Identification of in vitro cytochrome P450 modulators to detect induction by prototype inducers in the mallard duckling (Anas platyrhynchos)

Comparative Biochemistry and Physiology Part C 122 (1999) 273 – 281

Identification of in vitro cytochrome P450 modulators to detect induction by prototype inducers in the mallard duckling (Anas platyrhynchos) Amy E. Renauld b, Mark J. Melancon a,*, Lorraine M. Sordillo b b

a USGS Patuxent Wildlife Research Center, 12011 Beech Forest Road, Laurel, MD 20708 -4041, USA Department of Veterinary Science, The Pennsyl6ania State Uni6ersity, Uni6ersity Park, PA 16802, USA

Received 3 May 1998; received in revised form 28 August 1998; accepted 3 September 1998

Abstract Seven modulators of mammalian monooxygenase activity were screened for their ability to selectively stimulate or inhibit in vitro monooxygenase activities of hepatic microsomes from mallard ducklings treated with phenobarbital, b-naphthoflavone, 3,3%,4,4%,5-pentachlorobiphenyl or vehicle. Microsomes were assayed fluorometrically for four monooxygenases: benzyloxy-, ethoxy-, methoxy-, and pentoxyresorufin-O-dealkylase, in combination with each of the seven modulators. Four combinations: a-naphthoflavone and 2-methylbenzimidazole with benzyloxyresorufin, and Proadifen with methoxy- and ethoxyresorufin, respectively, were evaluated further. b-Naphthoflavone-treated groups were clearly distinguished from the corn oil vehicle control group by all of the assays and by the effects of the modulators in three of the four assay/modulator combinations. Enzyme activities of the phenobarbital and saline groups were statistically similar (P] 0.05) when assayed without modulator added, but each assay/modulator combination distinguished between these groups. The PCB-treated group was distinguished from the corn oil vehicle control group only for BROD activity, with or without the presence of modulator. Graphing of per cent modulation of BROD activity versus initial BROD activity provided the clearest distinction between all of the study groups. Identification of these selective in vitro modulators may improve detection and measurement of low level cytochrome P450 induction in avian species. Also, both the monooxygenase activities induced and the impacts of the modulators indicated differences between mammalian and avian cytochromes P450. © 1999 Elsevier Science Inc. All rights reserved. Keywords: a-Naphthoflavone; Cytochrome P450; Mallard duck; 2-Methylbenzimidazole; Proadifen; Microsomes; Monooxygenase; Dealkylase

1. Introduction The cytochromes P450 (P450s) are a superfamily of heme containing proteins that are responsible for the Abbre6iations: ANF, a-naphthoflavone; BNF, b-naphthoflavone; BROD, benzyloxyresorufin-O-dealkylase; EROD, ethoxyresorufinO - dealkylase; 2 - MB, 2 - methylbenzimidazole; 5,6 - DMB, 5,6 - dimethylbenzimidazole; 3-MC, 3-methylcholanthrene; MROD, methoxyresorufin-O-dealkylase; P450, cytochrome P450; PB, phenobarbital; PCB, polychlorinated biphenyl; PROD, pentoxyresorufin-Odealkylase. * Corresponding author. Tel.: +1 301 4975710; fax: +1 301 4975675.

metabolism of many xenobiotics. Specific P450s involved in xenobiotic metabolism can be induced by exposure to certain classes of environmental contaminants so that increased production of these forms can be used as a biomarker of contaminant exposure. More specifically, the mammalian 1A and 2B P450 subfamilies are induced by 3-methylcholanthrene-like (3MC) and phenobarbital-like (PB) inducers, respectively. The use of P450s as a biomonitoring tool has been developed successfully in many species [23,25,29]. Earliest applications of P450s as biomarkers of environmental contamination focused on fish species [4], with later application to mammalian and avian species [7,24,26].

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P450 levels are frequently assessed by assaying monooxygenase activities that are associated with particular P450s. Elevated metabolism of specific alkylsubstituted-resorufin substrates has been shown to reflect induction of specific P450 isozymes by prototype inducer treatment in mammals [2,3,19]. Although the same monooxygenase activities can be measured in avian species, the correlations of inducers to substrate specificity in avian species differ from those in mammalian species [10,19,22,26]. In assessment of P450-associated monooxygenase activities in environmental samples, the exposure to an inducing agent may be too low to cause the large increases in monooxygenase activity which are frequently generated in controlled-exposure laboratory studies, and exposure of different specimens may vary. The use of monooxygenase modulators was considered as an approach to increase the ability of monooxygenase assays to discriminate between constitutive and induced P450s in birds. Chemical compounds have been identified that specifically affect the activity of the induced rather than constitutive isozymes in mammals, and the inclusion of such compounds into monooxygenase assays therefore has been instrumental in verifying the induction of particular isozymes [20]. Some of the compounds that have been shown to specifically modulate mammalian or piscine P450 enzyme activity include flavones [14,27], imidazoles [13,16,17,31], beta-diethylaminoethyl diphenylpropylacetate (known as Proadifen and as SKF525A), [5] and metyrapone [21,30]. Previous reports of the effects of in vitro modulators of avian monooxygenase activity have been of limited scope [12,28]. There have been no reports of comparative effects of a variety of such modulators on multiple avian monooxygenases. The effectiveness of P450-associated monooxygenases as biomarkers in avian species would be improved by the ability to more readily differentiate induced enzyme activity from constitutive. This would be particularly important in field-collected samples where one frequently has to distinguish between submaximal induction and constitutive activity. Therefore, the effects of a number of modulators of mammalian cytochrome P450-associated monooxygenase activities on constitutive and induced mallard duckling monooxygenases were examined.

2. Materials and methods

2.1. Animals Mallard ducklings (Anas platyrhynchos) 1-week-old were purchased from Whistling Wings, (Hanover, IL). A total of 56 animals were divided evenly into seven groups and housed separately with unlimited access to water and commercial feed. At 3 weeks of age, the

animals were weighed and injected intraperitoneally with inducing agents in vehicle or their respective vehicles alone as controls, at a volume of 1 ml kg − 1 of body weight. Saline (0.9% sodium chloride) was the vehicle for PB (delivered as sodium phenobarbital) which was administered at 20 mg kg − 1 body weight PB (LPB group) and 50 mg kg − 1 body weight PB (HPB group). These groups were injected every 24 h for 3 consecutive days and sacrificed 1 day after the last treatment. Corn oil was the vehicle for BNF which was administered at 20 mg BNF kg − 1 body weight (LBNF group) and 100 mg BNF kg − 1 body weight (HBNF group), respectively, and for 3,3%,4,4%,5-pentachlorobiphenyl (PCB, 99+ % pure, Accustandard, New Haven, CT) (PCB126) which was administered at 0.25 mg kg − 1 body weight (PCB126 group). These groups were injected only once, and sacrificed 72 h later. At the time of sacrifice, the birds were euthanized by carbon dioxide asphyxiation then weighed. Their livers were removed, weighed, divided, placed in cryotubes containing glycerol, and flash frozen in liquid nitrogen. The samples were then stored at − 80°C until used.

2.2. Monooxygenase assays Initially, pooled microsomes from the saline, HPB and HBNF groups were assayed for four monooxygenase activities, benzyloxy-, ethoxy-, methoxy- and pentoxyresorufin-O-dealkylase (BROD, EROD, MROD and PROD), with the seven candidate modulators a-naphthoflavone (ANF), b-naphthoflavone (BNF), benzimidazole, 2-methylbenzimidazole (2-MB), 5,6-dimethylbenzimidazole (5,6-DMB), 2-methyl-1,2-di-3pyridyl-1-propanone (metyrapone), and Proadifen (beta-diethylaminoethyl diphenylpropylacetate). All of these compounds were purchased from Sigma (St. Louis, MO). Based on literature values for effective concentrations of these seven modulators ranges of six concentrations were selected for screening of each modulator. The concentration ranges were 1× 10 − 3 –3× 10 − 6 M for the benzimidazoles, 3×10 − 4 –1× 10 − 6 M for the naphthoflavones, and 1×10 − 4 –3× 10 − 7 M for Proadifen and metyrapone. For each of the 84 combinations of treatment group, substrate and modulator, assay activity was examined with vehicle and with the six concentrations of each modulator. Hepatic microsomes were prepared by differential centrifugation of liver homogenates, and benzyloxy-, ethoxy-, methoxy- and pentoxyresorufin-O-dealkylases were assayed in a fluorescence microwell plate scanner in a 260-ml incubation essentially as described previously [22] for a number of avian species. Benzyloxy-, ethoxy-, methoxy- and pentoxyresorufin were purchased from Molecular Probes, (Eugene, OR) and dissolved in dimethylsulfoxide for use as substrates in the fluorometric in vitro assays BROD, EROD, MROD

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and PROD, respectively. The protocol was modified to permit the addition of the modulators or vehicle. The volume of Tris buffer in the reaction well was reduced to accommodate the volumes of vehicles required, which were 50 ml 0.07 M HCl for the benzimidazoles, 20 ml methanol for the naphthoflavones, 50 ml potassium/sodium tartrate for metyrapone and 50 ml Tris buffer for Proadifen. All comparisons were made to assays run with the appropriate vehicle as control. Microsomal protein was determined using the method described by Lowry et al. [18]. Fluorescence produced in the monooxygenase assays was converted to picomoles of product by use of a standard curve, and enzyme activity was expressed as pmoles of product produced per minute per milligram of microsomal protein. Effects of each modulator were quantified by calculating the activity produced with the modulator present as a percent of the activity produced in absence of the modulator. We chose the four combinations, BROD with 2-MB, BROD with ANF, EROD with Proadifen, and MROD with Proadifen, to test for their selectivity among individual samples of each of the 56 birds representing seven treatment groups. These samples were assayed in triplicate with and without modulators, with enzyme activity measured as previously described. PROD activity was not evaluated further because of low activity levels and lesser modulator effects.

2.3. Statistical analysis Enzyme activity of individual bird samples in each of these three assays with or without modulator was analyzed for the effect of treatment and modulator. Effects of each modulator were quantified by calculating the activity assayed with the modulator present as per cent of the activity assayed in the absence of the modulator. All values were first examined by Bartlett’s test for equality of variances and log transformed when appropriate. Differences in P450-associated monooxygenase activities among treated ducklings and their respective vehicle-injected groups, and among treatment groups were determined for each assay/modulator combination by ANOVA and Tukey’s two-tailed test using GraphPad Prism (GraphPad Software, San Diego, CA). The line equations of per cent activity with modulator against initial enzyme activity were calculated using proc nlin in SAS (SAS Institute, Cary, NC).

3. Results BROD, EROD and MROD activities (pmol product min − 1 per mg microsomal protein) of the seven groups of mallard ducklings are given in Table 1. Although the variances differed significantly between groups for all

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three monooxygenase activities, BNF at both 20 and 200 mg kg − 1 body weight had such great effects that they were significantly different from the other groups for all three activities. However, because of the differences in variance between groups, all of the comparisons in Table 1 are based on log transformed values. Treatment of mallard ducklings with phenobarbital at 20 or 50 mg kg − 1 body weight did not produce BROD, EROD or MROD activities significantly different (P] 0.05) from those of the saline control group (Table 1). However, treatment with either concentration of BNF resulted in significantly greater (P5 0.05) hepatic microsomal BROD, EROD and MROD activities than the corn oil-treated control group (Table 1). Additionally, the HBNF group had significantly greater (P5 0.05) activity than the LBNF group in each of the assays (Table 1). Although the monooxygenase activities of the PCB126-treated birds appeared elevated, only the BROD activity was significantly different (P5 0.05) from the corn oil control group (Table 1). When evaluating the effects of addition of ANF or 2-MB to the BROD assay, the variances differed significantly between groups. Using log transformed data reduced the variances so all of the comparisons of the effects of ANF and 2-MB in Table 2 are based on log transformed values. Addition of ANF or 2-MB (Table 2) affected BROD activity much differently (P50.05) in saline and PB treatment groups, but did not differentiate between the two levels of PB treatment. As in the absence of these two modulators, BROD activities in the LBNF and HBNF treatment groups with ANF or 2-MB added were significantly different (P5 0.05) from that in the corn oil group. In contrast, although the Table 1 Hepatic microsomal BROD, EROD And MROD activity of induced and control mallard ducklings Treatment1

BROD2

EROD

MROD

Saline LPB3 HPB Corn oil LBNF4 HBNF PCB1265

8.3 91.3C 10.4 9 1.0C 9.0 91.3C 16.2 9 2.2C 182.1 918.5A 303.1 944.7A 56.3 9 9.6B

22.9 9 3.1C 37.5 9 2.8BC 43.9 99.2C 38.4 9 3.9BC 211.5 915.2A 366.0 959.8A 80.1 9 14.4B

20.6 92.3A 25.0 9 3.3B 28.3 9 3.1B 26.6 9 3.6B 86.0 9 7.3B 143.7 919.6A 39.4 9 7.5B

A,B,C

Different letters represent significant differences (PB0.05) between treatment groups within each assay.All data were log transformed and comparisons were done by Tukey’s test. 1 All treatments delivered in 1 ml kg−1 body weight. 2 Mean (n =8 ducklings) pmol product min−1 per mg microsomal protein 9 S.E. 3 LPB and HPB groups received three i.p. injections of 20 and 50 mg phenobarbital kg−1 body weight, respectively. 4 LBNF and HBNF groups received i.p. injections of 20 and 200 mg BNF kg−1 body weight, respectively. 5 PCB126 group received i.p. injection of 0.25 mg 3,3%,4,4%,5-pentachlorobiphenyl kg−1 body weight.

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Table 2 Effect of ANF and 2-MB on control and induced mallard duckling hepatic microsomal BROD activity Treatment1

Saline LPB HPB Corn oil LBNF HBNF PCB126

BROD activity2

BROD activity with ANF3

BROD activity with 2-MB4

Tukey group5

% of Baseline6

Tukey group

% of Baseline

Tukey group

C C C C A A B

86.5 9 17.9 144.19 14.4 185.9 9 13.1 52.7 9 9.4 6.3 9 1.4 5.79 0.5 24.7 9 6.4

B A A B D D C

74.3 99.8 164.5 99.7 245.1 933.7 45.3 91.3 11.69 1.3 9.69 0.6 26.3 9 6.3

B A A B D D C

1

All treatments delivered in 1 ml kg−1 body weight. Mean baseline activity (n=8 ducklings); activity expressed as pmol prod min−1 per mg microsomal protein (Table 1). 3 Addition of 0.003 mM ANF, calculated as % of baseline activity. 4 Addition of 3 mM 2-MB, calculated as % of baseline activity. 5 Different letters represent significant differences (P50.05) between treatments within each column. All data were log transformed and comparisons were done by Tukey’s test. 6 Mean activity (n =8) 9 S.E.; activity expressed as % of baseline. 2

assay without ANF or 2-MB added distinguished between the LBNF and HBNF treatment groups, inclusion of ANF or 2-MB in the assay reduced the microsomal BROD activities of the LBNF and HBNF groups similarly, so that they were not distinguishable (P ] 0.05, Table 2) by the effects of the modulators. Addition of ANF or 2-MB into the assay resulted in significant differences (P 5 0.05) between corn oil and PCB126 treatment groups (Table 2). Addition of 0.001 mM Proadifen to the EROD assay resulted in inhibition of saline control and LPB- and HPB-induced activities by differing extents to 75, 49 and 29%, respectively of original activity, thereby allowing the distinction (P 50.05) of all of these groups from each other (Table 3). The effect of Proadifen on EROD activity in all of the groups other than the PB groups was so similar that with Proadifen added the assay was no longer able to distinguish between BNF and PCB and control treatments. As with microsomal EROD activity, inclusion of Proadifen into the MROD assay resulted in significantly different (P 50.05) inhibition of control and LPB- and HPB-induced microsomal activities to 81, 44 and 29%, respectively (Table 3). These differences allowed each of the PB treatment groups to be distinguished from control treatment, and from each other. Also, as with microsomal EROD activity, inclusion of Proadifen into the MROD assay decreased discrimination between groups other than the PB groups.

4. Discussion

4.1. Induction of P450 We were interested in identifying compounds that

modulate P450 activity in the BROD, EROD, MROD or PROD assays for several reasons. Substrates in these assays have been shown to be metabolized preferentially in mammals by specific subfamilies of P450s [2,3,19] which, in turn, are induced by exposure to specific classes of environmental contaminants. By these characteristics, the P450s may serve for assessment of exposures or treatments. However, these assays often cannot conclusively detect induction even when animals are selected from areas of known contamination or have been treated intentionally with inducing agents. In our study, for example, differences in the levels of some of the P450-associated monooxygenase activities as measured by these assays were often not sufficient to distinguish inducer-treated from control ducklings. In the environment the situation is more complex because organisms of the same species collected in the same area may differ in their P450 status not just because of contaminant exposure differences, but also because of genetic differences and feeding differences, even when age, time of year and reproductive status are considered. Thus at a contaminated site there may be nominally uninduced animals, slightly induced animals and highly induced animals and it can be difficult to distinguish whether the monooxygenase activity of that group of animals is significantly different from that of a group from a reference location. The inability of these substrates to always clearly identify induction has been recognized by others [9,26]. In the three papers referenced for background for this series of monooxygenase substrates [2,3,19] MROD and EROD activities were highly related to treatment with 3-MC-type inducers, with little induction by PB. BROD and PROD activities were highly related to PB administration over 3-MC in rat and mouse, were little

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Table 3 Effect of proadifen on control and induced mallard duckling hepatic microsomal EROD and MROD activity Treatment1

Saline LPB HPB Corn oil LBNF HBNF PCB126

EROD activity2

EROD activity with Proadifen3

MROD activity2

MROD activity with Proadifen4

Tukey group5

% of Baseline6

Tukey group

Tukey group

% of Baseline

Tukey group

C BC C BC A A B

75.59 6.1 49.19 3.9 28.79 1.7 82.6 9 2.4 93.09 1.3 91.9 9 2.2 86.19 1.2

A B C A A A A

B B B B A A B

81.5 93.9 43.893.1 28.6 93.2 68.493.3 84.191.5 87.0 91.6 75.892.0

A C D B A A AB

1

All treatments delivered in 1 ml kg−1 body weight. Mean baseline activity (n=8 ducklings); activity expressed as pmol prod min−1 per mg microsomal protein (Table 1). 3 Addition of 0.001 mM Proadifen, calculated as % of baseline activity. 4 Addition of 0.003 mM Proadifen, calculated as % of baseline activity. 5 Different letters represent significant differences (P50.05) between treatments within each column. Enzyme activity values before addition of modulator were log transformed, but activity values after addition of modulator were not. Comparisons were done by Tukey’s test. 6 Mean activity (n =8) 9 S.E.; activity expressed as % of baseline. 2

affected by either PB or 3-MC administration in hamster, and responded more to 3-MC-type inducers than PB in quail. Aroclor 1254 increased these enzyme activities in every case where 3-MC-type or PB had done so individually. In studies of 14 avian species that utilized these same four monooxygenase activities BROD or PROD or both activities were frequently induced by 3MC-type inducing agents [22]. Most of nine avian species administered PB in these studies failed to demonstrate significant increases in BROD or PROD. In the present study the minimal induction of BROD and PROD activities, and significant induction of EROD activity produced in mallard ducklings by treatment with PB and BNF, respectively, are consistent with results obtained using other avian species [1,22]. In two studies with adult mallard ducks, BNF increased EROD [15,22], PROD [15,22], and BROD [22] activities. In the same studies PB failed to increase PROD activity, but increased EROD activity significantly in one of the studies [15] and caused a slight but insignificant increase in the other [22]. Administration of Aroclor 1254 to mallard ducks [8] elevated both EROD and PROD activities, but because Aroclor 1254 contains multiple types of inducers interpretation is limited. Although our results with mallard ducks agree with previous studies involving adult mallard ducks and other avian species, they differ from results in mammals where dramatic increases in BROD and PROD enzyme activities can be produced following PB-like induction. Mammalian P450s exhibit greater activity against some non-specific experimental substrates than do aves. Phenobarbital induction in aves is different than that in mammals as it appears to induce a different set of related P450 isozymes [10,11]. Our results indicate that these monooxygenase activities in mallard ducklings are

less responsive to PB induction than BNF induction. Additionally, the mallard duck PB-inducible P450s exhibited low activity against those resorufin ethers that, in contrast, are readily metabolized by mammalian PB-induced P450s. The lesser responsiveness of aves to some types of P450 inducers, lower activity against some experimental substrates, and differences in protein induction patterns can present a diagnostic dilemma when the objective is to identify induction and attribute it to specific exposures.

4.2. Effects of ANF and 2 -MB Differentiation of control and PB treatments by ANF was accomplished by enhancement of PB-induced microsomal BROD activity. Stimulation of the PB-induced microsomal activity by ANF was significant, with enzyme activity of the LPB and HPB groups being 144 and 186%, respectively, of baseline activity compared to a 13% inhibition of control activity. Stimulation of these isozyme specific P450 activities by ANF is thought to occur through a mechanism involving substrate activation [27]. As opposed to the stimulation of PB-induced microsomal activity by ANF, our results indicate that ANF significantly inhibits BNF- and PCB126-induced microsomal activity in the mallard duck. Inhibition of the CYP1A subfamily monooxygenase activities by ANF has been previously recognized in captive and wild bird species [12]. Fig. 1 presents graphically the impact of ANF on BROD activity for each of the ducklings studied. The calculated line equation is: % original BROD activity with ANF added =(893.9/original BROD activity)+ 10.0; r 2 = 0.83. When presented in this manner it is clear that CYP1A-related BROD

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Fig. 1. Effect of a-naphthoflavone on mallard duckling hepatic microsomal BROD activity. Graph of percent of original BROD activity with ANF added against the BROD activity without ANF addition. Line equation calculated as: % original BROD activity with ANF added = (893.9/ original BROD activity) +10.0; r 2 = 0.83.

activity is severely inhibited, BROD activity in PBtreated individuals is generally significantly stimulated, and constitutive BROD activity is inhibited or stimulated more moderately.

Similar to the effect of ANF, addition of 2-MB to the BROD assay also stimulated PB-induced microsomal activity so that the PB-treated groups were separated from each other and from the control group. However,

Fig. 2. Effect of 2-methylbenzimidazole on mallard duckling hepatic microsomal BROD activity. Graph of percent of original BROD activity with 2MB added against the BROD activity without 2MB addition. Line equation calculated as: % original BROD activity with 2MB added= (652.5/ original BROD activity) +21.5; r 2 = 0.82.

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Fig. 3. Effect of Proadifen on mallard duckling hepatic microsomal EROD and MROD activities. Column scatter graphs of the percent of original EROD and MROD activities with Proadifen added for each of the treatment groups.

HPB-induced microsomal activity was enhanced to a greater extent by 2-MB than ANF (245 and 186%, respectively). Studies involving the effect of benzimidazoles on P450 activity reveal that 2-MB has a higher affinity for binding PB-induced P450s than control P450s in rats [6]. Differences in affinity may explain the preferential modulation of PB-induced microsomes by 2-MB in our study. While many researchers have evaluated the inhibitory and stimulatory effects of benzimidazoles on P450-associated monooxygenase activities from fish and mammals [6,16,17,31], the stimulatory effect of 2-MB on PB-induced avian P450 activity has not, until now, been reported.

Our study revealed that, like ANF, 2-MB also can inhibit CYP1A-associated monooxygenase activity from BNF- and PCB126-treated mallard ducklings. Similarly, P450-associated monooxygenase inhibition by this compound has been recognized previously in mammals and fish [6,16]. Fig. 2 presents graphically the impact of 2-MB on BROD activity for each of the ducklings studied. The calculated line equation is: % original BROD activity with 2-MB added=(652.5/ original BROD activity) + 21.5; r 2 = 0.82. In this case also it is clear that CYP1A-associated BROD activity is severely inhibited, BROD activity in PB-treated individuals is generally significantly stimulated, and constitu-

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tive BROD activity is inhibited or stimulated more moderately. Although ANF and 2-MB had qualitatively similar effects on BROD activity from the variously treated ducklings, they were quantitatively different. It can be seen in Fig. 2 that there is less overlap between the effects of 2-MB on the BROD activity of the PBtreated, control and CYP1A inducer-treated groups than in the effects of ANF (Fig. 1). This would be advantageous in environmentally collected samples where P450 status can differ greatly between individual animals for reasons described above.

4.3. Effects of Proadifen Addition of Proadifen to the EROD and MROD assays discriminated between PB-induced monooxygenase activity and control activity. Proadifen has been shown to be a specific inhibitor of PB-induced, but not 3-MC induced or constitutive monooxygenase activity in rats [16], but its ability via EROD and MROD activities to differentiate between low levels of PB induction and controls is a novel observation. These activities generally are used only as indicators of CYP1A induction, because mammalian PB-induced microsomes exhibit little activity toward either ethoxyresorufin or methoxyresorufin [3]. In both assays, Proadifen differentially inhibited PB-induced monooxygenase activities with increasing doses of PB, thereby allowing separation of control from LPB, and LPB from HPB treatment. The impact of Proadifen can be seen more clearly from the column scatter graphs in Fig. 3. In contrast to the significant inhibitory effect of Proadifen on PB-induced monooxygenase activity, this modulator caused much less inhibition for the other treatment groups and thus was not effective at discriminating between control and BNF- and PCB126-induced monooxygenase activity. It can be seen from the column scatter graphs of the effect of Proadifen on the variously treated groups (Fig. 3) that Proadifen more clearly distinguished between the EROD than the MROD activities of these groups. The inhibition of EROD activity by Proadifen resulted in no overlap between the results with the PB-treated groups and any of the other groups. This would be very helpful in examining for PB-type induction. In addition to the use of these modulators to improve the ability to discriminate between groups of samples from different locations is the increased ability to determine whether an individual sample is induced. Thus, in spite of the difficulties introduced by contaminant exposure, genetic, and feeding differences, even when age, time of year and reproductive status are considered as mentioned earlier, we will be better able to discriminate between contaminant exposed and non-contaminant ex-

posed birds regardless of their absolute levels of monooxygenase activity.

4.4. Conclusions In conclusion, the fluorometric monooxygenase assays BROD, EROD, and MROD, with the selected modulators as appropriate were able to distinguish between types and levels of cytochrome P450 induction by PB, BNF or PCB126. The use of modulators of cytochrome P450-associated monooxygenase activities can improve the assessment of cytochrome P450 status in mallard ducks and facilitate the use of this biomarker for monitoring animal populations or environmental health. Future studies will examine the impact of these modulators on monooxygenase activity in other avian species and the application of these modulators to field-collected samples.

Acknowledgements The authors wish to acknowledge the assistance of Mary Paul and James Riggs for animal care, John Eisemann and Diane Beeman for enzyme assays, Jeff Hatfield for statistical help and Kinard Boone for preparing the figures. We also wish to acknowledge the critical review of the manuscript by Barnett Rattner and David Hoffman.

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