SAMI AHMAD, DAWN L. DUVAL, LEANNE C. WEINI-IOLD and RONALD S. PMtDINI Department of Biochemistry, University of Nevada-Reno, Reno, NV 89557-0014, U.S.A.
(Received 6 November 1990; revised and accepted 15 February 1991)
Abstract--A unique pattern of antioxidant enzymes exists in phytophagous insects for defense against endogenous and exogenous sources of toxic forms of oxygen, and data presented herein describe a profile of these enzymes in many tissues of larvae of the cabbage looper moth, Trichoplusia ni. The specific activities of the antioxidant enzymes were high in tissues of high metabolic activities, i.e. Malpighian tubules, hindgut, muscles and gonads. A unique finding was the high constitutive activity of a superoxide disrnutase (SOD) in hemocytes, probably consisting predominantly of the CuZn-SOD, which is analogous to the exclusive presence of this form of SOD in vertebrate erythrocytes and leukocytes. In all other tissues, the activity of Mn-SOD was higher than that of the CuZn-SOD which is converse to the pattern in vertebrate tissues. The glutathione peroxidase (GPOX) activity, present in all tissues and with highest levels in the gonads, did not seem to be the selenoprotein typical of the mammalian GPOX. Glutathione-S- transferase ((]ST) activity paralleled that of its glutathione peroxidase activity (GSTPX). The high GSTPX activity suggests that GSTPX and not GPOX, forms a sequential team with glutathione reductase (GR) to reduce deleterious lipid hydroperoxides and to reduce the oxidized glutathione, GSSG, back to GSH. Catalase (CAT) which decomposes H 2 O2 has very high activity apparently correlated with the low GPOX activity. Finally, the integumental epithelium, and the gut (combined sections) possessed higher amounts of antioxidant enzymes than other tissues. Thus, a physiological relationship may occur between the antioxidant enzyme levels in tissues of T. ni with particularly high metabolic activity and associated endogenous oxidative stress. In addition, another physiological role of these enzymes may be to protect from exogenous oxidative stress exerted by dietary redox-active pro-oxidants in the gut, and to the potential of photodynamically mediated oxygen toxicity in peripheral organs such as the integument.
Key Word Index: Activated oxygen; catalased glutathione peroxidase; glutathione reductase; glutathione transferase; hydroperoxides; lipid peroxidation; oxy-radicals; oxygen toxicity; singlet oxygen; superoxide dismutase; Trichoplusia ni
INTRODUCTION
All aerobic organisms are subject to oxidative stress exerted by the superoxide anion radical (O1-), which is generated by one-electron reduction of the ground- state of molecular oxygen (O2). Superoxide radical exists in equilibrium with the hydroperoxyl radical (HO~) and, in a free-radical cascade, these activated forms of 02 are converted to hydrogen peroxide (H202). In turn, H20, generates hydroxyl radicals ('OH) via the metal-catalyzed Haber-Weiss reaction (Fridovich, 1983). In addition, 02 is also activated in photosensitization and other reactions to the singlet molecular oxygen (1AGO 2 or IO2) (Singh, 1989). All activated forms of 02 cause deleterious reactions with DNA, RNA and proteins. However, the "OH radical and m O2 are the two most reactive forms of activated 02, and they peroxidize many unsaturated organic molecules such as polyunsaturated fatty acids 0aUFA), cholesterol and DNA. These deleterious reactions are implicated in pathologies such as can- cer, the process of aging, and cell death (Cerruti, 1985).
Lipid peroxidation is considered very injurious to cellular integrity and function in mammalian species. Tissues may be subject to direct oxidative damage by
lipid hydroperoxides (LOOH) or other organic per- oxides (e.g. endoperoxides and dioxetanes), and from more reactive breakdown products of peroxides such as malondialdehyde, or lipid peroxidizing radical (LO2) (Mannervik, 1985; Borg and Schaich, 1988). The potential is very high for insect susceptibility to lipid peroxidation because lipids, including choles- terol are essential components of cell membranes and have unique physiological functions. Cuticular lipids prevent desication, other lipids function as juvenile hormones and pheromones, and cholesterol is a precursor of ecdysteroid hormones (Downer, 1986).
We have investigated the role of antioxidant en- zymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX), giutathione-S- transferase (GST) and it's glutathione "peroxidase" activity (GSTPX), and glutathione reductase (GR), in affording herbivorous insects protection from en- dogenous and exogenous sources of toxic oxygen radicals generated by ingestion of pro-oxidant plant allelocbemicals. Constitutive enzyme levels and alter- ations in response to exogenous pro-oxidant stress were reported for cell-free larval extracts of the cabbage looper moth ( Trichoplusia ni ) (Ahmad et al., 1987, 1989; Ahmad and Pardini, 1988, 1989, 1990a; Weinhold et al., 1990). We have also investigated the
563
564 SAm AHMAD et al.
subcellular compa r t m en t a t i on o f these enzymes with the findings tha t in T. ni, all an t iox idant enzymes except for SOD are differently dis t r ibuted t han the pa t te rns described for m a m m a l i a n species ( A h m a d e t al., 1988a).
We now repor t the specific activities of the afore- ment ioned an t iox idan t enzymes and their relative activities present in var ious tissues of the cabbage looper. These da ta demons t ra te for the first t ime tha t a physiological re lat ionship exists between the meta- bolic activity pa t te rns of an insect 's tissues, hence to the endogenous oxidative stress, and the potent ia l for dietary pro-oxidant -media ted oxygen toxicity.
MATERIALS AND METHODS
Isolation o f tissues
Tissues were dissected from three batches of insects, each batch consisting of 30-35 midfifth-instar larvae (for three separate experiments) of a UC-Davis-UNR laboratory strain of cabbage loopers fairly uniform in mass, 166.7 + 11.1 (mean + SD) mg per larva. Dissections were performed in an ice-chilled 1.15% (w/v) KCI solution to isolate the following tissues; foregut, midgut, hindgut, fat body, salivary glands, Malpighian tubules, muscles, and gonads. The integument consisted of the epidermal tissue and cuticle, and the muscles were removed by scraping using a scalpel to detach as much tissue as possible. The degree of purity was high for discrete organ systems, but the muscles and amorphous mass of fat body were less pure. Tissues were drained of KC1 solution and were rinsed twice with 50raM potassium phosphate buffer, pH7.0, and were kept immersed in this buffer (homogenization buffer) at 4°C.
To extract the hemolymph, three batches of larvae, each batch consisting of 15-20 larvae, were punctured anteriorly as well as posteriorly without damaging the gut. The hemolymph oozed out by gentle swirling in a 10-ml flask in which the punctured larvae were placed together with 4 ml of ice-chilled homogenization buffer. The hemolymph was gently removed by a Pasteur pipette, and was kept at 4°C for further fractionation.
Enzyme source
Whole bodies of T. ni larvae and pools of each tissue isolated were homogenized in 50 mM potassium phosphate buffer, pH 7.0, in a glass homogenizer with a motor-driven teflon pestle for I min. The crude homogenates were cen- trifuged at 850g x 15min and the cell-free supernatants were removed. The supernatants were then mildly sonicated twice for I0 see to release the contents of the membrane- bound organelles (Ahmad et aL, 1988a). The sonicated supernatants, held on ice, were used directly as enzyme sources.
Hemolymph was divided into two portions; one portion was used directly for enzyme assays, and the other portion was centrifuged at 850g x 15 min to obtain cell-free plasma, and the pellet consisting primarily of hemocytes based on microscopic examination. The pellet was resuspended in 4 ml of the homogenizing buffer and was mildly sonicated to achieve a uniform suspension as well as to fracture the cellular membranes.
Enzyme assays
Antioxidant enzyme activities were assayed with follow- ing aliquots of the insects' enzyme extracts: CAT (2/~1); SOD, GST and GSTPX (20#1), GPOX (50/~1) and GR (40/~1). Where activity was negligible or not detectable up to 10-fold higher aliquots were used. The protein amounts were determined by the method of Lowry et aL (1951), with defatted bovine serum albumin as standard.
The activities of the antioxidant enzymes were assayed by the same procedures used in our earlier studies (Ahmad et al., 1987; Ahmad and Pardini, 1988, 1989; Pritsos et al., 1988a, 1988b, 1990; Weinhold et al., 1990). Nonetheless, a brief description of the assays is given here together with the basis for computing specific activities, and assessment of relative amounts of antioxidant enzymes present in the tissues.
SOD activity was measured by a sensitive colorimetric method of Oberly and Spitz (1984) which is a modification of the method of McCord and Fridovich (1969). As defined earlier by McCord and Fridovich (1969), the specific activity of SOD was expressed in units min -l mg protein -l at 25°C and pH 7.8.
CAT activity was assayed according to Aebi (1984) by monitoring the disappearance of the substrate, H202, at 240 nm. From the extinction coefficient of H202, 62.4 M -t cm -t, one unit of CAT activity was defined as 1/~mol H202 decomposed rain -~ mg protein -~ at 25°C and pH 7.0.
GST's conjugative activity with GSH was demonstrated with a model substrate, l-chloro-2,4-dinitrobenzene at 340 nm as described for mammalian (Habig and Jakoby, 1981) and insect enzymes (Weinhold et al., 1990). Using the extinction coefficient of 9.6mM -~ cm -~ for the CDNB conjugate, GST's conjugative activity was calculated as units, where one unit = 1/zmol CDNB conjugated min -l mg protein -l at 25°C and pH 6.5.
GSTPX or GST's peroxidase activity was measured by the procedure of Ahmad and Pardini (1988, 1989). The substrates were GSH and cumene hydroperoxide (cu- mOOH) and the reaction was coupled to that of GR (Sigma Chemical Co., Baker's yeast enzyme, 200 units mg pro- rein -~) which converted the GSSG formed to GSH using reducing equivalents from NADPH. The reaction could thus be monitored at 340 nm as the oxidation of NADPH to NADP +. One unit of GSTPX activity was defined as the oxidation of 1 nmol NADPH min -I mg protein -I at 25°C and pH 7.0. The extinction coefficient of NADPH used was 6.22 x 1 0 3 M - l c m - I .
GPOX activity was determined by the method of Strauss et al. (1980) with GSH and H202 as substrate. The reaction was coupled to that of GR as described for GSTPX assay. The enzyme activity was monitored as the amount of NADPH oxidized at 340nm. The specific activity was expressed in units where one unit equals the oxidation of 1 nmol NADPH min -I mg protein -I at 25°C and pH 7.0.
GR activity was measured by the procedure of Racker (1955) using GSSG as substrate and NADPH as reductant. One unit of GR activity was defined as the change of 0.001 OD at A~0 min -~ mg protein -~ at 25°C and pH 7.6.
From the specific activities and total protein contents of each larval tissue, the antioxidant enzyme content of each tissue was computed and expressed as percentage for en- zyme amount calculated for the whole-body supernatant.
Inhibition studies
Some limited enzyme inhibition studies were performed to differentiate the copper/zinc-containing SOD (CuZn-SOD, cytosolic) from manganese-containing SOD (Mn-SOD, mitochondrial). The CuZn-SOD but not the Mn-SOD is sensitive to inhibition by KCN whether the enzyme(s) source is a mammalian tissue, insect or plant (Weiser and Fridovich, 1973; Misra, 1979; Lee et al., 1981; Bird et al., 1986; Bowler et al., 1989). Selected tissues from the second experiment and hemolymph preparations from the third experiment were assayed for SOD activity in the presence of 1 mM KCN.
GSTPX and GPOX inhibitors, bromosulphopthalein (Singh et al., 1987), fl-mercaptoethanol and mercaptosucci- nate (Chaudiere et al., 1984) at 1 x l0 -~ mM concentration were used with tissue extracts exhibiting high activity (third experiment) to see if the activities of GST (particularly GSTPX) and GPOX overlapped.
Tissue specificity of antioxidant enzymes 565
Statistical protocols Data pertaining to three experiments (n = 3), with two or
four measurements from each experiment, were pooled for each enzyme and were analyzed by the analysis of variance (ANOVA). If the analysis exhibited significant difference by ANOVA, they were further analyzed by the Duncan's multiple range test (DMRT) to discern significant differ- ences among the means (~ --0.05).
RESULTS
Antioxidant enzymic activities of whole-body supernatant of T. ni. were in close agreement to the levels previously reported; only the GR level was higher but the difference was less than 2-fold (Ahmad et al., 1987, 1989, Ahmad and Pardini, 1988, 1989; Weinhold et al., 1990).
SOD activity
SOD was found in all tissues examined and its specific activity substantially varied. As can be seen in Fig. 1, significantly (P < 0.05) higher specific ac- tivity of SOD was found in the gonads; c. 6-fold higher activity than in the whole-body supernatant. High specific activities were also evident in several other tissues in the decreasing order: hindgut, muscles, Malpighian tubules, foregut and midgut. In the remaining tissues, the specific activity was relatively lower and in the hemolymph barely detectable. Figure 1 also shows that the epithelial tissue and combined sections of the gut were the major sites of SOD because of these tissues' higher protein contents. The combined SOD amounts of all tissues was 109%, which exceeded slightly the amount calculated from the whole-body supernatant data.
CAT activity
Unlike SOD, CAT activity was not present in all tissues. Salivary glands, Malpighian tubules, hemo- lymph and gonads lacked CAT activity (Fig. 2). The tissues possessing CAT activity displayed variability in specific activity which did not exceed by more than 35% that of the whole-body supernatant; only in the integument's epithelial tissue the relative specific ac- tivity was markedly lower. Again, as with SOD activity, the epithelial tissue and the entire gut had the highest CAT contents. The total yield of CAT activity from the tissues was 85%, relative to the amount anticipated from the data of whole-body supernatant.
GST activity
GST's CDNB-conjugative activity was detected in all tissues. Gonads, foregut and muscles had signifi- cantly (P < 0.05) higher specific activities than that found for this enzyme in the whole-body supernatant (Fig. 3). In the Malpighian tubules the specific ac- tivity was comparable to that of the whole-body supernatant, in other tissues it was less and the lowest specific activity was found in the hemolymph. The integumental epithelium and the gut had the highest relative GST contents. The amount of GST calcu- lated from tissue activities and protein amounts, exceeded by 20% the total based on similar calcu- lations from data of the whole-body supernatant.
GSTPX activity
The GSTPX, or peroxidase activity of GST, was also present in all tissues examined (Fig. 4). Based on the well-established stoichiometry of these enzymic reactions, the whole-body supernatant's GSTPX
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Tissues T issues Fig. 1. Specific (left panel) and relative (fight panel) activities of SOD in various tissues of fifth-instar larvae of T. hi. Means for specific activities of SOD are derived from triplicate assays (n = 3), each assay with two to three measurements. Activities are expressed in units rain-' mg protein-' as defined by McCord and Fridovich (1969). Tissue abbreviations are as follows: WB: whole-body supernatant; EP: integnmental epithelium; FG: foregnt; MG: hindgnt; FB: fat body; SG: salivary glands; MT: Malpighian tubules; MC: muscles; HL: hemolymph; and GD: gonads. ANOVA of specific activities; F(m2a0) = 21.75; P > F ffi 0.0001. For tissue, F(10) = 25.90; P > F = 0.0001; for enzyme, F(2 ) = 1.01; P > F = 0.3836. Specific activity means (data bars) not followed by the same lower-case letter are significantly different (P < 0.05)
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Fig. 2. Specific (left panel) and relative (right panel) activities of CAT in various tissues of fifth-instar larvae of T. ni. Means for specific activities are derived from triplicate assays (n = 3), each assay with two to three measurements. Specific activities are expressed in units, where 1 unit = l gmol H202 decomposed rain - img protein - ~. Tissue abbreviations are as for Fig. I. ANOVA of specific activities; Fo~ ~0) = 44.47; P > F = 0.0001. The same F and P values were obtained by a variance stabilizing transformation of data by dividing the values by 100 (due to this enzyme's variability of data points ranging from 0 to 498). For tissues, Fo0 ) --- 53.61; P < F = 0.001; for enzyme, F(:) = 0.40; P > F = 0.6780. Specific activity means (data bars) not followed by the same lower-case letter are significantly different (P < 0.05) by DMRT. No
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activity represented 18% of the total GST activity. Moreover , the specific activities of GSTPX were generally high in the same tissues which had high G S T activity (the only exception was the foregut). The specific activity o f GSTPX in the Malpighian tubules significantly (P < 0.05) surpassed the specific activities o f other tissues and the whole-body super-
natant. Substantial specific activities were found in gonads, midgut, salivary glands and muscles. In all other tissues the GSTPX specific activities were less than one-half of that in the whole-body supernatant, and in the hemolymph the activity was negligible. Despite low specific activity of the epithelial tissue, its higher protein content yielded the highest relative
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Fig. 3. Specific (left panel) and relative (right panel) activities of GST in various tissues of flfth-instar larvae of T. ni. Means for specific activities of GST are derived from triplicate assays (n -- 3), each assay with two to three measurements. Activities are expressed in units, where 1 unit -- l #mol CDNB conjugated rain-' mg protein -t. Tissue abbreviations are as for Fig. I. ANOVA of specific activities; Fo2~0 ) = 20.47; P > F ffi 0.0001. For tissues, F(m0) -- 24.32; P > F = 0.001; for enzyme, F(2) -- 1.23; P > F -- 0.3142. Specific activity means (data bars) not followed by the same lower-case letter are significantly different (P < 0.05)
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Fig. 4. Specific (left panel) and relative (right panel) activities of GSTPX in various tissues of fifth-instar larvae of T. ni. Means for specific activities of GST are derived from triplicate assays (n = 3), each assay with two to three measurements. Activities are expressed in units, where I unit = 1 nmol NADPH oxidized rain -~ mg protein -l. Tissue abbreviations are as for Fig. 1. ANOVA of specific activities; F,2.20) = 51.87; P >F=0.0001. For tissues F(m)= 62.06; P <F=0.0001; for enzyme, Ft2 ) =0.4177; P <F--0.4177. Specific activity means (data bars) not followed by the same lower-case letter are significantly different
(P < 0.05) by DMRT. The activity in HL was very trivial.
GSTPX activity of the total larval enzyme activity. In contrast, the combined GSTPX content of the intact gut was less than that of the integument. Nonetheless, the major sites for GSTPX are the epithelial tissue and the whole gut. Computat ions for the total larval GSTPX amounts showed the tissues surveyed yielded only 82.4% of the enzyme relative to the total amount expected from the whole-body supernatant data.
GPOX activity
The specific activity of a GSH-dependent GPOX- like enzyme was found to be low in all tissue prep- arations in contrast to higher specific activities observed for the GSH-dependent GSTPX. Figure 5 shows that at least in five tissues, i.e. gonads, Malpighian tubules, muscles and hindgut the specific activities were 9, 3.5, 1.8 and 1.4 fold higher (signifi-
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Fig. 5. Specific (left panel) and relative (right panel) activities of GPOX in various tissues of fifth-instar larvae of T. ni. Means for specific activities of GPOX are derived from triplicate assays (n = 3), each assay with two to throe measurements. Activities are expressed in units, where I unit = 1 nmol NADPH oxidized rain-i mg protein -l. Tissue abbreviations are as for Fig. 1. ANOVA of specific activities; F<12a0) = 395.21; P>F=O.O001. For tissues, F(m)=472.76; P>F=O.O001; for enzyme, F(2)=7.48; P > F = 0 . 0 0 3 8 (implying a significant difference among the triplicate assays). Specific activity means (data bars) not
followed by the same lower-case letter are significantly (P < 0.05) different by DMRT.
568 SAm ~ et al.
cant differences; P < 0.05) compared to the enzyme activity of the whole-body supernatant. On a relative scale, the epithelial tissue and the entire gut (sum- mation of data of the fore-, mid-, and hindgut) had nearly equal enzyme content, and represented the two major sites for this activity. The enzyme content computed for all tissues was 99%, as a relative yield to that assessed from the whole-body supernatant data.
GR activity
The GR activity of the whole-body supernatant was low, but an impressive 10-fold, significantly (P < 0.05) higher specific activity was found in the muscles (Fig. 6). Significantly higher (P < 0.05) ac- tivities were also recorded for gonads and Malpighian tubules, 5.5- and 3-fold higher activities, respectively. The specific activity of fat body GR was also high (but not significantly; P > 0.05). No GR activity was detected in the hemolymph. On a relative scale to whole-body supernatant, the GR content of tissues was more evenly distributed than observed for all other antioxidant enzymes; the four major sites of GR activity in decreasing order were, muscles, the complete gut, fat body, and the epithelial tissues. The total recovery of GR activity from the tissues was 82% relative to the amount expected from the whole- body supernatant data.
Resolution o f the hemolymph's low enzyme activities
When the hemolymph was fractionated by cen- trifugation, the source of trivial activities of SOD, GST, GSTPX and GPOX were found to be the hemocytes (Table 1). No activity was found in the cell-free plasma fraction. As can be seen from the data in Table 1, substantial specific activities were evident for all aforementioned enzymes. CAT or GR
activity was neither detected in the hemolymph, nor in the plasma or hemocyte fractions.
Inhibition o f SOD activity
The specific activity of SOD of some selected tissues was inhibited in the range 27-40% by 1 mM KCN. Specifically, the inhibition for the whole-body supernatant, hindgut, muscles and gonads was 40.4, 26.9, 40.1 and 31.5%, respectively (data are average of duplicate assays). In contrast to 9.4 units of SOD activity recorded for the hemocytes (Table 1), only 1.5 units (average of triplicate assays) of SOD activity was measured in the presence of I mM KCN; this amounts to 84% inhibition. These data clearly indi- cated that in majority of the tissues, the cyanide- sensitive CuZn-SOD represents a relatively minor portion of the overall SOD activity; the remainder enzyme may be the cyanide-insensitive Mn-SOD. However, in the hemocytes the predominant presence of CuZn-SOD is indicated.
Inhibition o f GSTPX and GPOX activities
The GST inhibitor, bromosulfopthalein, caused 77-90% inhibition of the peroxidase activity assayed with cumOOH as substrate. This supports the con- tention that the GSTPX activity represents the per- oxidase activity of GST rather than GPOX. This assumption is based on inhibition studies of the selective enzyme extracts, i.e. whole-body super- natant, midgut, Malpighian tubules and gonads, ex- hibiting 86.0, 76.9, 90.0 and 83.0% inhibition, respectively (data are average of duplicate assays).
The GSH-dependent peroxidase activity assayed with H202 as substrate showed negligible inhibition ( < 10%) with fl-mercaptoethanol or mercaptosucci- nate. With fl-mercaptoethanol, the activities were inhibited by 10.0, 7.1, 7.0 and 5.0% for the whole-
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Fig. 6. Specific (left panel) and relative (right panel) activities of GR in various tissues of fifth-instar larvae of T. ni. Means for specific activities of GR are derived from triplicate assays (n = 3), each assay with two to three measurements. ActiVities are expressed in units, where 1 unit = 1 nmol NADPH oxidized min - lm$ protein -t. Tissue abbreviations are as for Fig. 1. ANOVA of specific activities Fo2,20)= 44.51; P < F = 0.0001. For tissues, F¢,o) = 53.12; P < F = 0.0001; for enzyme, F(2 ) = 1.41; P > F = 0.2669. Specific activity means (data bars) not followed by the same lower-case letters are significantly (P < 0.05)
different by DMRT.
Tissue spedfidty of antioxidant enzymes
Table 1. Specific activities of the antioxidant enzymes of fifth-instar larvae of T. n / i n hemolymph and hemocytes
Enzyme; mean units + SD* Hemolymph fraction~' SOD CAT GST GSTPX GPOX GR
*Means are derived from triplicate assays (n = 3), each assay with two to three measurements. Enzyme units are defined as follows: SOD, specific activity rain -I mg protein -I as defined by McCord and Fridovich (1969); CAT, 1 unit ffi 1/tmol H202 dfcomposed min -I mg protein-m; GSTPX, GPOX and GR, 1 unit ffi 1 nmol NADPH oxidized rain - l mg protein -I.
~'No activity was detected in the cell-free plasma of the hemolymph. :[:ND = activity not detected.
569
body supernatant, hindgut, Malpighian tubules and gonads, respectively. In that order of tissues, 1 x 10 -4 mercaptosuccinate caused inhibition by 10.0, 7.1, 4.2 and 8.0% (all data are average of duplicate assays). The amount inhibited is within the error margin of the procedure employed. Thus, the GSH-dependent reduction of H202 did not appear to be due to a selenium-dependent GPOX typically found in ver- tebrates.
DISCUSSION
As reviewed recently (Ahmad and Pardini, 1990b) most previous studies of antioxidant enzymes were performed on homogenates of whole bodies of adult or larval insects. Only in the housefly, Musca domestica (L.) (Bird et al., 1986), and larvae of the bioluminescent elatrid, Pyraerinus termitilluminans (Colepicolo et al., 1986) were their body parts, i.e. head, thorax and abdominal segments assayed for SOD activity. The tissue specificity of antioxidant enzymes of T. ni and physiological relevance there- fore represent a previously neglected area.
The 02- radical is responsible for an oxygen- radical cascade in aerobic cells from processes such as autoxidations of redox-active molecules, catalytic cyc- ling of oxidoreductase, and electron transport sys- tems (Fridovich, 1983). Mitochondria have the most elaborate electron transport system where the poten- tial leakage of 02- is highest, e.g. ubiquinone is reduced to the ubisemiquinone free radical, which donates an electron to 02 to generate O2-. It follows that tissues rich in mitochondria, because of high energy demand, should have the high levels of SOD and other antioxidant enzymes to serve as chain- breakers of the oxygen-radical cascade. This explains the overall high specific activities of the enzymes in the Malpighian tubules, hindgut and muscles. The two former tissues are rich in mitochondria for active transport mechanisms associated with selective re- sorption of water, salts and useful organic molecules, and in muscles the demand is high for fuel conversion to ATP (Chapman, 1971).
Gonads also had high specific activities of the antioxidant enzymes. Being vital organs, they need protection from oxidative stress arising from rapid differentiation processes.
The presence of SOD in an insect's hemocytes was a unique discovery. A copper/zinc protein of un- known enzymic function called erythrocuprein, was first isolated in 1939 from bovine erythrocytes (Bannister and Bannister, 1985). Its enzymic func- tion, first discovered by McCord and Fridovich
(1969), is to dismutate O1- radicals t o H 2 0 2. Since then, SOD has been found in all aerobic prokaryotes and eukaryotes, including the erythrocytes and leuko- cytes of higher vertebrates.
Insect hemocytes are apparently similar to erythro- cytes and leukocytes in possessing predominantly the cyanide-sensitive CuZn-SOD. SOD has a protective role in erythrocytes, where activated oxygen species oxidatively denature proteins which are then de- graded by a proteinase complex (Pacifici et al., 1989). Other antioxidant enzymes will protect the cell from injury by deleterious peroxidations. On the other hand, in the phagocytic leukocytes a cytochrome-b- dependent NADH oxidase situated in the plasma membrane is responsible for generating 02- radicals in large amounts extracellulary which in part is con- v e t t e d t o n 2 0 2 a n d " O H radicals (Tauber and Babior, 1985). This represents a microbicidal action just prior to phagocytosis (Tauber and Babior 1985; Johnston, 1985). The natural function of antioxidant enzymes in the leukocytes, is to provide protection in the event that some oxyradicals leak into the cell interior.
Recent work has revealed a close homology be- tween hemocytes and leukocytes. Many hemocytes perform phagncytosis of bacteria, encapsulation of microorganism too large for phagocytosis, and con- tain immunoglobulins and recognition factors in the antigen-antibody reaction (Gupta, 1985; pers. com- mun., 1990). We therefore hypothesize that SOD in insect hemocytes may have a role similar to that ascribed for leukocytes. This hypothesis should stimulate research to determine if insect hemocytes have a 02- generating mechanism similar and/or parallel to that of leukocytes.
The Mn-SOD in eukaryotes is distinct from CuZn- SOD, yet both SODs catalyze the same reaction (Fridovich, 1983). The present finding of much higher Mn-SOD than CuZn-SOD in T. ni's tissues (based on KCN inhibition) supports an earlier finding of three- fold higher mitochondrial SOD activity than cytoso- lic SOD (Ahmad et al., 1988a). This pattern is different from mammalian tissues, where CuZn-SOD is in excess of the mitochondrial SOD (GeUer and Winge, 1984). According to Fridovich (1983), such an anomalous distribution of CuZn-SOD and Mn-SOD in some vertebrate tissues has been observed. In addition, a copper chelator, diethyldithiocarbamate (DETC) also inhibits the activity of CuZn-SOD but not Mn-SOD (Misra, 1979). In larvae of two lepidopteran species, the southern armyworm (Spodoptera eridania) and black swallowtail butterfly (Papilio polyxenes), DETC enhanced their suscepti- bility to quercetin (Pritsos et ai., 1991). Quercetin, a
570 SAMt AHMAD et al.
ubiquitous flavonoid, is a well-known O1- generator (Hodnick et al., 1986, 1989). In this study SOD was strongly inhibited because both insect species possess higher amounts of CuZn-SOD than Mn-SOD (Ahmad et al., 1988b, 1990). Pritsos et al. (1991) were also able to inhibit T. ni's larval SOD by DETC, yet toxicity of quercetin did not increase. These data also support our data that in T. ni tissues, the abundant form of SOD is the Mn-SOD.
Insects lack an organ analogous to the vertebrate liver, but many functions such as protein synthesis, carbohydrate and fat deposition, their interconver- sion and mobilization occurs in the fat body (Chap- man, 1971), while the gut is the major site for detoxification of plant allelochemicals and other xenobiotics (Ahmad et al., 1986). The specific activi- ties of all antioxidant enzymes assayed were moderate for fat body, except for high-activity GR. High GR activity may be important for the maintenance of high GSH levels; GSH participates in a variety of biochemical reactions aside from those as an antioxidant.
The conspicuous absence of CAT from many tissues that had high SOD levels, is puzzling. One possibility is that the minor GPOX activity may afford some protection in these tissues, in conjunction with dietary antioxidants such as ~-tocopherol.
The unresolved identity of a GPOX as an insect enzyme that reduces H202 (as well as organic per- oxides) was recently emphasized (Ahmad and Par- dini, 1990b). The constitutive levels of this obscure enzyme are generally very low in T. ni, S. eridania and P. polyxenes larvae (2-12 units/mg protein) compared to an average 300 units/mg protein of mammalian tissues (Ahmad et al., 1989). Nevertheless, higher specific activity observed in T. ni's Malpighian tubules and gonads, raises the prospect of even higher specific activities in tissues of S. eridenia and P. polyxenes. The presence of GPOX activity in M. domestica, and the essentiality of selenium for this activity was first claimed by Simmons et aL (1987). A similar phenomenon was observed for M. domestica in our studies (Ahmad et al., 1989), but only low levels of GPOX-like activity accompanied by low levels of tissue selenium in phytophagous insects were observed.
Simmons et al. (1989a) retracted their original claim and proposed that selenium administration gave rise to seleno-acids such as selenocysteine which may have mimicked the activity of GPOX. In the meantime, the enzyme responsible for reduction of organic peroxides in T. ni was found to be GSTPX (Ahmad and Pardini, 1988, 1989). Similar reports followed for M. domestica (Simmons et al., 1989b), S. eridenia, P. polyxenes and T. ni (Weinhold et al., 1990). The GPOX-like activity is not due to a typi- cally mammalian seleno-enzyme, and the existence of an unidentified GPOX remains unclear.
Another hydroperoxidase which accepts H202 as co-substrate is peroxidase (EC 1.11.1.7). Detected in Drosophila melanogaster (Armstrong et al., 1978; Nickla et al., 1983) and in other insect species, peroxidase is apparently involved in the cuticular tanning process (Hasson and Sugumaran, 1987). No activity of this enzyme was detectable in T. ni, however (Mitchell et al., 1991). We then explored if
this enzyme works as a complex with dehydroascor- bate reductase (EC 1.8.5.1) and GR for the removal of H202 as shown in a plant (Dalton et aL, 1986). However, no such activity was detected (S. Ahmad, unpubl., 1990). Jakoby (1985) and Gfinzler and Floh6 (1985) have claimed that GSTPX has no activity toward H202, although Mannervik (1985) suggested that it has negligible activity toward H2 02. Whether this enzyme can catalyze the reduction of H202 is a
debatable matter and can only be resolved with purified enzyme. Insects including T. ni have high GSTPX and CAT activities probably as an evolution- ary adaptation to meagre GPOX activity.
Unravelling the multiplicity of an enzmye is funda- mental to understanding their collective and strategic function(s) in a tissue. Recently, CAT of T. ni larvae was purified to an apparent homogeneity with a specific activity of 2.2 x 105 units/mg protein (Mitchell et al., 1991), which surpasses that of the CAT purified from D. melanogaster (Nahmias and Bewley, 1984). CAT activity in T. ni's tissues is apparently due to a single protein. No such infor- mation is available for other antioxidant enzymes, but both SOD and GSTPX are well known from other sources as multi-isozymic proteins (Bird et al., 1986; Lee et al., 1981; Mannervik, 1985).
Another way of characterizing the role of antioxi- dant enzymes in an organism is to compute the relative amounts of enzymes in each tissue which may specify likely site(s) for pro-oxidants that are either metabolically activated (e.g. redox-active phenolics and quinones such as quercetin), or light-activated pro-oxidants (e.g. 8-methoxypsoralen; 8-MOP). Since, the portal of pro-oxidant entry is gut, high antioxidant enzyme levels of this tissue will likely counteract the oxidative stress exerted by redox- active allelochemicals. T. ni larvae have a thin and virtually unpigmented cuticle which permits light penetration to the tissues. Moreover, they feed on plants of Apiaceae, celery (Apium graveolens) and wild parnsip (Pastinaca sativa), with the potential of ingesting 8-MOP. The larvae lack the capacity to detoxify 8-MOP to nonphototoxic metabolites (Lee and Berenbaum, 1989). It is not surprising therefore, that feeding occurs by behavioral avoidance to mini- mize the amount of 8-MOP ingested (Ahmad et al., 1987), or by avoidance of direct sunlight (Jones and Granett, 1982). The highest relative abundance of all antioxidant enzymes in the integumental epithelium, seems to augument behavioral avoidance to ensure that a photosensitive pro-oxidant is not activated by light.
In summary, a good physiological relationship has been demonstrated for tissue specificity of the antiox- idant enzymes of T. ni. The specific activities are higher in tissues which are high in metabolic activity, hence correlated to elevated endogenous oxidative stress. Moreover, the enzymes are more abundant in tissues where the potential is high for either metabol- ically activated or light-activated plant pro-oxidants.
Acknowledgements--This work was supported by an USDA competitive research grant 88-37153-3475, and is a contri- bution of the Natural Products Laboratory and the Neveda Agricultural Experiment Station. We thank Dr Y. O. Koh, Statistical Consultant, Graduate School, University of
Tissue specificity of antioxidant enzymes 571
Nevada, Reno, for the statistical analysis of data. We also thank C. N. Capps and R. S. MaeGill for valuable technical assistance, and Susan Hall for skilfully and rapidly typing the entire manuscript.
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