Braulio D. Jimenez

Interactions between polycyclic aromatic hydrocarbons and dissolved humic material: binding and dissociation

Binding of polycyclic aromatic hydrocarbons (PAHs) to dissolved humic material (DHM) was examined by using equilibrium dialysis and fluorescence techniques. There was a direct relationship between the hydrophobicity of the PAH and the affinity for binding to DHM. The binding affinity P/sub a/ for benzo(a)pyrene (BaP), benzanthracene, and anthracene decreased slightly as the concentration of DHM increased. The binding of BaP to DHM was completely reversible and the extent of reversibility was unrelated to the sorption time. The rate of binding of BaP to DHM, measured by the quenching to BaP fluorescence, was very rapid and was completed within 5-10 min. The results suggest that the presence of DHM, or other sorptive components of the dissolved organic pool, may affect binding to sediment or suspended particles and thus alter the fate and transport of organic contaminants in aquatic systems.

The use of bioindicators for assessing the effects of pollutant stress on fish

Abstract The use of bioindicators in environmental pollution studies involves monitoring a suite of selected stress responses at several levels of biological organization in order to (1) assess the effects of sublethal stress on fish, (2) predict future trends (early warning indicators) and (3) obtain insights into causal relationships between stress and effects at the community and ecosystem level. We have successfully applied this approach with redbreast sunfish (Lepomis auritus) in freshwater systems receiving inputs of complex contaminant mixtures containing polychlorinated biphenyls (PCBs), hydrocarbons, heavy metals and chlorine. Indicators such as mixed-function oxidase (MFO) enzymes and DNA damage have provided direct evidence of toxicant exposure, while condition indices and indicators related to lipid biochemistry and histopathology have reflected impaired lipid metabolism, immune and reproductive system dysfunction, and reduced growth potential. At higher levels of organization, stress-mediated effects have included changes in the richness and biotic integrity of fish communities.

Effect of dissolved humic material on accumulation of polycyclic aromatic hydrocarbons: Structure-activity relationships

Abstract The effect of binding of polycyclic aromatic hydrocarbons (PAHs) to dissolved humic material (DHM) on the uptake and bioaccumulation of PAHs is examined in the cladoceran Daphnia magna. To the extent that a PAH binds to DHM, which is related to its hydrophobicity, it becomes unavailable for uptake by the organisms. The structure-activity relationship developed here suggests that the high affinity for binding to DHM and the resultant decrease in bioavailability might mitigate the biological impact of those very hydrophobic contaminants having the greatest potential for bioaccumulation and transfer to man via food chains.

Analysis of adduct formation in the bluegill sunfish (Lepomis macrochirus) between benzo[a]pyrene and DNA of the liver and hemoglobin of the erythrocyte

Abstract Bluegill sunfish ( Lepomis macrochirus ) were exposed to a single dose of benzo[ a ]pyrene by i.p. injection and maintained at 13 or 20°C. Acid hydrolysis of DNA isolated from the liver and hemoglobin from the erythrocyte, 72-h posttreatment, released tetrols, which were separated and quantitated by high-performance liquid chromatography/fluorescence analysis. Examination of the tetrol profiles indicated that adduct formation with both macromolecules occurred via the anti - and syn -isomeric diol epoxide metabolites of benzo[ a ]pyrene. The majority of adducts in the DNA formed via the anti -diol epoxide (85%), which is similar to that reported for other organisms. However, adduct formation in hemoglobin was via both the anti - and syn -isomeric diol epoxides, which is different from that reported for mice. Furthermore, the amount of adduct formation in both macromolecules depended upon the temperatures at which the fish were maintained. Quantitation of the covalent interaction of genotoxic chemicals such as benzo[ a ]pyrene with cellular macromolecules can be a sensitive biological marker of early predictive value in assessing the significance of exposure to human health or ecosystem integrity.

Effects of hepatotoxicants on the induction of microsomal monooxygenase activity in sunfish liver by β-naphthoflavone and benzo[a]pyrene

Effects of chemically induced hepatic injury on biotransformation enzymes in fish were studied. Sunfish hybrids (Lepomis macrochirus x L. cyanellus) were dosed per os with allyl formate (ALF) and carbon tetrachloride (CCl4), and the induction of liver EROD (7-ethoxyresorufin O-deethylase) activity was subsequently challenged by injections of beta-naphthoflavone (BNF) and benzo[a]pyrene (B[a]P). Hepatotoxicity of chemical treatments was assessed using blood enzymes (ASAT, ALAT, and LDH) along with other biochemical variables. Both hepatotoxicants partially abolished the induction of EROD (maximally by 76-89%), and the decrease in induction was dose related. The cytosolic activity of glutathione S-transferase (GST) in the liver decreased in parallel with the decrease in EROD induction. Fish receiving high doses of ALF exhibited significantly less microsomal and blood plasma proteins and, occasionally, were jaundiced. These symptoms, however, were less sensitive indicators of hepatotoxicity than alterations in liver EROD and GST. Both ALF and CCl4 increased the activities of hepatic enzymes in the blood plasma, indicating cytotoxicity. In addition B[a]P, unlike BNF, also increased plasma activities of LDH and ALAT at a dose inducing liver EROD, implying simultaneous hepatotoxicity at high sublethal levels of this xenobiotic. These data suggest that hepatotoxic chemicals absorbed by fish may act antagonistically by decreasing the degree of induction of the cytochrome P450 system relative to the inherent capacity of inducing xenobiotic chemicals present in the environment. Therefore, when assessing the toxicological status of water using fish health biomarkers, it is advisable to measure a concert of metabolic and biochemical variables instead of any single biomarker.

Influence of environmental variables on the hepatic mixed-function oxidase system in bluegill sunfish, Lepomis macrochirus

Abstract 1. 1. MFO activity measured as 7-ethoxyresorufin O -deethylase (EROD) was assayed in hepatic microsomes of unfed bluegills acclimated to 4, 13, 18, or 26° C and injected once intraperitoneally with benzo(a)pyrene (BaP) at a dose of 1, 10 or 20 μg/g body weight. 2. 2. Fish acclimated to the higher temperature exhibited elevated EROD activity after the fifth day of injection; however, fish held at 4°C did not exhibit induction of this enzyme until 18 days following the initial injection and then only at the highest dose. 3. 3. Although the concentrations of BaP and metabolites in the liver 5 days after injection were similar for fish acclimated to three temperatures, EROD activities were greater for the fish acclimated to higher temperatures. 4. 4. Comparison of fed and unfed fish acclimated to 26°C revealed that at all doses higher concentrations of cytochromes P-450 and b 5 accumulated in hepatic tissue of fed fish; however, no difference was observed in EROD activities at any particular dose. 5. 5. The results of this work indicate that EROD activity increases as acclimation temperature and BaP dose increase.

Response of the mixed-function oxidase system to toxicant dose, food and acclimation temperature in the bluegill sunfish

Abstract The degree of induction of hepatic mixed function oxidase (MFO) enzymes in fish is modulated by environmental conditions. This study was designed to investigate the influence of water temperature, presence or absence of food, and exposure to benzo(a]pyrene (BaP) on the inductive response of bluegill sunfish ( Lepomis macrochirus ). The results show an increase in 7-ethoxyresorufin O -deethylase (EROD) activity with an increase in acclimation temperature and dose. This activity appears to be associated with a very small fraction of the total cytochrome P-450 induced. Major changes were observed in the 53- and 57-kilodaltons (kDa) electrophoretic bands.

Biological Markers in Animal Sentinels: Laboratory Studies Improve Interpretation of Field Data

A number of approaches have been used to evaluate the biological hazards of environmental pollution. Chemical analysis of the concentrations of toxic compounds in environmental media is clearly an important component of such an evaluation. Advanced analytical procedures are specific, quantitative, and exquisitely sensitive and precise. However, the biological significance of the chemical concentrations is not at all clear. We understand the toxic action of but a few of the thousands of chemicals in the environment and have almost no information on the toxicity of complex mixtures of chemicals. Furthermore, a chemical survey is a snapshot in time and space. Variations in concentrations over time resulting from intermittent releases of effluents by industries or from storm events, changes in winds, etc., cannot be accounted for without repeated analyses. Spatial patchiness of contaminant patterns also requires extensive and expensive sampling and chemical analyses.

Insulin-induced gene 33 mRNA expression in Chinese hamster ovary cells is insulin receptor dependent.

Gene 33 (g33) is a non‐tissue‐specific gene regulated in rat liver and hepatoma cells by insulin and other agents. It is thought to participate in the transition from quiescence to proliferation in mitogen‐treated cells. The mechanism(s) by which insulin exerts its action on g33 are not totally understood; it is unclear whether a functional insulin receptor is required for this action. In this study, we evaluate the mechanism for insulin induction of g33 mRNA in Chinese hamster ovary (CHO) cells transfected with the neomycin‐resistant plasmid (CHONeoB), human insulin receptor (CHONewIRa), and a kinase‐defective insulin receptor mutated at the ATP‐binding site (CHOK1018A). Transfected cells had higher levels of insulin binding than that of CHONeoB cells; insulin‐induced phosphorylation of the insulin receptor and its intracellular substrates were impaired in CHOK1018A cells. Maximal insulin induction of mRNAg33 occurred 3 h after hormonal exposure in all cell lines. The degree of insulin stimulation of g33 mRNA levels was four‐ to sixfold higher in CHONewIRa than in CHONeoB or CHOK1018A cells, which had minimal levels of insulin‐stimulated g33 mRNA levels. Half‐maximal stimulation of g33 mRNA levels was observed at 0.06 ± 0.01 nM in CHONewIRa cells, consistent with insulin interaction with its own receptor. Wortmannin, an inhibitor of phosphatidyl inositol 3‐kinase (PI3K), had some effects on insulin stimulation of g33 mRNA in CHO NewIRa cells. PD98059, an inhibitor of mitogen‐activated kinase kinase (MAPKK), and rapamycin, a p70 S6 kinase inhibitor, had minimal effect on insulin stimulation of g33 mRNA in all cells tested. By contrast, hydroxy‐2‐naphthalenylmethyl)phosphonic acid triacetoxymethyl ester (HNMPA(AM)3, a selective inhibitor of the insulin receptor tyrosine kinase, caused complete inhibition of insulin stimulation of g33 mRNA levels. These data indicate that the insulin receptor with intact kinase activity is required for insulin stimulation of g33 mRNA levels. They also suggest that AKT, a PI 3‐kinase downstream effector molecule, could mediate insulin stimulation of g33 mRNA. The mechanism(s) of insulin regulation of g33 expression downstream of receptor do not seem to rely entirely on the classic insulin receptor transduction pathway, as a minor effect was observed upon inhibition of MAPKK, suggesting that multiple pathways may be involved. J. Cell. Biochem. 77:432–444, 2000.

Pre-exposure to 3-methylcholanthrene increases benzo[a]pyrene adducts on DNA of bluegill sunfish

Abstract To determine how pre-induction of the hepatic mixed-function oxidase (MFO) system affects the formation of DNA adducts, bluegill sunfish, Lepomis macrochirus, were injected intraperitoneally with 3-methylcholanthrene (3-MC) 5 days prior to injection with benzo[a]pyrene (BaP). Another group of fish were injected only with BaP. On days 0, 1, 3 and 6 after BaP injection, liver microsomes were assayed for several MFO parameters, and BaP adducts in liver were quantified. Pre-exposure to 3-MC induced MFO activity to much higher levels, but did did not protect the DNA from formation of adducts of BaP. DNA from the liver of pre-exposed fish had twice as many BaP adducts. Multiple contaminants appear to act synergistically on the formation of DNA adducts, in spite of the higher MFO activity. Adverse effects from carcinogens in complex mixtures may be much greater than predicted from single-compound experiments.