3.1urine Environmental Research 34 (19921 139-145

C Y P 1 A 1 Protein and m R N A 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

A BSTRA C T

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 C Y P I A 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 o f 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 m R N A 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 o f 0"625-10"01tg [~-NF/liter, EROD activity, immunoreactive protein and hybridizable m R N A were increased bt a concentration-dependent manner. Environmental exposure o f 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 o f a 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 C Y P I A 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 m R N A 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 of f l -NF have shown that CYPIAI m R N A is maximally induced by approximately 24h and decreases to near control levels by 48 h post- injection.~'~' In order to use induction of m R N A 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

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C Y P I A 1 protein and rnRNA in teleosts 143

T A B L E 2

Summary of Laboratory Flow Through/~-NF (Largemouth Bass), Caged (Largemouth Bass), and Wild (Killifish) Fish Studies of Laboratory and Environmental Exposure

Treatment ERO D ~ hnmunoblot

Flowthrough ~ - N F

1 day, control 1 day, 0.28 mg, liter

4 day, control 4 day, 0"28 mg liter

Cage f ish 1 day, control I day, cage

3 day, control 3 day, cage

7 day, control 7 day, cagc

2"036 4- 0"968 100 4- 61 13"0394-2'014" 25454- 18

3"049 4- 0"562 100 4- 7 7"578 _+ 1.000" 347 4- 5

2"620 4- 0-266 I00 + 43 2"499 + 1'215 76 4- 50

3-065 _+ I-158 100 4- 39 14.495 _ 2"143 139 4- 21

10654-0'156 1004-9 6-715 + 1"635 236+67

Wihl fish

Location Date EROD hnmunohh~t h C YP1.41 mRN.4 ~

Tuckerton July 1989 0-292 + 1'875 4- 0'464 Newark July 1989 0.764 + + 6602 4- 0803

Tuckcrton Oct. 1989 0-449 + ND a Nc~ark Oct. 1989 1"251 + + + ND

° f!ROD=ethoxyresorutin-O-deethylase, expressed as pmol/min:mg microsomal protein for bass and nmol/min mg microsomal protein for killifish. h lmmunoblot refcrs to immunodetection with rabbit anti-trout LM4. ~ (CYPIAI) IgG, expressed as percent of control, while for killifish, + + + = strong reaction, + + = moderate reaction, and + = slight reaction. cCYPIAI mRNA refers to hybridization with pfP~450-3' cDNA, expressed as units of optical density. '~ ND = Not determined.

s a m e c o n c e n t r a t i o n s the C Y P 1 A I m R N A is increased o v e r t ime a n d the

i n d u c t i o n is s ignif icant , at least to 7 d a y s o f e x p o s u r e to f l -NF. This is in c o n t r a s t to the ear l ier i.p. in jec t ion s tudies , in wh ich m R N A dec reased by

4 8 h pos t - in jec t ion , a n d m a y be due to differences in u p t a k e a n d / o r

d i spos i t i on o f the inducer . T h e l ower E R O D act iv i ty at h igher B - N F c o n c e n t r a t i o n s cou ld be due to the p resence o f inhib i tors , i nc lud ing the h igh

c o n c e n t r a t i o n s o f f l - N F itself. I n h i b i t i o n o f E R O D ac t iv i ty has been s h o w n

144 Mary L. Haasch et al.

previously with polychlorinated biphenyls. 5 This emphasizes the im- portance of using more than one bioindicator to assess induction.

At the lower concentrations of 2.5 to 10-0t~g fl-NF/liter, EROD activity and immunoreactive CYP1AI protein were increased in a concentration- dependent manner over time (Table 1). Environmental exposure of largemouth bass placed in cages in water known to be contaminated with PCBs had increased (six-fold) EROD activity at 3 days and 7 days ITable 2t. Killifish taken from a TCDD-contaminated site had three-fold higher EROD activity and CYPIA1 m R N A and in addition, had increased immunoreactive protein, than killifish from a "clean" site (Table 2). Levels of CYPIAI m R N A were induced for at least 7 days after flow-through exposure to an inducer in rainbow trout, and were elevated at least three- fold in killifish that have been exposed to TCDD for generations. While m R N A measurement is a natural progression of the work that has been done using catalytic activity and immunodetect ion of protein, the establishment and rigorous testing of this method's reliability under a variety ofcircumstances is still in its infancy. These data indicate the efficacy of using immunodetection of CYPIAI protein and nucleic acid hybridiz- ation o f C Y P I A 1 m R N A as bioindicators of environmental contamination. In the future, kits may become available for the rapid measurement of CYPIAI m R N A in field situations, with the proper consideration of appropriate controls, which theoretically could make m R N A induction a quick, simple, low-cost and reliable bioindicator of environmental chemical contamination.

A C K N O W L E D G E M E N T S

This work was supported by US PHS Grants ES01080, ES04184 IJ.J.L) and ES01407 (K.R.C.)

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C YPIA 1 protein and mRNA in teteosts 145

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