in Fundulus heteroclitus muscle

1. 1. Kinetics of 14C-leucine incorporation into Fundulus heteroclitus muscle protein was observed as a function of physiological and environmental factors. 2. 2. Protein synthesis increased with temperature up to a critical point of 26–29°C beyond which it decreased sharply. 3. 3. Incorporation was reduced by: fasting, decreasing dissolved oxygen below 2·5 mg/l. and stress from insufficient water volumes. 4. 4. Leucine incorporation was increased by insulin, but not changed significantly by exercise, darkness, fish size or sex. 5. 5. Characteristic individual and group variability was encountered. 6. 6. Growth regulating factors and those influencing protein synthesis were compared to evaluate the feasibility of this technique for estimating the adequacy of an environment.

In vivo metallothionein and glutathione status in an acute response to cadmium in Mercenaria mercenaria brown cells

Brown cells that are found in the red glands of Mercenaria mercenaria accumulate, detoxify and excrete cadmium. Brown cell involvement in metal detoxification was due in part to endogenous glutathione (GSH) and protein sulfhydryl. Metallothionein (MT) and GSH have been shown to play an important role in metal detoxification in bivalve molluscs. This study showed that the protein sulfhydryl in brown cells of Mercenaria was in fact MT, that brown cell GSH functioned in acute protection against Cd2+ toxicity, that GSH provided the initial defense against Cd2+ toxicity prior to MT induction and that MT variants were unequal in response to Cd2+. During treatment of Mercenaria with 0.5 and 1.0 ppm Cd2+, brown cells were analyzed for MT by capillary electrophoresis and GSH colorimetrically after 0.25, 1, 2, 3, and 4 days. The data indicated that the cadmium-binding protein was MT with an apparent molecular weight of 9 kDa determined by gel filtration or 6 kDa as indicated by capillary electrophoresis. Glutathione appeared to prevail in the brown cell acute response to 0.5 ppm Cd2+, whereas MT appeared to prevail in the acute response to 1.0 ppm Cd2+. Capillary electrophoresis can be used to monitor and quantify MT and its variants in brown cells without need for prior separation of cytosolic components by chromatography. The change in MT-II was greater relative to the change in MT-I in the brown cell acute response to 0.5 ppm Cd2+, whereas the change in MT-1 was greater relative to the change in MT-II in the acute response to 1.0 ppm Cd2+. The variants of brown cell MT appeared to respond differentially to Cd2+ depending upon the Cd2+ treatment concentration.

A new fluorescence-yielding substrate for alkaline and acid phosphatase

Abstract In the search for an ultrasensitive method for the determination of alkaline phosphatase to be incorporated in an automated system, the ammonium salt of flavone 3-diphosphate was synthesized and found to be a stable, versatile, and sensitive substrate for assaying both acid and alkaline phosphatase. Flavone 3-monophosphate was also synthesized, and exhibited comparable sensitivity to the diphosphate. The enzymic cleavage of the mono- and diphosphate groups liberates 3-hydroxyflavone, which can be assayed by direct absorption at 410 mμ or, with greater sensitivity, fluorometrically at 510 mμ. Ultrahigh sensitivity can be achieved by forming the fluorescent metallic chelate of 3-hydroxyflavone with an excess of aluminum ions at a slightly acidic pH value. After an incubation period of 5 min, approximately 10−11 gm of phosphatase can be assayed by the chelate method. The substrate appears to have excellent cytohistochemical fluorescent stain applications. The fluorogenic phosphatase substrate 3-O-methylfluorescein phosphate (1), although more sensitive, lacks the desired stability in solution for automation. The substrate, β-naphthyl phosphate (2–4), has been investigated and was found in our laboratory to be less sensitive than flavone 3-diphosphate.

Using the DNA alkaline unwinding assay to detect DNA damage in laboratory and environmentally exposed cells and tissues

Abstract The DNA alkaline unwinding assay is being evaluated for use in the detection of DNA damage in marine animals exposed to environmental pollutants. In preliminary work, DNA unwinding methods were used with in vitro cell systems to demonstrate DNA strand breaks. Cultured mammalian fibroblasts and sperm from marine fish and invertebrates (Pseudopleuronectes, Arbacia and Mytilus) showed concentration-dependent increases in DNA strand breaks after brief exposures to alkylating agents. DNA unwinding methods were also used on DNA extracted from marine animals injected with genotoxicants and from animals exposed in situ at an estuarine site. Gills from blue mussels caged at the New Bedford Harbor Superfund Site (MA, USA) highly contaminated with many organic (e.g. PCBs) and inorganic contaminants, were also examined. A significant increase in DNA strand breaks was seen in gill tissues of animals held in the contaminated site for as little as 3 days. Although not as severe, an increase in strand breaks was also seen in animals held at the control site for 28 days. This apparent damage may have resulted from low-level contamination and/or from generally poor tissue condition. These results suggest that DNA alkaline unwinding results from controlled experiments using cells briefly exposed to toxicants can be used to identify DNA strand breaking agents. While whole-animal exposures to toxicants may affect DNA integrity, unwinding results may not specifically reflect genotoxicity.

Identification of deoxynucleoside-polyaromatic hydrocarbon adducts by capillary zone electrophoresis-continuous flow-fast atom bombardment mass spectrometry

Capillary zone electrophoresis coupled to continuous flow-fast atom bombardment mass spectrometry is shown to have utility for the detection and characterization of adducts formed by the covalent attachment of four polyaromatic hydrocarbon (PAH) and amino-PAH compounds to deoxyguanosine. Normal scanning provided structural information for a 1.3 ng injection of a model adduct, while 1.3 ng of each of a mixture of adducts was sufficient to determine their molecular weights by monitoring the constant neutral loss of deoxyribose. Exploitation of this loss in the multiple reaction monitoring mode resulted in the detection of low picogram amounts of target adducts in mixtures.

Mechanism for the differential induction of mutation by S9 activated benzo[a]pyrene employing either a glucose-6-phosphate-dependent NADPH-regenerating system or an isocitrate-dependent system

A significant difference in mutation frequency has been observed in CHO cells exposed to benzo[a]pyrene with alternative activation systems. Each system employed rat-liver S9 homogenate with one using isocitrate dehydrogenase to provide reduced NADP, while the other method uses glucose-6-phosphate dehydrogenase. Total aryl hydrocarbon hydroxylase (AHH) activity was greater for the isocitrate dependent system, however, this yielded a lower level of HGPRT mutants. It was ascertained that this reduced mutation frequency may result from sequestering of B[a]P substrate by crystals in the medium, possibly calcium phosphate, which decreased the effective substrate concentration. This sequestration enhances B[a]P internalization, which would explain the dichotomy between the AHH values and the mutation frequency data. The production of specific B[a]P metabolites was also examined by reverse phase HPLC quantitation of extracts of solutions in which the two activation systems were used. The levels of 7,8 dihydroxybenzo[a]pyrene produced by the glucose-6-phosphate protocol were consistently greater than with isocitrate. This may also be a contributing mechanism for elevating the mutation frequency with this procedure. These results demonstrate several interactions between test compound, cells, and metabolizing system which must be considered with in vitro activation systems.