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Studies on aromatic hydrocarbon quinone metabolism and DT-Diaphorase function in liver of fish species
Marine Environmental Research
Vol. 42, No. 1-4, pp. 317-321, 195’6 Copyright 0 1996Elsevier Science Ltd
0141-1136(95)00042-9
Printed in Great Britain. All rights reserved 0141-1136/96/S15.00+0.00
ELSEVIER
Studies on Aromatic Hydrocarbon Quinone Metabolism and DT-Diaphorase Function in Liver of Fish Species Philippe Lemaire,a Joachim Sturve,b Lars F6rlinb & David R. Livingstone”* “NERC Plymouth Marine Laboratory, Plymouth PLl 2PB, UK bDepartment of Zoophysiology, University of Giiteborg, S-413 90 Gateborg,
Sweden
ABSTRACT Studies were carried out to examine the role of aromatic hydrocarbon (AH)-quinone metabolism and DT-diaphorase (quinone oxidoreductase; EC 1.6.99.2; DTD) function in pollution-caused oxidative damage in fish. Redox cycling of quinones to produce reactive oxygen species (ROS) was studied by oxygen consumption and oxidation of the hydroxyl radical scavenger 2-keto-4-methiolbutyric acid. NADHdependent redox cycling by hepatic microsomes offlounder (Platichthys flesus) was seen for I ,I-benzoquinone, I ,2- and 1 ,I-naphthoquinones, phenanthroquinone and anthraquinone. Combined with previous similar results for duroquinone, menadione and benzo[a]pyrene (BaP) quinones (Lemaire et al., 1994), this demonstrates a general potential for AH-quinone-mediated ROS production. NADPH-dependent ‘H-BaP metabolism by hepatic microsomes of P. flesus produced 54% dials, 45% phenols and trace levels of quinones, whereas cumene hydroperoxide-dependent metabolism produced up to 67% quinones (metabolites resolved by HPLC). Exposure to AH-contaminated sediment can lead to enhanced hepatic lipid peroxidation (Livingstone et al., 1993) raising the possibility of an AH-mediated toxicity cycle of peroxidation leading to enhanced quinone and ROS production leading to more peroxidation and oxidative damage. Hepatic DTD puriJied almost to homogeneity from trout (Oncorhynchus mykiss; Sturve et al., in prep.) also catalysed NADHdependent AH-quinone-mediated ROS production, indicating a possible function for this enzyme in quinone toxicity. Copyright 0 1996 Elsevier Science Ltd
Hepatic neoplasms and other pathologies in fish have been correlated with both experimental and field exposure to various organic contaminants, including in particular aromatic hydrocarbons (AHs; Livingstone et al., 1994). Whereas oxidative DNA damage has been observed in such pathologies (Malins et al., 1990), and a potential has been demonstrated for xenobiotic-mediated reactive oxygen species (ROS) formation (Lemaire *Author
to whom correspondence
should
be addressed. 317
318
P. Lemaire et al.
et al., 1994) leading to enhanced DNA (Nishimoto et al., 1991) and possibly lipid (Livingstone et al., 1993) oxidative damage, little is known of the role of AHs in such processes. Studies were carried out to examine the ROS-generating properties of AHquinones and the production of benzo[a]pyrene (BaP) quinones by hepatic microsomes of flounder (Platichthys Jesus). Additionally, the detoxication-toxication role in AH-quinone metabolism of DT-diaphorase (quinone oxidoreductase; EC 1.6.99.2; DTD) from rainbow trout (Oncorhynchus mykiss) was also examined. Adult P.Jlesus (200-300 g) were caught locally in Plymouth waters, and adult 0. mykiss (100 g) were obtained from Anthens Fiskodling AB, Giiteborg. Hepatic microsomes of P. fresus were prepared in 0.15 M KCl, pH 7.5, containing 20% w/v glycerol as described in Lemaire et al. (1994). Redox cycling of AH-quinones and ROS generation were studied by oxygen consumption (measured by Clark electrode) and iron-EDTA-supported oxidation of the hydroxyl radical scavenging agent 2-keto-4-methiolbutyric acid (KMBA) to ethylene (measured gas chromatographically) at 25”C, using an NADH concentration of 0.3 mM (Garcia Martinez et al., 1995). Metabolism of 3H-BaP was carried out at 25°C in the presence of either 0.2 mM NADPH or 0.1 or 1 mM cumene hydroperoxide (CHP), and free polar metabolites resolved by HPLC (Lemaire et al., 1993). DTD was assayed by both NADH-dependent dicoumarol-inhibitable dichlorophenolindophenol (DCPIP) reductase and dicoumarol-inhibitable menadione reductase activities (Fiirlin et al., 1995). Hepatic cytosolic DTD was purified almost to homogeneity (as judged by SDS-PAGE) from 0. mykiss by the cibacron blue affinity chromatographic method of Prochaska (1988; Sturve et al., in prep.). Protein was measured by the method of Lowry et al. (195 1). Values are presented as means f SEM. Various rates of NADH-dependent enhanced oxygen consumption (Fig. l(A)) and ROS production (Fig. l(B)) were observed for hepatic microsomes of P.$esus for a range of one- to five-ring AH-quinones. Rates were highest for the naphthoquinones and phenanthroquinone. Oxygen consumption roughly paralled ROS production but differences were also seen, viz. oxygen consumption was higher for 1,Znaphthoquinone than for 1,Cnaphthoquinone but rates of KMBA oxidation were similar. The differences in rates of ROS generation for different AH-quinones most likely relate to the substrate specificities of the microsomal flavoprotein reductases catalysing the redox cycling (Lemaire & Livingstone, 1994). Combined with previous findings for enhanced KMBA oxidation by tetramethylbenzoquinone (duroquinone), 2-methyl- 1,4naphthoquinone (menadione) and BaP 1,6-, 3,6- and 6,12-quinones by hepatic microsomes of P. fzesus and perch (Perca jhviatilis; Lemaire ef al., 1994), overall the results indicate a general potential for AH-quinone-mediated ROS production in fish liver. Cytochrome P450 can catalyse the metabolism of xenobiotics by NADPH-dependent mono-oxygenation or hydroperoxide-dependent peroxidation. NADPH-dependent BaP metabolism by hepatic microsomes of P. Jesus produced 53.7 f 3.9% diols, 44.3 f 4.4% phenols and trace amounts of quinones, whereas CHP-dependent metabolism produced from 55.1 f 3.1% (0.1 mM CHP) to 66.8 f 3.8% (1 mM CHP) quinones. Given that exposure of flatfish (dab, Limanda limandu) to AH-contaminated sediment resulted in increased lipid peroxidation (Livingstone et al., 1993), this raises the possibility that such lipid hydroperoxide products could serve as substrates for cytochrome P450 leading to enhanced quinone production from AHs. This in turn could lead to an AH-mediated toxicity cycle of enhanced ROS production leading to more peroxidation and oxidative damage (Livingstone et al., 1994).
Studies on (AH)-quinone
and DT-diaphorase
319
a
Fig. 1. Rates of NADH-dependent oxygen consumption (A) and iron-EDTA-supported ROS generation (KMBA oxidation to ethylene) (B) by hepatic microsomes of P.fIesus in the presence of 0.5 mM AH-quinone; values are means f SEM (n = 3), rates represent AH-stimulated minus basal rates, -> not measured. AH-quinones are shown in order of increasing molecular size (number of benzene rings) and presence of methyl groups.
320
P. Lemaire
et al.
Fig. 2.
Rates of NADH-dependent iron-EDTA-supported ROS generation (KMBA oxidation to ethylene) by purified hepatic DT-diaphorase from 0. mykiss in the absence (no quinone) and presence of 0.5 mM AH-quinone; values are means %SEM (n = 3).
DTD is proposed to act as an antioxidant enzyme by catalysing the two-electron reduction of quinones to hydroquinones, so preventing redox cycling and ROS generation (Lind et al., 1982). However, recent evidence indicates that DTD in liver of fish may actually stimulate ROS production (Hasspieler & Di Giulio, 1992). Purified hepatic DTD from 0. mykiss (defined by its DCPIP and menadione reductase activities being completely inhibited by 5 ,uM dicoumarol) catalysed NADH-dependent AH-quinone mediated ROS production for several quinones (Fig. 2), indicating a possible function for this enzyme in quinone toxicity. The maximal one-electron reductive activity of the purified enzyme using 1,Cnaphthoquinone as substrate (2.57 f 0.01 pmol/min/mg protein) was significant compared to its maximal two-electron reductive activity using DCPIP as substrate (12.2 ~mol/min/mg protein). However, this phenomenon appears to be substrate specific, since with menadione as substrate the one-electron reductive activity (0.2 i 0.01 pmol/min/mg protein) was much less than the two-electron reductive activity (56.1 mmol/ min/mg protein), indicating that the toxication-detoxication role of DTD may depend on the particular quinone being metabolized.
ACKNOWLEDGEMENTS This work was carried out under the tenure of the European Environmental Research Organization and European Science Foundation postdoctoral fellowships to P. Lemaire and a Ph.D. studentship to J. Sturve.
Studies on (AH)-quinone
and DT-diaphorase
321
REFERENCES Forlin, L., Lemaire, P. & Livingstone, D. R. (1995). Mar. Environ. Res., 39, 201-204. Garcia Martinez, P., Winston, G. W., Metosh-Dickey, C., O’Hara, S. C. M. & Livingstone, D. R. (1995). Toxicol. Appl. Pharmacol., 131, 332-341. Hasspieler, B. M. & Di Giulio, R. T. (1992). Toxicol. Appl. Pharmacof., 114, 156161. Lemaire, P., Den Besten, P. J. & Livingstone, D. R. (1993). Poly. Arom. Comp., 3, Suppl., 1133-40. Lemaire, P. & Livingstone, D. R. (1994). .I. Biochem. Toxicol., 9, 87-95. Lemaire, P., Matthews, A., Fiirlin, L. & Livingstone, D. R. (1994). Archs Environ. Contam. Toxicol., 26, 191-201. Lind, C., Hochstein, P. & Ernster, L. (1982). Archs Biochem. Biophys., 216, 175-185. Livingstone, D. R., Lemaire, P., Matthews, A., Peters, L. D., Bucke, D. & Law, R. J. (1993). Mar. Pollut. BUN., 26, 602-606. Livingstone, D. R., Fiirlin, L. & George, S. (1994). In Sutcliffe, D. W. (ed.) Water Quality and Stress Indicators in Marine and Freshwater Systems: Linking Levels of Organisation. Freshwater Biological Association, Ambleside, UK, pp. 154-171. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L. & Randall, R. J. (1951). .I. Biol. Chem., 193, 265-275. Malins, D. C., Ostrander, G. K., Haimanot, R. & Williams, P. C. (1990). Carcinogenesis, 11, 1045-1047. Nishimoto, M., Roubal, W. T., Stein, J. E. & Varanasi, U. (1991). Chem.-Biol. Inter., 80, 317-326. Prochaska, H. J. (1988). Archs Biochem. Biophys., 267, 529-538.
Vol. 42, No. 1-4, pp. 317-321, 195’6 Copyright 0 1996Elsevier Science Ltd
0141-1136(95)00042-9
Printed in Great Britain. All rights reserved 0141-1136/96/S15.00+0.00
ELSEVIER
Studies on Aromatic Hydrocarbon Quinone Metabolism and DT-Diaphorase Function in Liver of Fish Species Philippe Lemaire,a Joachim Sturve,b Lars F6rlinb & David R. Livingstone”* “NERC Plymouth Marine Laboratory, Plymouth PLl 2PB, UK bDepartment of Zoophysiology, University of Giiteborg, S-413 90 Gateborg,
Sweden
ABSTRACT Studies were carried out to examine the role of aromatic hydrocarbon (AH)-quinone metabolism and DT-diaphorase (quinone oxidoreductase; EC 1.6.99.2; DTD) function in pollution-caused oxidative damage in fish. Redox cycling of quinones to produce reactive oxygen species (ROS) was studied by oxygen consumption and oxidation of the hydroxyl radical scavenger 2-keto-4-methiolbutyric acid. NADHdependent redox cycling by hepatic microsomes offlounder (Platichthys flesus) was seen for I ,I-benzoquinone, I ,2- and 1 ,I-naphthoquinones, phenanthroquinone and anthraquinone. Combined with previous similar results for duroquinone, menadione and benzo[a]pyrene (BaP) quinones (Lemaire et al., 1994), this demonstrates a general potential for AH-quinone-mediated ROS production. NADPH-dependent ‘H-BaP metabolism by hepatic microsomes of P. flesus produced 54% dials, 45% phenols and trace levels of quinones, whereas cumene hydroperoxide-dependent metabolism produced up to 67% quinones (metabolites resolved by HPLC). Exposure to AH-contaminated sediment can lead to enhanced hepatic lipid peroxidation (Livingstone et al., 1993) raising the possibility of an AH-mediated toxicity cycle of peroxidation leading to enhanced quinone and ROS production leading to more peroxidation and oxidative damage. Hepatic DTD puriJied almost to homogeneity from trout (Oncorhynchus mykiss; Sturve et al., in prep.) also catalysed NADHdependent AH-quinone-mediated ROS production, indicating a possible function for this enzyme in quinone toxicity. Copyright 0 1996 Elsevier Science Ltd
Hepatic neoplasms and other pathologies in fish have been correlated with both experimental and field exposure to various organic contaminants, including in particular aromatic hydrocarbons (AHs; Livingstone et al., 1994). Whereas oxidative DNA damage has been observed in such pathologies (Malins et al., 1990), and a potential has been demonstrated for xenobiotic-mediated reactive oxygen species (ROS) formation (Lemaire *Author
to whom correspondence
should
be addressed. 317
318
P. Lemaire et al.
et al., 1994) leading to enhanced DNA (Nishimoto et al., 1991) and possibly lipid (Livingstone et al., 1993) oxidative damage, little is known of the role of AHs in such processes. Studies were carried out to examine the ROS-generating properties of AHquinones and the production of benzo[a]pyrene (BaP) quinones by hepatic microsomes of flounder (Platichthys Jesus). Additionally, the detoxication-toxication role in AH-quinone metabolism of DT-diaphorase (quinone oxidoreductase; EC 1.6.99.2; DTD) from rainbow trout (Oncorhynchus mykiss) was also examined. Adult P.Jlesus (200-300 g) were caught locally in Plymouth waters, and adult 0. mykiss (100 g) were obtained from Anthens Fiskodling AB, Giiteborg. Hepatic microsomes of P. fresus were prepared in 0.15 M KCl, pH 7.5, containing 20% w/v glycerol as described in Lemaire et al. (1994). Redox cycling of AH-quinones and ROS generation were studied by oxygen consumption (measured by Clark electrode) and iron-EDTA-supported oxidation of the hydroxyl radical scavenging agent 2-keto-4-methiolbutyric acid (KMBA) to ethylene (measured gas chromatographically) at 25”C, using an NADH concentration of 0.3 mM (Garcia Martinez et al., 1995). Metabolism of 3H-BaP was carried out at 25°C in the presence of either 0.2 mM NADPH or 0.1 or 1 mM cumene hydroperoxide (CHP), and free polar metabolites resolved by HPLC (Lemaire et al., 1993). DTD was assayed by both NADH-dependent dicoumarol-inhibitable dichlorophenolindophenol (DCPIP) reductase and dicoumarol-inhibitable menadione reductase activities (Fiirlin et al., 1995). Hepatic cytosolic DTD was purified almost to homogeneity (as judged by SDS-PAGE) from 0. mykiss by the cibacron blue affinity chromatographic method of Prochaska (1988; Sturve et al., in prep.). Protein was measured by the method of Lowry et al. (195 1). Values are presented as means f SEM. Various rates of NADH-dependent enhanced oxygen consumption (Fig. l(A)) and ROS production (Fig. l(B)) were observed for hepatic microsomes of P.$esus for a range of one- to five-ring AH-quinones. Rates were highest for the naphthoquinones and phenanthroquinone. Oxygen consumption roughly paralled ROS production but differences were also seen, viz. oxygen consumption was higher for 1,Znaphthoquinone than for 1,Cnaphthoquinone but rates of KMBA oxidation were similar. The differences in rates of ROS generation for different AH-quinones most likely relate to the substrate specificities of the microsomal flavoprotein reductases catalysing the redox cycling (Lemaire & Livingstone, 1994). Combined with previous findings for enhanced KMBA oxidation by tetramethylbenzoquinone (duroquinone), 2-methyl- 1,4naphthoquinone (menadione) and BaP 1,6-, 3,6- and 6,12-quinones by hepatic microsomes of P. fzesus and perch (Perca jhviatilis; Lemaire ef al., 1994), overall the results indicate a general potential for AH-quinone-mediated ROS production in fish liver. Cytochrome P450 can catalyse the metabolism of xenobiotics by NADPH-dependent mono-oxygenation or hydroperoxide-dependent peroxidation. NADPH-dependent BaP metabolism by hepatic microsomes of P. Jesus produced 53.7 f 3.9% diols, 44.3 f 4.4% phenols and trace amounts of quinones, whereas CHP-dependent metabolism produced from 55.1 f 3.1% (0.1 mM CHP) to 66.8 f 3.8% (1 mM CHP) quinones. Given that exposure of flatfish (dab, Limanda limandu) to AH-contaminated sediment resulted in increased lipid peroxidation (Livingstone et al., 1993), this raises the possibility that such lipid hydroperoxide products could serve as substrates for cytochrome P450 leading to enhanced quinone production from AHs. This in turn could lead to an AH-mediated toxicity cycle of enhanced ROS production leading to more peroxidation and oxidative damage (Livingstone et al., 1994).
Studies on (AH)-quinone
and DT-diaphorase
319
a
Fig. 1. Rates of NADH-dependent oxygen consumption (A) and iron-EDTA-supported ROS generation (KMBA oxidation to ethylene) (B) by hepatic microsomes of P.fIesus in the presence of 0.5 mM AH-quinone; values are means f SEM (n = 3), rates represent AH-stimulated minus basal rates, -> not measured. AH-quinones are shown in order of increasing molecular size (number of benzene rings) and presence of methyl groups.
320
P. Lemaire
et al.
Fig. 2.
Rates of NADH-dependent iron-EDTA-supported ROS generation (KMBA oxidation to ethylene) by purified hepatic DT-diaphorase from 0. mykiss in the absence (no quinone) and presence of 0.5 mM AH-quinone; values are means %SEM (n = 3).
DTD is proposed to act as an antioxidant enzyme by catalysing the two-electron reduction of quinones to hydroquinones, so preventing redox cycling and ROS generation (Lind et al., 1982). However, recent evidence indicates that DTD in liver of fish may actually stimulate ROS production (Hasspieler & Di Giulio, 1992). Purified hepatic DTD from 0. mykiss (defined by its DCPIP and menadione reductase activities being completely inhibited by 5 ,uM dicoumarol) catalysed NADH-dependent AH-quinone mediated ROS production for several quinones (Fig. 2), indicating a possible function for this enzyme in quinone toxicity. The maximal one-electron reductive activity of the purified enzyme using 1,Cnaphthoquinone as substrate (2.57 f 0.01 pmol/min/mg protein) was significant compared to its maximal two-electron reductive activity using DCPIP as substrate (12.2 ~mol/min/mg protein). However, this phenomenon appears to be substrate specific, since with menadione as substrate the one-electron reductive activity (0.2 i 0.01 pmol/min/mg protein) was much less than the two-electron reductive activity (56.1 mmol/ min/mg protein), indicating that the toxication-detoxication role of DTD may depend on the particular quinone being metabolized.
ACKNOWLEDGEMENTS This work was carried out under the tenure of the European Environmental Research Organization and European Science Foundation postdoctoral fellowships to P. Lemaire and a Ph.D. studentship to J. Sturve.
Studies on (AH)-quinone
and DT-diaphorase
321
REFERENCES Forlin, L., Lemaire, P. & Livingstone, D. R. (1995). Mar. Environ. Res., 39, 201-204. Garcia Martinez, P., Winston, G. W., Metosh-Dickey, C., O’Hara, S. C. M. & Livingstone, D. R. (1995). Toxicol. Appl. Pharmacol., 131, 332-341. Hasspieler, B. M. & Di Giulio, R. T. (1992). Toxicol. Appl. Pharmacof., 114, 156161. Lemaire, P., Den Besten, P. J. & Livingstone, D. R. (1993). Poly. Arom. Comp., 3, Suppl., 1133-40. Lemaire, P. & Livingstone, D. R. (1994). .I. Biochem. Toxicol., 9, 87-95. Lemaire, P., Matthews, A., Fiirlin, L. & Livingstone, D. R. (1994). Archs Environ. Contam. Toxicol., 26, 191-201. Lind, C., Hochstein, P. & Ernster, L. (1982). Archs Biochem. Biophys., 216, 175-185. Livingstone, D. R., Lemaire, P., Matthews, A., Peters, L. D., Bucke, D. & Law, R. J. (1993). Mar. Pollut. BUN., 26, 602-606. Livingstone, D. R., Fiirlin, L. & George, S. (1994). In Sutcliffe, D. W. (ed.) Water Quality and Stress Indicators in Marine and Freshwater Systems: Linking Levels of Organisation. Freshwater Biological Association, Ambleside, UK, pp. 154-171. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L. & Randall, R. J. (1951). .I. Biol. Chem., 193, 265-275. Malins, D. C., Ostrander, G. K., Haimanot, R. & Williams, P. C. (1990). Carcinogenesis, 11, 1045-1047. Nishimoto, M., Roubal, W. T., Stein, J. E. & Varanasi, U. (1991). Chem.-Biol. Inter., 80, 317-326. Prochaska, H. J. (1988). Archs Biochem. Biophys., 267, 529-538.