CYP1A1 protein and mRNA in teleosts as an environmental bioindicator: laboratory and environmental studies

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  • 3.1urine Environmental Research 34 (19921 139-145

    CYP1A1 Protein and mRNA in Teleosts as an Environmental Bioindicator: Laboratory and

    Environmental Studies

    Mary L. Haasch," Ellen M. Quardokus," Leslie A. Suthertand," Mark S. Goodrich," Ruth Prince, ~' Keith R. Cooper b &

    John J. Lech"

    Medical College of Wisconsin, Department of Pharmacology and Toxicology, NIEHS Aquatic Biomedical Research Core Center, Great Lakes Research Facility,

    Milwaukee, Wisconsin 53226, USA h Joint Graduate Program in Toxicology, Rutgers University, New Jersey 08854, USA


    Teleosts arc exposed, in the environnwnt, to a number of chemicals that are capable of in~htcing hepatic c3"tochrome P450 monooxygcnase activity. This imhwtion has been described as a most sensitive biological bulicator ~" the presence of certain chtsses ~[" chemicals ht water. The concept of a hioindicator, as applied here, is ~h'rived from the idea that a toxic t~ff'ect will he man(J'ested at the suhcelhdar level he/'ore ~[]'ects will he apparent at higher levels ~?]'biological organization. Laboratory and environmental imhtction of hepatic cvtochrome P450 CYP IA I (P4501AI) was invest~ffated using c'atalvtic activity, intmunodetection and nucleic acid hyhrk#zation. Rainbow trout atul largemouth bass were exposed, under flow-through cont6tions, to fl- naphth~?flavone ([I-NF), a known C YP I A 1 in~hwer, at concentrations ranging from 0"625 to 500 ltg fl-NF/liter for periods of l to 21 days. A t c'oncentrations of 50-500~tg fl-NF/liter, ethoxyresoru.[in-O-deethylase ( EROD) activity and immunoreactive C YPIAI leveL~" were inch.'ed, hut the induction was inversely related to the [I-NF concentration. At these same concentrations, hyhridizable C YPIA 1 mRNA was increased at all conc'entJ ations over time and was imhtced at least up to 7 days of fl-NF treatment. A t concentrations of 0"625-10"01tg [~-NF/liter, EROD activity, immunoreactive protein and hybridizable mRNA were increased bt a concentration-dependent manner. Environmental exposure of largemouth bass. plac'ed in cages in water known to he contaminated with polychlorinated biphenyls, had elevated (six-foht) EROD activity at 3 and 7 days. Killifish taken from a TCDD-contaminated site had three-fold higher EROD activity atul C YPIA 1 mRNA, as well as

    139 Marine Environ. Res. 0141-1136/92/$05.00 1992 Elsevier Science Publishers Ltd, England. Printed in Great Britain

  • 140 Mary L. Haasch et al.

    increased immunoreactice protein, than killifish from a "clean'site. These data indicate the efficacy o fusing nucleic acid hyhridization of C YP I A 1 m RNA as a hioindicator of enrironmental contamination.

    The ever-increasing need to assess the possible adverse effects of environmental contaminants to both humans and the biota has prompted the development of early warning sentinels which will identify and define areas of contamination in critical domestic and industrial water supplies. As the name implies, an early warning system should detect the presence of toxic chemicals betbre detrimental health effects occur ~ to either the biota or to humans. It is widely accepted that the consequences of a toxic insult, such as exposure to environmental contaminants, causes detectable, recognizable changes in the blood and tissues of fish and other aquatic organisms. A change in the blood or tissues must manifest itself at the molecular/cellular level before effects will be apparent at higher levels of biological organization. The concept ofa bioindicator, as applied here, is derived from the idea that an ability to detect toxicological changes at the subcellular level would constitute a sensitive, early warning, biological indicator of environmental chemical contamination.

    Many chemicals have the ability to cause an increase in one or more cytochrome P450 isozymes in various species. Induction of hepatic cytochrome P450 has been suggested as an early warning system because of its sensitivity. 2 Earlier stt, dies involved the nleasurement of hepatic monooxygenase (MO) catalytic enzyme activity. 3 Potential problems with these studies are that a P450 isozyme from a particular family in different species may have different substrate specilicities a or that catalytic activity may be inhibited, s More recently, the specitic isozyme (CYPIAI derived from CYPIAI mRNA, see below) induced by the model compound fi- naphthollavone (fl-N FI was purified and antibodies (rabbit anti-trout LM.,b IgG = anti-trout CYPIAI lgGt were prepared allowing immunodetection to be used to quantitate the specific protein in both marine and freshwater fish species. 6-~ The methods of detection have now been further refined to include the development of a complementary DNA (cDNA), 9 designated pfP~450-3' (known to belong to the CYP IA I gene family'j, for the measurement of the mRNA which codes for the induced protein. This eDNA can be used to measure the induction of mRNA in a variety of species, including different teleosts and lower vertebrates, and has been shown to have the potential to be applied as an early warning system in the monitoring of water quality. ~ Previous studies using an intraperitoneal injection off l -NF have shown that CYPIAI mRNA is maximally induced by approximately 24h and decreases to near control levels by 48 h post- injection.~'~' In order to use induction of mRNA as a bioindicator it was

  • C YPIA 1 protein and rnRNA in teleosts 141

    necessary to show that CYP1AI mRNA would remain at induced levels over time, using waterborne exposures. This data is a necessary prerequisite to the development of the induction of CYPIA1 mRNA levels as a bioindicator of chemical contamination.

    The present investigations involved laboratory studies, using rainbow trout (Oncorhynchus mykiss) and largemouth bass (Micropterus sahnoides) to assess flow-through exposure to known concentrations of inducer, and field study validation, using caged largemouth bass and wild-captured killifish (Fum&hts heteroclitus). Induction of hepatic MO was determined using nucleic acid hybridization to measure induction of CYPIA1 mRNA, immunodetection of induced CYPIA1 protein, and spectrofluorometric determination of induced catalytic activity, for comparison of the various bioindicator tools. Largemouth bass were chosen for the caged fish experiment because of their relative hardiness and ability to withstand the conditions of the river water exposure (adjacent to Milwaukee Harbor, Kinnickinnic River, Wisconsin). The killifish were chosen as the wild species based on previous experience and on their limited home range, which allows them to be sampled from areas either known to be contaminated or areas known not to be contaminated.

    Rainbow trout were exposed, by flow-through exposure, to con- centrations ranging from 0.625 to 500 l~g fl-NF/liter water (10-15-~C), using dimethvlformamide as carrier, for periods ranging from 1 day to 21 days. Largemouth bass were exposed to 0"28 mg fl-N F/liter water (22-25~C), using dimethvlformamide as carrier. These fish were sampled at 1 day and 4 days, with controls sampled from clean laboratory water at identical time points. Caged largemouth bass (n = 4) were placed in modified animal carriers and lowered to an appropriate depth in an area known to be contaminated with PCBs (Kinnickinnic River, adjacent to the Great Lakes Research Facility, Milwaukee, Wisconsin) for 1,3, or 7 days of river water exposure, using clean water laboratory animals as controls. This area has been reported to contain high levels of PCBs in both the fish and water and sediment samples taken upstream from the exposure site had PCB concentrations between 5 and 20 ppm on a dry weight basis (study by D. Edgington, pers. comm. to M. Melancon, see Ref.). Also, this river water has been shown to cause induction of catalytic activity in carp and bullheads.~a Killifish were captured from natural populations, in the summer and fall of 1989, using minnow traps placed in estuaries at Tuckerton, New Jersey (a clean site) and Newark, New Jersey {a tetrachlorodibenzo-p-dioxin-contaminated site).

    The results show that at concentrations of fl-NF ranging from 50 to 500/~g/liter, the catalytic enzyme activity (EROD) and immunoreactive protein (CYP1AI) are induced but that the magnitude of the induction is inversely related to the concentration offl-NF used, over time, while at these

  • 142 Mary L. Haasch et a l .

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