Grantee Research Project Results

1997 Progress Report: Linkage of Stress Proteins and Toxic Alterations A Laboratory and Field Investigation

EPA Grant Number: R823297
Title: Linkage of Stress Proteins and Toxic Alterations A Laboratory and Field Investigation
Investigators: Hinton, David E. , Sanders, Brenda M. , Washburn, Barbara S.
Institution: University of California - Davis , California State University - Long Beach
EPA Project Officer: Hahn, Intaek
Project Period: July 1, 1995 through June 1, 1998
Project Period Covered by this Report: July 1, 1996 through June 1, 1997
Project Amount: $474,656
RFA: Exploratory Research - Environmental Biology (1995) RFA Text |  Recipients Lists
Research Category: Biology/Life Sciences , Human Health , Aquatic Ecosystems

Objective:

The applicants seek to conduct advanced, exploratory, investigative environmental research that focuses on the effects of pollutants on the environment. The research will test the hypothesis that the accumulation of the major heat inducible stress proteins, stress-70 and chaperonin (cpn60), reflects contaminant induced damage to tissues and subcellular compartments. We shall take an integrative (molecular biology, physiology, and comparative & environmental pathobiology) approach by determining the effects of 2 polyaromatic hydrocarbons (benzo(a)pyrene and -napthoflavone) and copper on whole animal, cellular, and sub-cellular levels of biological organization and link these to the induction and accumulation of stress proteins in rainbow trout (Oncorhynchus mykiss) and medaka (Oryzias latipes). The first two years of the study will involve testing this relationship in laboratory studies under conditions of acute, acute with recovery, and chronic exposures at various life stages of the fish. We seek to identify quantifiable changes in function and structure brought about by the exposure using biochemical, immunochemical, and histochemical techniques. The linkage between toxicity and stress protein production will be determined by localizing the production of stress-70 and cpn60. The third year of the study will apply the most relevant methods to evaluate a copper- contaminated field site and an upstream reference site inhabited by rainbow trout. Field testing of our results will allow us to account for potential confounding results such as parasites or variable nutritional status that are not seen in a laboratory setting. Results of these studies will: a) identify target tissues of toxicity; b) delineate functional manifestations of toxicity; c) evaluate the utility of using stress proteins by themselves or in conjunction with other biomarkers as an indicator of exposure to key classes of compounds; and d) advance the state of knowledge in the environmental sciences and technology by producing high quality scientific and technical articles in refereed journals.

Progress Summary:

A. Medaka.
The hsp70 response to heat-shock was characterized in eight developmental stages of a representative teleost, the medaka (Oryzias latipes). Hsp70 was not inducible in stage 11 embryos (early gastrulation), and these embryos proved less tolerant to heat stress than embryos at later stages (19 and higher). Although all stages after hatching (larvae, juvenile, adult) were able to raise a stress protein response, they were more susceptible to heat-shock than embryonal stages (with the exception of stage 11 embryos). Constitutive levels of hsp 70 were elevated just before (stage 35)- and after (24-36 hour old larvae) - hatching.

B. Trout.
The hsp70 and tissue toxic responses to metal stressors were studied in trout (Oncorhynchus mykiss) exposed to 10 ppb of copper plus three different concentrations of zinc (0 ppb, 20 ppb, and 45 ppb). Fish were sampled weekly for four weeks. At weeks one to three, no significant difference in hsp70 expression was found in gill, liver, and muscle exposed to 10 ppb copper (Cu)+ 0 ppb zinc (Zn), 10 ppb Cu + 20 ppb Zn, and 10 ppb Cu + 45 ppb Zn. Trout exposed to 10 ppb Cu + 45 ppb Zn for four weeks showed significantly increased hsp70 expression in the liver. However hsp70 analyses of gill and muscle showed no statistically significant difference to control values. Histopathologic analysis of gill and liver revealed 1) increased incidence of single cell necrosis and aneurysm in gill at 4 weeks after exposure to 10 ppb Cu, 10 ppb + 20 ppb Zn, and 10 ppb Cu + 45 ppb Zn, and 2) no significant changes were seen in liver at 4 weeks after the exposure when compared to control.

C. Field validation of stress protein in Asian clam.
As part of several collaborative projects involving the analysis of a range of biomarkers, our laboratory is investigating the suitablility of hsp70 protein(s) in Asian clam (Potamocorbula amurensis) as a biomarker for field application. Our efforts involve two studies conducted in northern San Francisco Bay: 1) monthly collection of clams from 4 sites following a metal contamination gradient; 2) 7 day in situ exposure of clams at two sites at Mare Island Naval Shipyard. Site-specific differences in levels of two hsp70 proteins, hsp70 and hsp76, were measured in May and June 1997. After in situ exposure at Mare Island Naval Shipyard, both hsp70 and hsp76 levels were significantly reduced in clams from site R. However, given a brief heat-shock, hsp70 protein levels were significantly higher in clams from site R than in controls. Results indicate that inhibition as well as induction of hsp70 proteins may be sensitive indicators of effect in P. amurensis. Further evaluation of ongoing projects will yield valuable information on links between different biomarker responses indicative of exposure and effect.

Conclusions:

Ability of medaka to induce hsp 70 is dependent on embryonic developmental stage. Tolerance to a temperature of 400C is reduced in stages not showing induction of hsp70. Embryonic stages able to induce expression of hsp70 after heat shock developed and hatched normally.

Induction of hsp70 was seen in liver of trout exposed to 10 ppb Cu + 45 ppb Zn for 4 weeks with no histopathological changes. While histopathologic changes were seen in the gill, no hsp70 expression was found. Results indicate that increased expression of hsp70 proteins may protect tissues from pathological changes.

Results from field experiments with Asian clam (P. amurensis) show that inhibition as well as induction of hsp70 proteins may be sensitive indicators of effect. Further evaluation of ongoing projects will yield valuable information on links between different biomarker responses indicative of exposure and effect.

Additional information:

Immunohistochemical localization of hsp70 (StressGen, SPA-810) was performed on 3 week-old and 3 month-old medaka exposed to 37?C water for 30 min. No detectable changes was seen in gill, liver, and muscle of 3 week-old medaka when compared to the control. Enhanced hsp70 expression was seen in muscle of 3-month-old medaka when compared to control. Further refinement of the protocol will optimize hsp70 localization. This immunohistochemical protocol will eventually be used in localizing hsp70 expression in medaka and trout exposed to toxic stressors.

Exposure to DEN: To compare and contrast stress protein responses with other biomarker type responses, 100 medaka were exposed to 300 ppm Diethylnitrosamine (DEN)for 48 hours and 100 medaka were treated as control. Ten female and 10 male livers of both treatments were collected for hsp70 and histopathologic analyses at the following exposure times: 6h, 24h, 48h and 120h (72h + 48h post-exposure). In addition, at 72h post-exposure, another 10 male and 10 female of the DEN-treated fish were heat-shock at 37?C for 15 minutes. Livers were then collected 6 hours after heat-shock. Medaka liver samples are currently being analyzed for hsp70 expression and histopathology.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

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Publications

Type	Citation	Project	Document Sources
Journal Article	Werner I, Hinton DE. Field validation of hsp70 stress proteins as biomarkers in Asian clam (Potamocorbula amurensis): is downregulation an indicator of stress? Biomarkers 1999;4(6):473-484.	R823297 (1997) R825433 (Final)	Abstract from PubMed Abstract: Informa-Abstract Exit

Supplemental Keywords:

Medaka, trout, Asian clam, Hsp 70, histopathology,, RFA, Toxics, Ecosystem Protection/Environmental Exposure & Risk, Ecosystem/Assessment/Indicators, Ecosystem Protection, exploratory research environmental biology, Chemical Mixtures - Environmental Exposure & Risk, HAPS, Ecological Effects - Environmental Exposure & Risk, Ecological Effects - Human Health, Ecological Indicators, fate, marine ecosystem, heat stress, stressors, hydrocarbon, hydrological, PAH, pathobiological, field validation, hydrocarbons, toxic chemicals, toxic environmental contaminants, fish , Benzene (including benzene from gasoline), exposure assessment, rainbow trout

Progress and Final Reports:

Original Abstract

1996

Final