Propiconazole, Clotrimazole - American Chemical Society

Chapter 10

Modulation of Teleost CYP1A1 Activity by EBI Fungicides (Propiconazole, Clotrimazole) and Interaction with an Organophosphate Insecticide Downloaded by PENNSYLVANIA STATE UNIV on September 17, 2012 | http://pubs.acs.org Publication Date: November 1, 2000 | doi: 10.1021/bk-2001-0771.ch010

1

2

S. L . Levine , J. S. McClain , and J. T. Oris

2

1

Department of Veterinary Science, Pennsylvania State University, University Park, P A 16802 Department of Zoology, Miami University, Oxford, O H 45056 2

This review examines mechanisms by which clotrimazole and a technical grade formulation of propiconazole (TGP) modulate cytochrome P4501A1 (CYP1A1) expression and mechanisms by which TGP may enhance the acute toxicity of the organophosphate insecticide parathion. In vitro, clotrimazole was a mixed-type noncompetitive inhibitor and TGP was a noncompetitive inhibitor of rainbow trout hepatic microsomal CYP1A1 catalytic activity. In vivo, analytical grade formulations of clotrimazole and propiconazole caused inhibition of CYP1A1 catalytic activity but did not influence hepatic CYP1A1 gene expression. In contrast, rainbow trout hepatic CYP1A1 expression following waterborne exposures to TGP exhibited a mixed-pattern response. The mixed-pattern response was characterized by inhibition of CYP1A1 catalytic activity and concurrent induction of CYP1A1 mRNA levels. Induction of CYP1A1 expression by TGP was determined to result from AhR-active compounds within the formulation. Although exposure to TGP can modulate CYP1A1 expression, it remains to be determined whether modulation of CYP1A1 is related to the enhancement of acute parathion toxicity to fathead minnows.

Introduction Over the past two decades a number of ergosterol biosynthesis inhibiting (EBI) imidazole and triazole derivatives have been approved for use in agricultural applications. These fungicides were designed to inhibit cytochrome P450-mediated ergosterol biosynthesis and are used as systemic foliar fungicides [1]. A technical 130

© 2001 American Chemical Society

In Pesticides and Wildlife; Johnston, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

Downloaded by PENNSYLVANIA STATE UNIV on September 17, 2012 | http://pubs.acs.org Publication Date: November 1, 2000 | doi: 10.1021/bk-2001-0771.ch010

131 grade formulation of the antifungal triazole compound propiconazole (Figure 1) has been shown to be a potent inducer of cytochrome P450 drug metabolizing isoforms in mammalian species [2,3], bobwhite quail [3,4], and recently in rainbow trout (Oncorhyncus mykiss) [5]. Currently, technical grade propiconazole (TGP) is widely used on banana plantations and TGP is aerially applied 40 or more times per year in an attempt to control the fungal disease Black Sigatoka [6]. Concerns have been raised in the scientific and popular press over potential toxic interactions between antifungal imidazole/triazole compounds and organophosphate (OP) insecticides [7,8]. Presently, the opportunity exists for concurrent application of TGP along and different OP insecticides on banana plantations [9]. As a result of high precipitation rates, irrigation practices, and an unenclosed design, a large quantity of pesticide applications on banana plantations is lost to surface runoff, leaching, and erosion [6]. Aquatic systems are often incorporated into drainage systems to allow excess water to runoff into local streams and rivers for additional restriction of fungal growth [9,10]. Therefore, aquatic ecosystems near areas of frequent applications could receive high inputs of TGP. In a recent analysis of pesticide concentrations in Costa Rican banana plantations, propiconazole concentrations in nearby streams were as high as 24 μg/L during the dry season [11]. During periods of high precipitation, propiconazole concentrations in aquatic ecosystems could reach concentrations higher than 24 μg/L. In a comprehensive study which surveyed pesticide concentrations in aquatic ecosystems of Central America, propiconazole was -found to be the most widely distributed pesticide [12]. Propiconazole was present in 60% of the samples collected in effluents, 56% of the samples collected in creeks, 43% of the samples collected from mainrivers,and some riverine sediment samples reached 130 μg/kg [12]. Because of the potential for interactions between TGP and other pesticides, there is the need to develop a biomarker for TGP exposure. One candidate biomarker for TGP exposure is cytochrome P4501A1 (CYP1A1) expression. This chapter summarizes recent studies which: 1) Characterized the influence of a model EBI compound (clotrimazole; Figure 1) and TGP on CYP1A1 expression; 2) Examined whether CYP1A1 expression could be used as a biomarker of TGP exposure; and 3) Explored the hypothesis that a mechanistic relationship exists between modulation of cytochrome P450 activity by TGP and enhanced toxicity of OP insecticides to fish. This hypothesis was initially tested with the EBI compounds prochloraz and penconazole with red-legged partridge [13-14]. These studies demonstrated that some EBI compounds can enhance the toxicity of OP insecticides.

Figure 1. Structure of clotrimazole and propiconazole. In Pesticides and Wildlife; Johnston, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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Regulation of CYP1A1 Expression and its use as a Biomarker The molecular mechanisms involved in induction CYP1A1 expression have been extensively studied in mammalian species. Induction of CYP1A1 expression in mammalian and teleost species is initiated with the binding of the aryl hydrocarbon receptor (AhR) by a ligand. Well-characterized AhR ligands include aromatic hydrocarbons, polychlorinated biphenyls, dibenzodioxins, and dibenzofuran congeners [15]. In mammalian models, the ligand activated AhR translocates to the nucleus [16], binds with the AhR nuclear translocator protein, and then dimerizes to specific DNA sequences termed dioxin responsive elements (DREs) in the regulatory region of the gene [17]. A similar induction process is believed to occur in fish [18], Currently, there is little information on the functionality of the teleost AhR in terms of binding affinities for nontraditional agonists, particularly at environmentally relevant concentrations. The primary role of the CYP1A1 enzyme is to enhance the elimination of water insoluble compounds [19]. A single atom of oxygen is added to the foreign molecule resulting in the addition of a hydroxyl group which is the first phase for increasing the water solubility and hence the excretability of the compound. The CYP1A1 enzyme accounts for the majority of benzo[a]pyrene (BaP) hydroxylase activity in mammals [20] and fish [19]. Induction of CYP1A1 expression in fish liver and gill has been successfully used to identify exposure BaP [21] and other planar environmental contaminants [22]. However, the use of CYP1A1 expression and other cytochrome P450 genes as a biomarker for TGP and many other pesticides/fungicides requires further investigation. Mechanisms of CYP1A1 Inhibition by Clotrimazole and Propiconazole EBI imidazole and triazole compounds have been shown to either act partially or exclusively as noncompetitive inhibitors of CYP1A1 catalytic activity. Initial work with hepatic microsomes isolated from gizzard shad (Dorosoma cepedianum) and difference spectroscopy demonstrated that clotrimazole can directly bind to the heme of cytochromes P450 [23]. In these experiments, addition of 0.1 μΜ clotrimazole to the reference cuvette resulted in the formation of a type II binding spectrum (Figure 2, curve 2). The type II spectrum was characterized by a gain in absorbance at 427 nm and a loss of absorbance between 400 to 412 nm. Addition of clotrimazole to a final concentration of 1 μΜ resulted in an increase in the magnitude of the type II binding spectrum (Figure 2, curve 3). The isobetic point for the two binding curves occurred at 417 nm which is approximately the same isobetic point found with mammalian hepatic microsomes. Bubbling the reference cuvette for 30 sec with CO and reducing with sodium dithionite displaced clotrimazole allowing for recovery of the cytochrome P450 difference spectra (Figure 2, curve 4). Recovery of the cytochrome P450 difference spectra demonstrated clotrimazole reversibly binds to the heme of cytochromes P450. In a similar manner, propiconazole was found to directly bind to the heme of cytochromes P450 and can be displaced by CO [5].


133 0.0404

ABS


0.020

-0.010. 390

500 Wavelength (nm]

Figure 2. Type II binding spectra for gizzard shad hepatic microsomes after the addition of clotrimazole to the reference cuvette (curves 2 and 3) and recovery of the CO difference spectrum (curve 4). Scan line 1 represents equal absorbance between cuvettes. (Reproduced from reference 23, Copyright 1997 Environmental Toxicology and Chemistry.) Although, by definition, coordination with the heme of cytochromes P450 is a noncompetitive mechanism, CYP1A1 kinetic assays were performed with rainbow trout hepatic microsomes to determine if inhibition of CYP1A1 catalytic activity, indexed as ethoxyresorufin-O-deethylase (EROD) activity, was either purely noncompetitive or noncompetitive with a competitive component. Clotrimazole caused a decrease in apparent V values and a increase in apparent K values [23,24], which is indicative of a noncompetitive mixed-type inhibitor (Figure 3a) [25]. Noncompetitive mixed-type inhibitors have different affinities for the free enzyme and the enzyme-substrate complex (Kj Φ Kjg) [25]. Secondary plots indicated that Kj and Kjg values were significantly different; the value for Kj was calculated to be 3 ± 3 nM and the value for Kjg was calculated to be 1900 ± 5 nM [24]. Kjg being greater than Kj indicates that clotrimazole has a greater affinity for the free enzyme over the enzyme substrate complex and also indicates that the noncompetitive component is the dominant mechanism of inhibition [25]. Increases in propiconazole concentration also caused apparent V values to decrease but propiconazole did not cause a significant effect on apparent K values (Figure 3b) [5]. The lack of significant differences in apparent K values, along with a concentration-dependent decrease in apparent V values, is indicative of purely noncompetitive inhibition of EROD activity. The IC50 values for clotrimazole and propiconazole were 0.51 ± 0.01 μΜ and 4.7 ± 0.1 μΜ, respectively [23,5]. Clotrimazole's mixed-pattern of inhibition allows it to be a more effective CYP1A1 inhibitor compared to propiconazole [23,24]. m a x

m

m a x

m

m

m a x


134


a.

b.

ΟμΜ

ΐσ2μΜ

10-ι μΜ

ΟμΜ

10-2 Μ μ

10-1 μΜ

Figure 3, Inhibition of EROD activity + SEM by (a) clotrimazole (b) propiconazole. Values with an asterisk are significantly different from control values (μΜ) (ρ < 0.05). (Figure 3b is reproduced from reference 5, Copyright 1999 Environmental Toxicology and Chemistry.) In Vivo Effects of Propiconazole on CYP1A1 Expression In Vivo, TGP modulated hepatic CYP1A1 expression at the transcriptional and catalytic levels [5]]. Rainbow trout given 24 h waterborne exposures to concentrations > 19 μg TGP/L demonstrated a mixed-pattern response. The mixedpattern response was characterized by induction of CYP1A1 gene expression but inhibition of EROD activity (Figure 4a). Although EROD activity was elevated following exposure to 90 μg TGP/L, the level of EROD activity was lower than expected based on CYP1A1 mRNA levels. Induction of CYP1A1 mRNA levels suggested that TGP contained a compound(s) that was capable of activating AhRmediated CYP1A1 expression. Since 19 μg TGP/L caused induction of CYP1A mRNA levels along with significant inhibition of EROD activity after 24 h of exposure, this concentration was chosen to examine the influence of TGP on hepatic CYP1A1 expression over a 120 h exposure [5]. A 120 h exposure to 19 μg TGP/L produced a biphasic response with CYP1 A l mRNA levels and EROD activity (Figure 4b). The biphasic response at the level of gene expression was characterized by initial induction of CYP1A1 mRNA levels after 24 h of exposure but a return to basal levels between 72 and 120 h of exposure. The biphasic response at the catalytic level was characterized by initial inhibition of EROD activity after 24 h of exposure followed by induction of EROD activity after 72 h of exposure. The decrease of CYP1A1 mRNA levels between 72 and 120 h of exposure may have resulted from first-pass metabolism of an AhR ligand(s) in TGP as it partitioned through the gill membrane. In other words, high deactivation rates of AhR-active compounds by the gill between 72 and 120 h of exposure caused a decrease in the amount of AhR-active compounds that reached the liver through the systemic circulation. Cytochrome P450 monooxygenase systems in gills may have toxicological significance because they are in direct contact with chemicals dissolved in the water, and these chemicals must pass directly through the branchial epithelium before entering the circulation. In a recent investigation, our lab


135


demonstrated that hepatic CYP1A1 mRNA levels also returned to basal levels between 72 and 120 h of continuous BaP-exposure, however, gill CYP1A1 mRNA levels remained maximally induced between 72 and 120 h of exposure [21]. It was hypothesized that significant first-pass metabolism of BaP by the gill, as BaP partitioned into the systemic circulation, modulated hepatic CYP1A1 expression during continuous exposures. Since TGP can induce CYP1A1 expression, measurement of CYP1A1 mRNA levels in a tissue, such as gill, which is in direct contact with the environment could potentially be used to identify exposure to TGP in the field.

[TGP] ML)

Time (h)

Figure 4. (a) Concentration-response and (b) time-response for rainbow trout hepatic CYP1A1 mRNA + SEM and EROD activity + SEM. Values with an asterisk represent values that are significantly different compared to control values (p < 0.05). (Figures are reproduced from reference 5, Copyright 1999 Environmental Toxicology and Chemistry.) Unidentified Compounds in TGP Cause CYP1A1 Induction To determine if induction of hepatic CYP1A1 mRNA levels resulted from propiconazole itself being an AhR ligand, or from the presence of an unidentified AhR ligand(s) in TGP, rainbow trout received intraperitoneal (i.p.) injections with either TGP (90.2% purity) or with AGP (99.9% purity). Fish that were treated with 100 mg TGP/kg had an 1100% increase in CYP1 A l mRNA levels and a, lower but, significant 500% increase in EROD activity (Figure 5a) [5]. However, 100 mg AGP/kg did not affect CYP1A1 mRNA levels, but caused a greater than 80% inhibition of EROD activity (Figure 5a) [5]. Comparable results to those reported with AGP, were obtained with rainbow trout that were treated with 50 mg clotrimazole (99.9% purity)/kg [24]. Analytical grade formulations of these compounds only caused inhibition of EROD activity and did not influence CYP1A1 gene expression. The inability of either analytical grade clotrimazole or AGP to activate rainbow trout CYP1A1 gene expression, is consistent with the assumption that TGP contains an unidentified compound(s) which is an AhR ligand. An additional comparison between AGP and TGP was conducted with a recombinant mammalian HepG2/40-6 cell line [5]. The HepG2/40-6 cell line was In Pesticides and Wildlife; Johnston, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.


136 created by stably integrating a plasmid that contains the firefly luciferase gene that is under the control of several DREs which respond to AhR agonists with a concentration-, time- and AhR-specific induction of luciferase activity [26]. HepG2/40-6 cells were incubated with either carrier alone, AGP, TGP, or 2,3,7,8tetrachlorodibenzo-/?-dioxin (TCDD) as a positive control in a 24-well plate format for 4 h. Incubations with AGP at a maximum concentration of 100 μΜ caused only a slight increase in DRE-dependent luciferase activity, however, incubations with TGP concentrations ranging from 2 to 100 μΜ caused a concentration-dependent increase in luciferase activity (Figure 5b). Induction of luciferase activity in the HepG2/40-6 cell line and induction of EROD activity in vivo by TGP implies ligand mediated activation of the AhR, but does not directly confirm it. In order to assess the ability of these formulations to transform the AhR into its active DNA-binding form, gel retardation analysis using guinea pig hepatic cytosol was conducted [5]. Although this assay does not directly confirm the ability of an AhR agonist to induce CYP1A1 expression, the positive correlation between induction of luciferase activity in the HepG2/40-6 cell line and the ability of TGP to stimulate AhR transformation and DNA-binding demonstrated that TGP contains an AhR agonist (Figure 5c).

Figure 5. (a) Modulation of hepatic CYP1A1 expression by TGP and AGP; EROD activity is expressed as pmol/min/mg protein and the inset shows slot blots for CYP1A mRNA levels, (b) Influence of AGP, TGP, and TCDD on DRE-driven luciferase activity. Values are expressed as relative light units (RLU) and values with In Pesticides and Wildlife; Johnston, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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an asterisk are different from controls (p < 0.05) (c) Gel mobility shift induced by 1 nM TCDD, 100 μΜ AGP, and 100 μΜ TGP. The arrow indicates the retarded band induced by ligand-AhR-DRE complex. (Figures 5a and 5c are reproduced from reference 5, Copyright 1999 Environmental Toxicology and Chemistry.)

In our initial work with TGP, reverse-phase HPLC with spectrophotometric analysis (λ = 268 nm) was used to detect the presence of AhR-active compounds within TGP that were not found in AGP [5]. It was hypothesized that the compounds that were detected at 268 nm may contain AhR-active compounds. To isolate TGP fractions which contain AhR ligands, 50 μι of 100 mM TGP was injected onto a C18 reverse-phase column (4.6 X 150 mm, 5 micron) and eluted with a linear gradient from 30% acetonitrile:70% water to 70% acetonitrile:30% water over 45 min at 30 °C with a flow rate of 1.0 mL/min [5]. Fractions were collected at 1 min intervals, vacuum concentrated, resuspended in 5 μΐ, of DMSO, and then added to the HepG2/40-6 cells in a 24 well-plate format and luciferase activity was measured 4 h post-exposure. Many of the TGP fractions contain an AhR active compound(s) (Figure 6). Fractions 6, 16, 19, 36, and 42 showed the highest induction of DREdriven luciferase activity. Fractions 29-30 contained concentrations greater than 0.1 M propiconazole and propiconazole at a concentrations greater than 200 μΜ were toxic to the HepG2/40-6 cell line. Therefore, to test for the presence of AhR-active compounds in fractions 29-31 a concentration response experiment was conducted with concentrations ranging from 1 μΜ to 100 μΜ TGP.

Figure 6. Identification of TGP fractions which cause induction of luciferase activity in the HepG2/40-6 cell line. The dotted line represents relative luciferase activity (RLU) and the solid line is the chromatograph for TGP.


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The fractions containing propiconazole (fractions 29-31) do not appear to contain a significant amount of an AhR ligand(s) (Figure 7a). The concentrationresponse with the combined propiconazole fractions 29-31, were in agreement with results obtained with 100 μΜ AGP (Figure 5a). To further characterize the inducing potential of AhR active compounds within TGP, several fractions which induced luciferase activity in Figure 6 were combined and retested in the HepG2/40-6 cells in a 24 well-plate format. The selected fractions produced a concentration-dependent increase in luciferase activity (Figure 7b).

a. 2η

% of Fractions

Figure 7. (a) Influence of combined fractions 29-31 on relative luciferase activity (RLU) in the HepG2/40-6 cell line. Values with an asterisk are different from 0% controls (p < 0.05). (b) HepG2/40-6 concentration-response for combined TGP fractions that contained AhR-active compounds in Figure 6. Interactions between EBI Fungicides and Cytochrome P450 Substrates Preliminary studies were conducted with clotrimazole to determine if pretreatment with this compound could influence BaP-bioconcentration. BaP is primarily metabolized by CYP1A1, but many other CYP isoforms are capable of



139 metabolizing BaP to more water soluble products [19]. To assess the influence of clotrimazole on BaP-bioconcentration, gizzard shad were injected with 50 mg clotrimazole (99.9% purity)/kg and 48 h later given waterborne exposures to approximately 1 μg BaP/L [23]. Gizzard shad that were pre-treated with clotrimazole had significant inhibition of EROD activity, compared to fish exposed to BaP alone, and bioconcentrated 8- and 11-times more parent-BaP after 1 and 3 d of BaPexposure, respectively (Table 1). Parent-BaP was separated from BaP-metabolites by reverse phase HPLC with an ion-pairing reagent [23]. It was not determined whether clotrimazole altered the pattern of BaP metabolites or relative proportions of BaP metabolites. However, this work demonstrated that this class of compounds can alter the metabolic fate of a cytochrome P450 substrate during a concurrent exposure.

Table 1. Effect of Clotrimazole on BaP Bioconcentration BaP exposure

Treatment: BaP [Whole fish parent-BaP] (Mg/ kg )

Id

3.1±0.2

3d

2.8 ± 0.3

a

a

Treatment: BaP + C L O T [Whole fish parent-BaP] (Mg/kg) b

26.1 ± 6.7 29.9 ±10.1

Treatment groups (BaP or BaP + clotrimazole (CLOT)) with the same letters were not significantly different (p < 0.05). (Reproduced from reference 23, Copyright 1996 Environmental Toxicology and Chemistry.)

Previously, it was demonstrated with the EBI fungicides prochloraz and penconazole can enhance malathion toxicity to red-legged partridge, and enhancement resulted from increased cholinesterase inhibition [13, 14], Increased cholinesterase inhibition was believed to have resulted from induction of a novel cytochrome P450 isoform that is capable of desulphuration of malathion to its toxic product malaoxon. Under the same experimental conditions, TGP did not enhance malathion toxicity [14]. It was speculated by this group that TGP did not enhance malathion toxicity because cytochrome P450 inhibition by TGP dominated over cytochrome P450 induction by TGP at the dose of 200 mg/kg. This may be a plausible interpretation of the results, since a mixed-pattern response occurred following injections with rainbow trout that received 100 mg TGP/kg [5]. Although it is presently not known which cytochrome P450(s) are responsible for the desulfuration reaction of OPs, the CYP1A, CYP2B, and CYP3A isoforms of mammalian species are known to catalyze desulphuration reactions. Therefore, there may be drug metabolizing cytochrome P450 isoforms may be involved in OP activation [27, 28]. A proposed mechanism for activation of the OP parathion by a cytochrome P450 isoform which is shown in Figure 8.



140

OEt.

Figure 8. Proposed mechanism of cytochrome P450 activation of parathion. We recently re-tested the hypothesis that induction of cytochrome P450 activity by an EBI imidazole/triazole would enhance the toxicity of an OP insecticide by increasing activation rates relative to inactivation rates [13, 14]. Our results demonstrated that TGP can enhance the acute toxicity of parathion to fathead minnows (Pimphales promelas). This enhancement was uncovered by comparing survival distributions of groups of fish that were pre-exposed to TGP followed by exposure to parathion with survival distributions of groups of fish that were exposed to parathion alone (Figure 9). Survival distributions were compared with the KaplanMeier (product limit) test [29]. The concentrations of TGP used in the survival analysis experiments were higher than reported field concentrations for propiconazole. The target exposure concentration of 125 μg TGP/L was chosen as a worst case scenario for propiconazole concentrations during times of high precipitation. Survival times were reduced for fish pre-exposed to 138 ± 9 μg TGP/L followed by exposure to 1.9 ± 0.1 mg parathion/L compared to fish that were exposed to 1.9 ± 0.1 mg parathion/L alone (Figure 7). Although exposure to 1.9 mg parathion/L alone was acutely toxic to fathead minnows, body burdens near this concentration have been measured in freshwater species of fish in El Salvador.

glOOj-^ 1.9 mg parathion/L 138 μ9 TGP/L; 1.9 mg parathion/L

< c ο

Έ 50 ο Q.

2 Q.

0

24

48

72 Time (h)

120

144

Figure 9. Survival distributions for fathead minnows pre-exposed to TGP and then exposed to 1.9 ± 0.1 mg parathion/L or to 1.9 ± 0.1 mg parathion/L alone. (Reproduced from reference 30, Copyright 1999 Pesticide Biochemistry and Physiology.)



141 Apparently these species of fish are less sensitive to parathion toxicity compared to fathead minnows [12]. The early life stage maximum allowable toxicant concentration (MATC) for fathead minnows exposed to TGP is above concentrations used in the acute toxicity study. The MATC value for fathead minnows spans the range of 430 to 970 μg/L [30]. Time-to-death experiments were consistent with in vitro data that demonstrated that hepatic microsomes isolated from TGP-treated fathead minnows (79 μg TGP/L for 48 h) had a higher rate of paraoxon formation relative to untreated microsomes [30]. The rate of paraoxon formation for control microsomes was 7.0 ± 2.6 pmol/min/mg protein and the rate of paraoxon formation for TGP-treated microsomes was 16.5 ± 0.2 pmol/min/mg protein which was significantly higher relative to the control microsomes [31]. Formation of the deactivation product pnitrophenol was below the detectable limit and thus it was not possible to index activatiomdeactivation ratios. In a study examining the influence of TGP on parathion metabolism with mammalian microsomes, it was reported that TGP did not affect parathion induced inhibition of serum cholinesterase activity [32]. In spite of exposure to TGP enhancing parathion activation rates in vitro, there was a concomitant increase in parathion deactivation rates in vitro, thereby, producing an overall decrease in the activation:deactivation ratio [32]. Although it has been shown that TGP can modulate CYP1A1 expression in rainbow trout and enhance acute parathion toxicity to fathead minnows, the participation of CYP1A1 and a CYP3A-type isoform in Nile tilapia toward malathion metabolism was indirectly ruled out [33]. CYP1A1 and CYP3A induction in Nile tilapia did not alter malathion toxicity or effect AChE activity. In light of the results of the former study, induction of CYP1A1 expression in fathead minnows may not have been the mechanism by which parathion toxicity was enhanced by TGP. It is possible that increased paraoxon formation was not responsible for the enhanced acute toxicity of parathion, but rather, pre-exposure to TGP enhanced the acute toxicity of parathion to fathead minnows by increasing parathion clearance rates. To date, only a few cytochrome P450 isoforms in species of fish have been shown to be inducible with model substrates. These cytochrome P450s include CYP1A1 in a variety of species [34], CYP2K1 and CYP2M1 in bluegill and channel catfish [35] and a CYP3A-type isoform in Nile tilapia [33]. Although only a few inducible cytochrome P450 isozymes have been characterized with teleosts, other inducible forms may exist. Therefore, it is possible that teleosts, like rats, have an inducible novel cytochrome P450 isoform(s) that is capable of parathion activation following exposure to TGP. Summary The first portion of this chapter described the mechanisms by which clotrimazole and propiconazole modulate in vitro and in vivo CYP1A1 expression. Analytical grade formulations of clotrimazole and propiconazole were noncompetitive inhibitors of CYP1A1 catalytic activity and did not influence CYP1A1 gene expression. However, a technical grade formulation of propiconazole



142 was shown to produce a mixed-pattern response with respect to CYP1A1 expression. The mixed-pattern response was characterized by induction of CYP1A1 mRNA levels but inhibition of CYP1A1 catalytic activity. Induction of CYP1A1 expression by TGP was caused by unidentified AhR-active compounds within the tested TGP formulation. Although, TGP can inhibit CYP1A1 catalytic activity, CYP1A1 mRNA levels in gill tissue could potentially be used to identify exposure to TGP concentrations > 20 μg/L. This chapter also reviewed a study which examined the potential for interaction between TGP and the OP insecticide parathion. TGP was shown to interact with parathion. The interaction between TGP and parathion may have resulted from induction of a novel cytochrome P450 isoform that was responsible for desulphuration of parathion. Although this is a possible mechanism, additional analysis is required. Future studies will address whether enhancement of acute parathion toxicity by TGP resulted either from propiconazole, from an unidentified compound(s) in the TGP formulation, from TGP influencing parathion clearance rates, or from a combination of any or all of these mechanisms. Although future studies are required to determine the mechanism by which TGP enhances the acute toxicity of parathion, the results reviewed in this chapter point out that toxic interactions can occur between these two compounds.

Acknowledgments The authors would like to extend their thanks to the former Ciba-Geigy Corporation for supplying the technical grade formulation of propiconazole used in these studies. This research was funded by an EPA Doctoral Fellowship awarded to SLL.

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Propiconazole, Clotrimazole - American Chemical Society

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