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Ontogenic Expression of Detoxication Enzymes in an Australian Marsupial, the Brushtail Possum (Trichosurus vulpecula)
Comp. Biochem. Physiol. Vol. 118B, No. 1, pp. 239–247, 1997 Copyright 1997 Elsevier Science Inc. All rights reserved.
ISSN 0305-0491/97/$17.00 PII S0305-0491(97)00035-7
Ontogenic Expression of Detoxication Enzymes in an Australian Marsupial, the Brushtail Possum (Trichosurus vulpecula) Robyn M. Bolton and Jorma T. Ahokas Key Centre for Applied and Nutritional Toxicology, RMIT-University, GPO Box 2476V, Melbourne, Vic. 3001, Australia ABSTRACT. Marsupials and eutherians display vastly different reproductive strategies. Marsupials are characterised by the production of altricial neonates with little functional capacity. An investigation of the ontogenic expression of phase I (mixed function oxidase) and phase II (glutathione transferase) enzyme systems in the marsupial, the brushtail possum was undertaken. Enzyme expression in the youngest age group studied (60 days old) was between 5% and 10% of the adult level. A gradual increase in expression was then observed until a significant 3-fold increase to adult levels of expression of cytochrome P450, cytochrome b 5 and glutathione transferase content and ECOD and AE activity was observed in brushtail possum young between the ages of 150 6 15 and 180 6 15 days. The expression of EROD activity reached adult levels by the age of 150 6 days, while the expression of NADPH-cytochrome c reductase activity was delayed and adult levels had not yet been achieved by the oldest group studied (.200 days). The ontogenic expression of detoxication enzymes was significantly delayed in the marsupial in comparison to eutherians. Adult levels were achieved during the weaning period, suggesting that dietary xenobiotics act as a regulatory mechanism in the developmental expression of these enzymes in the brushtail possum. comp biochem physiol 118B;1:239–247, 1997. 1997 Elsevier Science Inc. KEY WORDS. Brushtail possum, development, glutathione transferase, mammalian evolution, marsupial, mixed function oxidase, ontogenic enzyme expression, xenobiotic metabolism
INTRODUCTION Eutherian and marsupial mammals differ markedly in their mode of reproduction and development. Marsupials are generally characterised by a short gestation period in which limited intrauterine development is achieved. The most striking characteristic of marsupial development is the immaturity of the young at birth. In contrast to eutherians, in which the birth weight of the litter is 15% of the maternal body mass, the birth weight of a marsupial litter is 0.1% of the maternal body mass (13). Limited development prior to birth in marsupials is thought to be a result of the uniformity in the period of intrauterine organogenesis and involves restriction of both the preattachment and organogenesis phases of gestation (26). As a result, most of the organ systems in marsupials at birth are either absent or at a primitive Address reprint requests to: Jorma Ahokas, Key Centre for Applied and Nutritional Toxicology, RMIT-University, GPO Box 2476V, Melbourne, Vic. 3001, Australia. Tel. (03) 9660 2650; Fax (03) 9663 6087; E-mail: [email protected]. Abbreviations–MFO, mixed function oxidase; GST, glutathione transferase (EC 2.5.1.18); ECOD, ethoxycoumarin O-deethylase; EROD, ethoxyresorufin O-deethylase; AE, aldrin epoxidation. Received 7 November 1996; revised 5 March 1997; accepted 13 March 1997.
stage of development (12). In contrast, eutherian mammals undertake the major part of their development in the uterus, and at birth are at a more advanced stage of development than any marsupial (14). Further development of marsupials occurs in the pouch during the lactation period and is generally characterised by an initial slow growth rate followed by increased developmental rate at weaning (8,28). With regard to most organs and functions, marsupials and eutherians develop along similar pathways (38), with the most significant difference occurring in the rate and timing of development of functional capacity (8). Little is known about how such differences in the rate of development of organs and biochemical systems effect the ability of marsupials to metabolise xenobiotics during the extended developmental period. Bentley and Shield (3) studied metabolism and kidney function in the pouch young of the quokka (Setonix brachyurus) and concluded that quokka young do not reach the same stage of functional development as neonatal eutherians until 120 days after birth. Only one study reporting an investigation of the ontogeny of xenobiotic metabolism in the developing marsupial has been previously published. McManus et al. (19) investigated the development of the hepatic mixed function oxidase (MFO) system in the quokka and first observed adult levels of expression
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of MFO enzymes in young between the ages of 155 and 255 days. In contrast, adult levels of MFO enzymes are observed within 30 to 40 days of birth in most eutherians investigated (10,16,36). This study investigates the ontogenic expression of detoxication enzymes representing both phase I (MFO enzymes) and phase II (glutathione transferase) metabolic pathways in the marsupial, the brushtail possum. This folivorous marsupial gives birth to a single young after 17.5 days of gestation (25). Neonates remain attached to the maternal teat for approximately 94 days after birth. Young remain in the pouch until about 140 to 150 days old, after which they continue to ride on the mother’s back and to suckle from the maternal teat for another 30 to 60 days (37). Weaning commences at around the time of first pouch exit (although it may start as early as 130 days old) and is completed at approximately 230 days old (31). MATERIALS AND METHODS Animals Young brushtail possums were trapped along with their mothers in a semi-rural area of native forest 60 km eastnorth-east of Melbourne (Department of Conservation Permit RP-93-062, Royal Melbourne Zoological Gardens Project Number HS/12/93), and were transported to the laboratory with the mother in hessian sacks or darkened cages. Pouch young were weighed, and measurement of head length was made to enable estimation of age (17). Both mother and young were anaesthetised using carbon dioxide inhalation. Anaesthesia was maintained during surgery with ether inhalation. Liver perfusion and organ removal and storage was carried out as described previously (4,5). Measurement of Hepatic MFO Content and Activity in Brushtail Possum Pouch Young Differential centrifugation was employed to prepare the hepatic microsomal fraction. Measurement of microsomal cytochrome P450 and cytochrome b 5 content was made via carbon monoxide- and NADH-difference spectra, respectively. Hepatic microsomal ECOD and EROD activity were determined using spectrofluorimetric assays with the substrates 7-ethoxycoumarin and 7-ethoxyresorufin, respectively. NADPH-cytochrome c reductase activity was determined via spectrophotometric assay with cytochrome c as the substrate. Aldrin epoxidation activity was measured using gas chromatographic quantification of the assay product, dieldrin. All assays were optimised for the brushtail possum and detailed assay conditions have been described previously (4). Purification of Glutathione Transferases Hepatic cytosol (100,000 g supernatant) was prepared via differential centrifugation. Purification of glutathione trans-
ferases was achieved via affinity chromatography of individual cytosols as described previously (5). Briefly, cytosol was applied to two affinity columns, containing S-hexyl glutathione-linked Sepharose and glutathione-linked Sepharose, arranged in tandem. After sample loading, the columns were detached from each other and developed separately. Following appropriate washing, bound GSTs were eluted from both columns using mM Tris-HCI containing 30 mM glutathione, pH 9.6.
Quantitation of Hepatic Glutathione Transferase Content Soluble hepatic glutathione transferase content was determined using HPLC analysis of affinity purified GST fractions, enabling quantification of GST subunits. HPLC analysis was performed as described previously (5). Integration of peak area was undertaken using an SPD-M6A UV-VIS spectrophotometric detector with an associated computer package (Shimadzu, Kyoto, Japan). Quantitation of GST content was achieved using a standard curve (4 points, 1– 15 mg Possum GST 1-1).
Statistical Analysis Pouch young were arranged according to age into 6 groups, consisting of pouch young of 60 6 15 days old (51 to 75 days old, n 5 4), 90 6 15 days old (82 to 103 days old, n 5 10), 120 6 15 days old (120 to 128 days old, n 5 6), 150 6 15 days old (139 to 165 days old, n 5 5), 180 6 15 days old (173 to 190 days old, n 5 6) and pouch young greater than 200 days old (n 5 6). The growth curves based on head length (17) become unreliable after 200 days of age, however, young in the .200 day group were trapped around 30 days after those in the 180-day group, and were most likely around 210 days of age. Due to the small number of pouch young available for study (resulting from wildlife permit restrictions,) pouch young of both sexes were pooled in each age group. Sexspecific patterns in cytochrome P450 expression are usually associated with the onset of puberty (41). All sub-adult young investigated in this study were prepubescent. Pouch young MFO content and activity and hepatic GST content were compared to values determined in previous studies for adult brushtail possums from the same population (5 adult males and 5 adult females, sexes pooled) (4,5). A one-way ANOVA using individual data was employed to compare age groups. Cochran’s C test was used to test for homogeneity of variance between the groups and data log transformed if heterogeneous. Whenever a significant result (p 2 0.05) was found in the ANOVA, a multiple-range test (Tukey Compromise LSD test, based on 95% confidence intervals for group means) was conducted to distinguish differences between category means. Liver to body
Ontogenic Expression of Brushtail Possum Detoxication Enzymes
FIG. 1. Development of liver to body weight ratio with age
in the brushtail possum. Values are shown as mean 1 SE. Maximum and minimum ages of pouch young are shown for each age group. Groups shown without a common letter are significantly different ( p # 0.05).
weight ratios were arcsine transformed prior to statistical analysis. RESULTS Development of Hepatic Parameters Ontogenic changes in liver to body weight ratio are shown in Fig. 1. This parameter was shown to increase gradually across the age groups studied, with significant differences found between the two youngest groups and the adult possums only. Development of hepatic microsomal protein content with age is shown in Fig. 2. A constant increase in hepatic microsomal protein content was shown to occur until adult levels were attained. Significant increases occurred between age groups around the time of weaning and adult levels were achieved by 150 6 15 days old. Determination of an appropriate parameter for expression of enzyme concentration or activity in developmental studies is an issue of some contention in the literature. The most common parameters used are concentration/activity relative to microsomal protein and concentration/activity relative to microsomal protein and concentration/activity relative to liver weight. Both methods have been shown to potentially underestimate true activity when used in developmental studies (2,30). Expression of the development of enzyme activity per liver weight may underestimate enzyme activity because foetal SER may be less efficiently pelleted by standard centrifugation techniques (2). Changes in relative hepatic cell type during ontogeny may also influence the developmental profile of activity expressed on a liver weight basis. Expression of enzyme activity as a function of microsomal protein
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FIG. 2. Development of microsomal protein content with age in the brushtail possum. Values are shown as mean 1 SE. Maximum and minimum ages of pouch young are shown for each age group. Groups shown without a common letter are significantly different ( p # 0.05).
does not account for changes in ER morphology. Since changes in the ratio of smooth to rough ER have been shown to occur in the hepatocytes of developing mammals (30), a resultant overall decrease in ribosomal protein per unit microsomal fraction may result in a dilutional effect of enzymatic values when expressed as specific activity. Expressing MFO activity and content in terms of liver weight has the advantage of indicating the individual animal’s overall hepatic capacity for metabolism. This parameter is relevant to the current investigation of the capability of a developing marsupial for xenobiotic metabolism and thus potentially its susceptibility (or lack thereof) to environmental chemicals. In addition, microsomal protein content was shown to increase significantly during brushtail possum development (Fig. 2), while liver to body weight ratio showed only a slight increase with age (Fig. 1), and, therefore, provides a more suitable reference parameter for indication of enzyme activity. Thus, while statistical analysis of the results when calculated as specific activity showed a similar overall picture, data are expressed relative to liver weight for the purposes of the discussion. Development of Hepatic MFO Components Hepatic cytochrome P450 expression (Fig. 3) was approximately 5% of adult levels in the youngest age group investigated. A steady increase was observed until at 120 6 15 days old hepatic cytochrome P450 content was 12% of adult levels. Between the 120 6 15 and 150 6 15 day old age groups, a significant increase in cytochrome P450 content was observed (12 to 29% of adult level). A rapid and significant increase was observed in the 180 6 15 day old
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FIG. 5. Development of hepatic NADPH-cytochrome c reFIG. 3. Development of hepatic cytochrome P450 content
with age in the brushtail possum. Values are shown as mean 1 SE. Maximum and minimum ages of pouch young are shown for each age group. Groups shown without a common letter are significantly different ( p # 0.05).
group (in comparison to the 150 6 15 days age group), and adult levels of hepatic cytochrome P450 content were attained at this development stage. The development of hepatic cytochrome b 5 content occurred along a similar pattern to cytochrome P450 and is shown in Fig. 4. In the 60 6 15 days age group, hepatic cytochrome b 5 content was 4% of adult values. This significantly increased to around 12% of adult level between the
ductase activity with age in the brushtail possum. Values are shown as mean 1 SE. Maximum and minimum ages of pouch young are shown for each age group. Groups shown without a common letter are significantly different ( p # 0.05).
ages of 90 6 15 and 120 6 15 days old. A further significant increase occurred at 150 6 15 days old (26% of adult level). Again, a rapid and significant increase in hepatic cytochrome b 5 content occurred at 180 6 15 days old (in comparison to the 150 6 15 days age group), at which age adult levels were achieved. Hepatic NADPH-cytochrome c reductase activity developed later in ontogeny in comparison to cytochrome P450 and cytochrome b 5 content, although the developmental pattern was similar (Fig. 5). Activity at around 12% of adult level was observed in pouch young between the ages of 60 6 15 and 120 6 15 days old. A significant increase (to 28% of adult level) was then observed at 150 6 15 days old. A further significant increase at 180 6 15 days old was found, constituting an increase to around 50% of adult activity. Activity increased to 65% of the adult level in the . 200day-old group, however, this value was significantly less than the adult level. Thus, hepatic NADPH-cytochrome c reductase activity reached adult levels in brushtail possums at an age greater than 200 days old. Development of Hepatic MFO Enzyme Activity
FIG. 4. Development of hepatic cytochrome b5 content with
age in the brushtail possum. Values are shown as mean 1 SE. Maximum and minimum ages of pouch young are shown for each age group. Groups shown without a common letter are significantly different ( p # 0.05).
Hepatic ECOD activity in the developing brushtail possum is shown in Fig. 6a. Activity was easily detectable in the youngest possums investigated and constituted about 10% of the adult level. Although ECOD activity increased to around 37% of the adult level by the age of 150 6 15 days old, a significant increase between groups was not observed until 180 6 15 days old. At this point, ECOD activity rapidly increased to adult levels (from 37% of adult level at 150 6 15 days old). ECOD activity, expressed in relation
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FIG. 7. Development of hepatic EROD activity in the brushFIG. 6. Development of hepatic ECOD activity in the brush-
tail possum. (a) Development of ECOD activity when considered on a liver weight basis. (b) Development of ECOD activity in relation to hepatic cytochrome P450 content. Values are shown as mean 1 SE. Maximum and minimum ages of pouch young are shown for each age group. Groups shown without a common letter are significantly different ( p # 0.05).
to cytochrome P450 content, decreased over the age groups studied (Fig. 6b). The development of hepatic EROD activity in the brushtail possum is shown in Fig. 7a. EROD activity increased from 6 to 9% of adult levels in the three youngest age groups studied to 32% of the adult level at 150 6 15 days old. This value and those of age groups 180 6 15 (45% of adult level) and .200 days old (47% of the adult level) were not significantly different from adult EROD activity, although lack
tail possum. (a) Development of EROD activity when considered on a liver weight basis. (b) Development of EROD activity in relation to hepatic cytochrome P450 content. Values are shown as mean 1 SE. Maximum and minimum ages of pouch young are shown for each age group. Groups shown without a common letter are significantly different ( p # 0.05).
of significance is most likely due to the high variability in the adult values, which in turn is due to the significant sex differences observed in EROD activity in this group of adult brushtail possums. No significant difference during development was found in hepatic EROD activity expressed relative to cytochrome P450 content (Fig. 7b). The developmental increase in aldrin epoxidation activity in the brushtail possum is shown in Fig. 8a. Activity of one animal in the youngest age was shown to be 7% of the adult level, however, no activity was detected in the other members of this age group. Hepatic aldrin epoxidation ac-
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FIG. 9. Development of hepatic glutathione transferase con-
tent in the brushtail possum. The total hepatic concentration of Possum GST 1-1 was determined in individual cytosolic samples using HPLC analysis of the affinity-purified fraction. Values are shown as mean 1 SE. Maximum and minimum ages of pouch young are shown for each age group. Groups shown without a common letter are significantly different ( p # 0.05).
Purification of Glutathione Transferase
FIG. 8. Development of hepatic aldrin epoxidation activity in the brushtail possum. (a) Development of aldrin epoxidation activity when considered on a liver weight basis. (b) Development of aldrin epoxidation activity in relation to hepatic cytochrome P450 content. Values are shown as mean 1 SE. Maximum and minimum ages of pouch young are shown for each age group. Groups shown without a common letter are significantly different ( p # 0.05). *In three of the four animals in the youngest age group no activity was detected. The results for these animals in relation to cytochrome P450 content are not shown, and the group was not included in statistical analysis.
tivity remained at around 10% of the adult level until it increased to around 22% at 150 6 15 days old. A significant increase then occurred, and at 180 6 15 days old, adult levels of hepatic aldrin epoxidation activity were attained. Aldrin epoxidation activity showed some variability during development when expressed as a function of cytochrome P450 content (Fig. 8b).
A previous study has shown that adult brushtail possums express a predominant hepatic alpha class GST isoenzyme (Possum GST 1-1) that constitutes 2 to 5% of the total cytosolic protein (5). Purification of glutathione transferases from the hepatic cytosol of developing brushtail possums also resulted in the isolation of the same GST isoenzyme from all young investigated, with no new GST isoenzymes observed. Quantitation of Hepatic Glutathione Transferase Content During Development The increase in hepatic glutathione transferase (Possum GST 1-1) content during development of the brushtail possum is shown in Fig 9. Between the ages of 60 6 15 days and 150 6 15 days old, a stable hepatic GST content was observed. A significant (3-fold) increase to adult hepatic GST levels was observed between the ages of 150 6 15 and 180 6 15 days old. DISCUSSION The ontogenic expression of three components of the MFO system, cytochrome P450, cytochrome b 5 and cytochrome P450 reductase (measured via its NADPH-cytochrome c reductase activity), was investigated in the brushtail possum. Both cytochrome P450 and cytochrome b 5 content in the
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liver increased gradually between the ages of 60 6 15 and 150 6 15 days (Figs. 3 and 4). A significant and rapid increase in the hepatic content of these MFO components occurred between 150 6 15 and 180 6 15 days of postnatal development. In contrast, the developmental expression of hepatic NADPH-cytochrome c reductase activity (Fig. 5) lagged behind that of cytochrome P450 and cytochrome b 5 content. Although significant increases in NADPH-cytochrome c reductase activity were observed between each of the three age groups between 120 6 15 and 180 6 15 days old, adult expression of activity had not yet been achieved in the most developed age group studied (.200 days old). Thus, maximum hepatic NADPH-cytochrome c reductase activity is developed after 200 days of postnatal development in the brushtail possum and may have a rate limiting effect on the function of the MFO system in possums at earlier development stages (20,22). The developmental expression of hepatic ECOD and aldrin epoxidation activity (Fig. 6a and Fig. 8a) followed a similar pattern to that observed with cytochrome P450 content. ECOD and AE activity remained low prior to 150 6 15 days of postnatal development. A significant increase to adult levels of ECOD and AE activity occurred between the 150 6 15 and 180 6 15 days old age groups. Expression of ECOD and AE activity as a function of cytochrome P450 content (Fig. 6b and Fig. 8b) showed that activity per cytochrome P450 content was slightly greater in the two youngest age groups studied and remained relatively stable thereafter. This indicates that the increase in ECOD and AE activity with age is associated with a concurrent and relative increase in hepatic cytochrome P450 content, and thus with increased expression of the cytochrome P450 isoenzymes responsible for this activity. In other mammalian species, ECOD activity has been measured in a broad range of cytochrome P450 families, with isoenzymes from the CYP1, CYP2 and CYP3 families showing varying levels of ECOD activity (32). While the mammalian cytochrome P450 isoenzymes displaying aldrin epoxidation activity are not well defined, AE activity is thought to be relatively specific for the CYP2B subfamily (39). The significantly greater ratio of ECOD and AE activity to cytochrome P450 content observed in the youngest age groups may represent a change in the ratio of the cytochrome P450 isoenzymes represented by ECOD and AE activity in relation to other cytochrome P450 isoenzymes. The development of the expression of hepatic EROD activity in the brushtail possum (Fig. 7a) occurs earlier than that observed for ECOD and AE activity. A more gradual increase in expression of EROD activity with age was shown, and adult levels were first observed in the 150 6 15 days old age group. This finding is in contrast to that observed in eutherians in which EROD activity predominates in neonates and decreases with age (15). In eutherian mammals previously investigated, EROD activity is associated with cytochrome P450 isoenzymes CYP1A1 and CYPIA2
(15). Expression of EROD activity as a function of cytochrome P450 content (Fig. 7b) showed no significant change during ontogenesis, indicating that the increasing cytochrome P450 content of the liver includes increasing expression of the isoenzyme(s) responsible for EROD activity. These findings clearly demonstrate that the MFO system of the brushtail possum develops during, and in some cases subsequent to, pouch life, and correlate well with the observations of McManus et al. (19) for another marsupial, the quokka. Unfortunately, the age at which enzyme expression is first observable could not be determined as animals younger than 60 days of age were unavailable for study due to wildlife permit restrictions. Similarly to the quokka, the brushtail possum reaches adult levels of MFO components and their activity after significant postnatal development. Indeed, for both marsupials, 5 to 7 months of postpartum development was necessary before adult xenobiotic metabolism capacity was attained. In contrast, most eutherians reach a similar level of functional capacity after a number of days or weeks of postpartum development (11,24). For example, Crestiel et al. (9) reported that cytochrome P450 content of 20-day-old foetal rats was 5% of the adult level. In the brushtail possum, this level of cytochrome P450 development was observed 60 days after parturition. Twelve hours after parturition in the rat, 30% of adult cytochrome P450 content was observed (9), while a similar level required around 150 days of postnatal development in the brushtail possum. The ontogenic expression of hepatic glutathione transferase content was investigated in the brushtail possum. Similarly to adult animals, a single GST isoenzyme (Possum GST 1-1) was detected in the affinity fraction purified from all pouch young investigated. Quantitation of hepatic Possum GST 1-1 content during development (Fig. 9) revealed that hepatic content of this GST isoenzyme was unchanging and approximately 30% of the adult level between 60 6 15 and 150 6 15 days of age. A significant 3-fold increase to adult levels occurred between the ages of 150 6 15 and 180 6 15 days old. Qualitative changes in GST isoenzyme profile during development have been observed in humans (33,34) and rats (1,7,35). A general ontogenic pattern of expression of the different GST classes has emerged in which expression of alpha and mu class GSTs increases during postnatal development. Significant hepatic expression of pi class GSTs is observed during gestation, but declines as birth approaches. Changes in GST isoenzyme profile were not observed in the brushtail possum, since a single alpha class GST isoenzyme was detected and purified. Most eutherians studied display adult levels and GST profiles within 2 months of birth (29,35). In the brushtail possum, a similar level of expression was attained after 5 to 6 months of postnatal development. The development of capacity for the metabolism of xeno-
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biotics showed a significant increase between the age of 150 6 15 and 180 6 15 days in the brushtail possum, an age span that correlates with the period following the commencement of weaning in this marsupial. Developmental patterns in which significant increases in xenobiotic metabolism function occur at weaning have been shown in many mammals (6,22,23,27,36,40). The inducibility of the MFO system by plant allelochemicals and other dietary xenobiotics is well characterised (21) and Possum GST 1-1 has been suggested to be important in the detoxication of allelochemicals (and their oxidised metabolites) present in the Eucalyptus spp. diet of the brushtail possum (5). Thus exposure to dietary xenobiotics upon weaning may act as an exogenously derived regulator of expression of these enzymes. Lactational transfer of dietary xenobiotics, as shown by McLennan et al. (18), may explain the low levels of detoxication enzymes expressed by pre-weaning brushtail possums. The features responsible for the differences between eutherian and marsupial mammals in the development of metabolic function are most likely of both physiological and behavioural origin. The rate of development of detoxication enzyme expression varies between eutherian species and the hypothesis of Neims et al. (20) that this is influenced by the relative state of maturity of the young at birth is supported by the present findings. The ontogenic stage at which expression of xenobiotic metabolising capacity is first developed and reaches maximum levels in marsupials is most likely influenced by both the rate of attainment of functional capacity of organs, as well as by exogenous factors such as foodborne xenobiotics and maternal hormones, as well as a myriad of regulatory factors yet to be investigated and defined. This study highlights the different rate of developmental expression of detoxication enzymes in a marsupial in comparison to eutherians. Similarly to many eutherian developmental patterns, the phase I and II enzyme systems studied in the brushtail possum first develop during postpartum differentiation of the liver and then increase in level and activity to adult levels during weaning. However, due to the differences in reproductive strategy and rate of development in the marsupial, physiological development (such as development of functional organs) as well as life history events (such as weaning) occur after a significant period of lactation and postpartum development. As a result of this slower rate of development, marsupials may be considerably more susceptible to environmental chemical exposure during their relatively extended development in comparison to eutherian mammals. Clearly, a better understanding of the mechanisms regulating the developmental expression of detoxication enzymes in marsupials is of great importance to understanding the potential for environmental chemicals to impact developing marsupials. Important future directions include the investigation of enzyme expression at the gene and transcription level, as well as investigation of neonatal and foe-
tal marsupials. This study has provided a much needed fundamental basis for further investigation. RMB is a recipient of a John Storey Memorial Scholarship, RMIT (1991) and an Australian Postgraduate Research Award (1992– 1994). The research was supported by the Australian Research Council.
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28. Russell, E.M. Patterns of parental care and investment in marsupials. Biol. Rev. 57:423–486;1982. 29. Serafini, M.T.; Arola, L.; Romeu, A. Glutathione and related enzyme activity in the 11-day rat embryo, placenta and perinatal rat liver. Biol. Neonate 60:236–242;1991. 30. Short, C.R.; Kinden, D.A.; Smith, R. Fetal and neonatal development of the microsomal monooxygenase system. Drug Metab. Rev. 5:1–42;1976. 31. Smith, M.J.; Brown, B.K.; Frith, H.J. Breeding of the brushtailed possum, Trichosurus vulpecula (Kerr), in New South Wales. CSIRO Wildl. Res. 14:181–193;1969. 32. Soucek, P.; Gut, I. Cytochromes P450 in rats: Structures, functions, properties and relevant human forms. Xenobiotica 22:83–103;1992. 32. Strange, R.C.; Davis, B.A.; Faulder, C.G.; Cotton, W.; Bain, A.D.; Hopkinson, D.A.; Hume, R. The human glutathione Stransferases: Developmental aspects of the GSTI, GST2, and GST3 loci. Biochem. Genet. 23:1011–1028;1985. 34. Strange, R.C.; Howie, A.F.; Hume, R.; Matharoo, B.; Bell, J.; Hiley, C.; Jones, P.; Beckett, G.J. The developmental expression of alpha-, mu- and pi-class glutathione S-transferases in human liver. Biochem. Biophys. Acta 993:186–190;1989. 35. Tee, L.B.G.; Gilmore, K.S.; Meyer, D.; Ketterer, B.; Vandenberghe, Y.; Yeoh, G.C.T. Expression of glutathione S-transferase during rat liver development. Biochem. J. 282:209–218; 1992. 36. Tredger, J.M.; Chhabra, R.S.; Fouts, J.R. Postnatal development of mixed-function oxidation as measured in microsomes from the small intestine and liver of rabbits. Drug Metab. Dispos. 4:17–24;1976. 37. Tyndale-Biscoe, C.H. Reproductive physiology of possums and gliders. In: Smith, A.; Hume, I. (eds). Possums and Gliders. Chipping Norton: Surrey Beatty and Sons; 1984:79–87. 38. Tyndale-Biscoe, C.H.; Janssens, P.A. (eds). The Developing Marsupial. Models for Biomedical Research; 1988. 39. Wolff, T.; Greim, H.; Huang, M.-T.; Miwa, G.T.; Lu, A.Y.H. Aldrin epoxidation catalyzed by purified rat-liver cytochromes P-450 and P-448. High selectively for cytochrome P-450. Eur. J. Biochem. 111:545–551;1980. 40. Yaffe, S.J.; Juchau, M.R. Perinatal pharmacology. Ann. Rev. Pharmacol. 14:219–238;1974. 41. Zangar, R.C.; Springer, D.L.; Buhler, D.R. Alterations in cytochrome P-450 levels in adult rats following neonatal exposure to xenobiotics. J. Toxicol. Environ. Health 38:43–55;1993.
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ISSN 0305-0491/97/$17.00 PII S0305-0491(97)00035-7
Ontogenic Expression of Detoxication Enzymes in an Australian Marsupial, the Brushtail Possum (Trichosurus vulpecula) Robyn M. Bolton and Jorma T. Ahokas Key Centre for Applied and Nutritional Toxicology, RMIT-University, GPO Box 2476V, Melbourne, Vic. 3001, Australia ABSTRACT. Marsupials and eutherians display vastly different reproductive strategies. Marsupials are characterised by the production of altricial neonates with little functional capacity. An investigation of the ontogenic expression of phase I (mixed function oxidase) and phase II (glutathione transferase) enzyme systems in the marsupial, the brushtail possum was undertaken. Enzyme expression in the youngest age group studied (60 days old) was between 5% and 10% of the adult level. A gradual increase in expression was then observed until a significant 3-fold increase to adult levels of expression of cytochrome P450, cytochrome b 5 and glutathione transferase content and ECOD and AE activity was observed in brushtail possum young between the ages of 150 6 15 and 180 6 15 days. The expression of EROD activity reached adult levels by the age of 150 6 days, while the expression of NADPH-cytochrome c reductase activity was delayed and adult levels had not yet been achieved by the oldest group studied (.200 days). The ontogenic expression of detoxication enzymes was significantly delayed in the marsupial in comparison to eutherians. Adult levels were achieved during the weaning period, suggesting that dietary xenobiotics act as a regulatory mechanism in the developmental expression of these enzymes in the brushtail possum. comp biochem physiol 118B;1:239–247, 1997. 1997 Elsevier Science Inc. KEY WORDS. Brushtail possum, development, glutathione transferase, mammalian evolution, marsupial, mixed function oxidase, ontogenic enzyme expression, xenobiotic metabolism
INTRODUCTION Eutherian and marsupial mammals differ markedly in their mode of reproduction and development. Marsupials are generally characterised by a short gestation period in which limited intrauterine development is achieved. The most striking characteristic of marsupial development is the immaturity of the young at birth. In contrast to eutherians, in which the birth weight of the litter is 15% of the maternal body mass, the birth weight of a marsupial litter is 0.1% of the maternal body mass (13). Limited development prior to birth in marsupials is thought to be a result of the uniformity in the period of intrauterine organogenesis and involves restriction of both the preattachment and organogenesis phases of gestation (26). As a result, most of the organ systems in marsupials at birth are either absent or at a primitive Address reprint requests to: Jorma Ahokas, Key Centre for Applied and Nutritional Toxicology, RMIT-University, GPO Box 2476V, Melbourne, Vic. 3001, Australia. Tel. (03) 9660 2650; Fax (03) 9663 6087; E-mail: [email protected]. Abbreviations–MFO, mixed function oxidase; GST, glutathione transferase (EC 2.5.1.18); ECOD, ethoxycoumarin O-deethylase; EROD, ethoxyresorufin O-deethylase; AE, aldrin epoxidation. Received 7 November 1996; revised 5 March 1997; accepted 13 March 1997.
stage of development (12). In contrast, eutherian mammals undertake the major part of their development in the uterus, and at birth are at a more advanced stage of development than any marsupial (14). Further development of marsupials occurs in the pouch during the lactation period and is generally characterised by an initial slow growth rate followed by increased developmental rate at weaning (8,28). With regard to most organs and functions, marsupials and eutherians develop along similar pathways (38), with the most significant difference occurring in the rate and timing of development of functional capacity (8). Little is known about how such differences in the rate of development of organs and biochemical systems effect the ability of marsupials to metabolise xenobiotics during the extended developmental period. Bentley and Shield (3) studied metabolism and kidney function in the pouch young of the quokka (Setonix brachyurus) and concluded that quokka young do not reach the same stage of functional development as neonatal eutherians until 120 days after birth. Only one study reporting an investigation of the ontogeny of xenobiotic metabolism in the developing marsupial has been previously published. McManus et al. (19) investigated the development of the hepatic mixed function oxidase (MFO) system in the quokka and first observed adult levels of expression
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of MFO enzymes in young between the ages of 155 and 255 days. In contrast, adult levels of MFO enzymes are observed within 30 to 40 days of birth in most eutherians investigated (10,16,36). This study investigates the ontogenic expression of detoxication enzymes representing both phase I (MFO enzymes) and phase II (glutathione transferase) metabolic pathways in the marsupial, the brushtail possum. This folivorous marsupial gives birth to a single young after 17.5 days of gestation (25). Neonates remain attached to the maternal teat for approximately 94 days after birth. Young remain in the pouch until about 140 to 150 days old, after which they continue to ride on the mother’s back and to suckle from the maternal teat for another 30 to 60 days (37). Weaning commences at around the time of first pouch exit (although it may start as early as 130 days old) and is completed at approximately 230 days old (31). MATERIALS AND METHODS Animals Young brushtail possums were trapped along with their mothers in a semi-rural area of native forest 60 km eastnorth-east of Melbourne (Department of Conservation Permit RP-93-062, Royal Melbourne Zoological Gardens Project Number HS/12/93), and were transported to the laboratory with the mother in hessian sacks or darkened cages. Pouch young were weighed, and measurement of head length was made to enable estimation of age (17). Both mother and young were anaesthetised using carbon dioxide inhalation. Anaesthesia was maintained during surgery with ether inhalation. Liver perfusion and organ removal and storage was carried out as described previously (4,5). Measurement of Hepatic MFO Content and Activity in Brushtail Possum Pouch Young Differential centrifugation was employed to prepare the hepatic microsomal fraction. Measurement of microsomal cytochrome P450 and cytochrome b 5 content was made via carbon monoxide- and NADH-difference spectra, respectively. Hepatic microsomal ECOD and EROD activity were determined using spectrofluorimetric assays with the substrates 7-ethoxycoumarin and 7-ethoxyresorufin, respectively. NADPH-cytochrome c reductase activity was determined via spectrophotometric assay with cytochrome c as the substrate. Aldrin epoxidation activity was measured using gas chromatographic quantification of the assay product, dieldrin. All assays were optimised for the brushtail possum and detailed assay conditions have been described previously (4). Purification of Glutathione Transferases Hepatic cytosol (100,000 g supernatant) was prepared via differential centrifugation. Purification of glutathione trans-
ferases was achieved via affinity chromatography of individual cytosols as described previously (5). Briefly, cytosol was applied to two affinity columns, containing S-hexyl glutathione-linked Sepharose and glutathione-linked Sepharose, arranged in tandem. After sample loading, the columns were detached from each other and developed separately. Following appropriate washing, bound GSTs were eluted from both columns using mM Tris-HCI containing 30 mM glutathione, pH 9.6.
Quantitation of Hepatic Glutathione Transferase Content Soluble hepatic glutathione transferase content was determined using HPLC analysis of affinity purified GST fractions, enabling quantification of GST subunits. HPLC analysis was performed as described previously (5). Integration of peak area was undertaken using an SPD-M6A UV-VIS spectrophotometric detector with an associated computer package (Shimadzu, Kyoto, Japan). Quantitation of GST content was achieved using a standard curve (4 points, 1– 15 mg Possum GST 1-1).
Statistical Analysis Pouch young were arranged according to age into 6 groups, consisting of pouch young of 60 6 15 days old (51 to 75 days old, n 5 4), 90 6 15 days old (82 to 103 days old, n 5 10), 120 6 15 days old (120 to 128 days old, n 5 6), 150 6 15 days old (139 to 165 days old, n 5 5), 180 6 15 days old (173 to 190 days old, n 5 6) and pouch young greater than 200 days old (n 5 6). The growth curves based on head length (17) become unreliable after 200 days of age, however, young in the .200 day group were trapped around 30 days after those in the 180-day group, and were most likely around 210 days of age. Due to the small number of pouch young available for study (resulting from wildlife permit restrictions,) pouch young of both sexes were pooled in each age group. Sexspecific patterns in cytochrome P450 expression are usually associated with the onset of puberty (41). All sub-adult young investigated in this study were prepubescent. Pouch young MFO content and activity and hepatic GST content were compared to values determined in previous studies for adult brushtail possums from the same population (5 adult males and 5 adult females, sexes pooled) (4,5). A one-way ANOVA using individual data was employed to compare age groups. Cochran’s C test was used to test for homogeneity of variance between the groups and data log transformed if heterogeneous. Whenever a significant result (p 2 0.05) was found in the ANOVA, a multiple-range test (Tukey Compromise LSD test, based on 95% confidence intervals for group means) was conducted to distinguish differences between category means. Liver to body
Ontogenic Expression of Brushtail Possum Detoxication Enzymes
FIG. 1. Development of liver to body weight ratio with age
in the brushtail possum. Values are shown as mean 1 SE. Maximum and minimum ages of pouch young are shown for each age group. Groups shown without a common letter are significantly different ( p # 0.05).
weight ratios were arcsine transformed prior to statistical analysis. RESULTS Development of Hepatic Parameters Ontogenic changes in liver to body weight ratio are shown in Fig. 1. This parameter was shown to increase gradually across the age groups studied, with significant differences found between the two youngest groups and the adult possums only. Development of hepatic microsomal protein content with age is shown in Fig. 2. A constant increase in hepatic microsomal protein content was shown to occur until adult levels were attained. Significant increases occurred between age groups around the time of weaning and adult levels were achieved by 150 6 15 days old. Determination of an appropriate parameter for expression of enzyme concentration or activity in developmental studies is an issue of some contention in the literature. The most common parameters used are concentration/activity relative to microsomal protein and concentration/activity relative to microsomal protein and concentration/activity relative to liver weight. Both methods have been shown to potentially underestimate true activity when used in developmental studies (2,30). Expression of the development of enzyme activity per liver weight may underestimate enzyme activity because foetal SER may be less efficiently pelleted by standard centrifugation techniques (2). Changes in relative hepatic cell type during ontogeny may also influence the developmental profile of activity expressed on a liver weight basis. Expression of enzyme activity as a function of microsomal protein
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FIG. 2. Development of microsomal protein content with age in the brushtail possum. Values are shown as mean 1 SE. Maximum and minimum ages of pouch young are shown for each age group. Groups shown without a common letter are significantly different ( p # 0.05).
does not account for changes in ER morphology. Since changes in the ratio of smooth to rough ER have been shown to occur in the hepatocytes of developing mammals (30), a resultant overall decrease in ribosomal protein per unit microsomal fraction may result in a dilutional effect of enzymatic values when expressed as specific activity. Expressing MFO activity and content in terms of liver weight has the advantage of indicating the individual animal’s overall hepatic capacity for metabolism. This parameter is relevant to the current investigation of the capability of a developing marsupial for xenobiotic metabolism and thus potentially its susceptibility (or lack thereof) to environmental chemicals. In addition, microsomal protein content was shown to increase significantly during brushtail possum development (Fig. 2), while liver to body weight ratio showed only a slight increase with age (Fig. 1), and, therefore, provides a more suitable reference parameter for indication of enzyme activity. Thus, while statistical analysis of the results when calculated as specific activity showed a similar overall picture, data are expressed relative to liver weight for the purposes of the discussion. Development of Hepatic MFO Components Hepatic cytochrome P450 expression (Fig. 3) was approximately 5% of adult levels in the youngest age group investigated. A steady increase was observed until at 120 6 15 days old hepatic cytochrome P450 content was 12% of adult levels. Between the 120 6 15 and 150 6 15 day old age groups, a significant increase in cytochrome P450 content was observed (12 to 29% of adult level). A rapid and significant increase was observed in the 180 6 15 day old
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FIG. 5. Development of hepatic NADPH-cytochrome c reFIG. 3. Development of hepatic cytochrome P450 content
with age in the brushtail possum. Values are shown as mean 1 SE. Maximum and minimum ages of pouch young are shown for each age group. Groups shown without a common letter are significantly different ( p # 0.05).
group (in comparison to the 150 6 15 days age group), and adult levels of hepatic cytochrome P450 content were attained at this development stage. The development of hepatic cytochrome b 5 content occurred along a similar pattern to cytochrome P450 and is shown in Fig. 4. In the 60 6 15 days age group, hepatic cytochrome b 5 content was 4% of adult values. This significantly increased to around 12% of adult level between the
ductase activity with age in the brushtail possum. Values are shown as mean 1 SE. Maximum and minimum ages of pouch young are shown for each age group. Groups shown without a common letter are significantly different ( p # 0.05).
ages of 90 6 15 and 120 6 15 days old. A further significant increase occurred at 150 6 15 days old (26% of adult level). Again, a rapid and significant increase in hepatic cytochrome b 5 content occurred at 180 6 15 days old (in comparison to the 150 6 15 days age group), at which age adult levels were achieved. Hepatic NADPH-cytochrome c reductase activity developed later in ontogeny in comparison to cytochrome P450 and cytochrome b 5 content, although the developmental pattern was similar (Fig. 5). Activity at around 12% of adult level was observed in pouch young between the ages of 60 6 15 and 120 6 15 days old. A significant increase (to 28% of adult level) was then observed at 150 6 15 days old. A further significant increase at 180 6 15 days old was found, constituting an increase to around 50% of adult activity. Activity increased to 65% of the adult level in the . 200day-old group, however, this value was significantly less than the adult level. Thus, hepatic NADPH-cytochrome c reductase activity reached adult levels in brushtail possums at an age greater than 200 days old. Development of Hepatic MFO Enzyme Activity
FIG. 4. Development of hepatic cytochrome b5 content with
age in the brushtail possum. Values are shown as mean 1 SE. Maximum and minimum ages of pouch young are shown for each age group. Groups shown without a common letter are significantly different ( p # 0.05).
Hepatic ECOD activity in the developing brushtail possum is shown in Fig. 6a. Activity was easily detectable in the youngest possums investigated and constituted about 10% of the adult level. Although ECOD activity increased to around 37% of the adult level by the age of 150 6 15 days old, a significant increase between groups was not observed until 180 6 15 days old. At this point, ECOD activity rapidly increased to adult levels (from 37% of adult level at 150 6 15 days old). ECOD activity, expressed in relation
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FIG. 7. Development of hepatic EROD activity in the brushFIG. 6. Development of hepatic ECOD activity in the brush-
tail possum. (a) Development of ECOD activity when considered on a liver weight basis. (b) Development of ECOD activity in relation to hepatic cytochrome P450 content. Values are shown as mean 1 SE. Maximum and minimum ages of pouch young are shown for each age group. Groups shown without a common letter are significantly different ( p # 0.05).
to cytochrome P450 content, decreased over the age groups studied (Fig. 6b). The development of hepatic EROD activity in the brushtail possum is shown in Fig. 7a. EROD activity increased from 6 to 9% of adult levels in the three youngest age groups studied to 32% of the adult level at 150 6 15 days old. This value and those of age groups 180 6 15 (45% of adult level) and .200 days old (47% of the adult level) were not significantly different from adult EROD activity, although lack
tail possum. (a) Development of EROD activity when considered on a liver weight basis. (b) Development of EROD activity in relation to hepatic cytochrome P450 content. Values are shown as mean 1 SE. Maximum and minimum ages of pouch young are shown for each age group. Groups shown without a common letter are significantly different ( p # 0.05).
of significance is most likely due to the high variability in the adult values, which in turn is due to the significant sex differences observed in EROD activity in this group of adult brushtail possums. No significant difference during development was found in hepatic EROD activity expressed relative to cytochrome P450 content (Fig. 7b). The developmental increase in aldrin epoxidation activity in the brushtail possum is shown in Fig. 8a. Activity of one animal in the youngest age was shown to be 7% of the adult level, however, no activity was detected in the other members of this age group. Hepatic aldrin epoxidation ac-
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FIG. 9. Development of hepatic glutathione transferase con-
tent in the brushtail possum. The total hepatic concentration of Possum GST 1-1 was determined in individual cytosolic samples using HPLC analysis of the affinity-purified fraction. Values are shown as mean 1 SE. Maximum and minimum ages of pouch young are shown for each age group. Groups shown without a common letter are significantly different ( p # 0.05).
Purification of Glutathione Transferase
FIG. 8. Development of hepatic aldrin epoxidation activity in the brushtail possum. (a) Development of aldrin epoxidation activity when considered on a liver weight basis. (b) Development of aldrin epoxidation activity in relation to hepatic cytochrome P450 content. Values are shown as mean 1 SE. Maximum and minimum ages of pouch young are shown for each age group. Groups shown without a common letter are significantly different ( p # 0.05). *In three of the four animals in the youngest age group no activity was detected. The results for these animals in relation to cytochrome P450 content are not shown, and the group was not included in statistical analysis.
tivity remained at around 10% of the adult level until it increased to around 22% at 150 6 15 days old. A significant increase then occurred, and at 180 6 15 days old, adult levels of hepatic aldrin epoxidation activity were attained. Aldrin epoxidation activity showed some variability during development when expressed as a function of cytochrome P450 content (Fig. 8b).
A previous study has shown that adult brushtail possums express a predominant hepatic alpha class GST isoenzyme (Possum GST 1-1) that constitutes 2 to 5% of the total cytosolic protein (5). Purification of glutathione transferases from the hepatic cytosol of developing brushtail possums also resulted in the isolation of the same GST isoenzyme from all young investigated, with no new GST isoenzymes observed. Quantitation of Hepatic Glutathione Transferase Content During Development The increase in hepatic glutathione transferase (Possum GST 1-1) content during development of the brushtail possum is shown in Fig 9. Between the ages of 60 6 15 days and 150 6 15 days old, a stable hepatic GST content was observed. A significant (3-fold) increase to adult hepatic GST levels was observed between the ages of 150 6 15 and 180 6 15 days old. DISCUSSION The ontogenic expression of three components of the MFO system, cytochrome P450, cytochrome b 5 and cytochrome P450 reductase (measured via its NADPH-cytochrome c reductase activity), was investigated in the brushtail possum. Both cytochrome P450 and cytochrome b 5 content in the
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liver increased gradually between the ages of 60 6 15 and 150 6 15 days (Figs. 3 and 4). A significant and rapid increase in the hepatic content of these MFO components occurred between 150 6 15 and 180 6 15 days of postnatal development. In contrast, the developmental expression of hepatic NADPH-cytochrome c reductase activity (Fig. 5) lagged behind that of cytochrome P450 and cytochrome b 5 content. Although significant increases in NADPH-cytochrome c reductase activity were observed between each of the three age groups between 120 6 15 and 180 6 15 days old, adult expression of activity had not yet been achieved in the most developed age group studied (.200 days old). Thus, maximum hepatic NADPH-cytochrome c reductase activity is developed after 200 days of postnatal development in the brushtail possum and may have a rate limiting effect on the function of the MFO system in possums at earlier development stages (20,22). The developmental expression of hepatic ECOD and aldrin epoxidation activity (Fig. 6a and Fig. 8a) followed a similar pattern to that observed with cytochrome P450 content. ECOD and AE activity remained low prior to 150 6 15 days of postnatal development. A significant increase to adult levels of ECOD and AE activity occurred between the 150 6 15 and 180 6 15 days old age groups. Expression of ECOD and AE activity as a function of cytochrome P450 content (Fig. 6b and Fig. 8b) showed that activity per cytochrome P450 content was slightly greater in the two youngest age groups studied and remained relatively stable thereafter. This indicates that the increase in ECOD and AE activity with age is associated with a concurrent and relative increase in hepatic cytochrome P450 content, and thus with increased expression of the cytochrome P450 isoenzymes responsible for this activity. In other mammalian species, ECOD activity has been measured in a broad range of cytochrome P450 families, with isoenzymes from the CYP1, CYP2 and CYP3 families showing varying levels of ECOD activity (32). While the mammalian cytochrome P450 isoenzymes displaying aldrin epoxidation activity are not well defined, AE activity is thought to be relatively specific for the CYP2B subfamily (39). The significantly greater ratio of ECOD and AE activity to cytochrome P450 content observed in the youngest age groups may represent a change in the ratio of the cytochrome P450 isoenzymes represented by ECOD and AE activity in relation to other cytochrome P450 isoenzymes. The development of the expression of hepatic EROD activity in the brushtail possum (Fig. 7a) occurs earlier than that observed for ECOD and AE activity. A more gradual increase in expression of EROD activity with age was shown, and adult levels were first observed in the 150 6 15 days old age group. This finding is in contrast to that observed in eutherians in which EROD activity predominates in neonates and decreases with age (15). In eutherian mammals previously investigated, EROD activity is associated with cytochrome P450 isoenzymes CYP1A1 and CYPIA2
(15). Expression of EROD activity as a function of cytochrome P450 content (Fig. 7b) showed no significant change during ontogenesis, indicating that the increasing cytochrome P450 content of the liver includes increasing expression of the isoenzyme(s) responsible for EROD activity. These findings clearly demonstrate that the MFO system of the brushtail possum develops during, and in some cases subsequent to, pouch life, and correlate well with the observations of McManus et al. (19) for another marsupial, the quokka. Unfortunately, the age at which enzyme expression is first observable could not be determined as animals younger than 60 days of age were unavailable for study due to wildlife permit restrictions. Similarly to the quokka, the brushtail possum reaches adult levels of MFO components and their activity after significant postnatal development. Indeed, for both marsupials, 5 to 7 months of postpartum development was necessary before adult xenobiotic metabolism capacity was attained. In contrast, most eutherians reach a similar level of functional capacity after a number of days or weeks of postpartum development (11,24). For example, Crestiel et al. (9) reported that cytochrome P450 content of 20-day-old foetal rats was 5% of the adult level. In the brushtail possum, this level of cytochrome P450 development was observed 60 days after parturition. Twelve hours after parturition in the rat, 30% of adult cytochrome P450 content was observed (9), while a similar level required around 150 days of postnatal development in the brushtail possum. The ontogenic expression of hepatic glutathione transferase content was investigated in the brushtail possum. Similarly to adult animals, a single GST isoenzyme (Possum GST 1-1) was detected in the affinity fraction purified from all pouch young investigated. Quantitation of hepatic Possum GST 1-1 content during development (Fig. 9) revealed that hepatic content of this GST isoenzyme was unchanging and approximately 30% of the adult level between 60 6 15 and 150 6 15 days of age. A significant 3-fold increase to adult levels occurred between the ages of 150 6 15 and 180 6 15 days old. Qualitative changes in GST isoenzyme profile during development have been observed in humans (33,34) and rats (1,7,35). A general ontogenic pattern of expression of the different GST classes has emerged in which expression of alpha and mu class GSTs increases during postnatal development. Significant hepatic expression of pi class GSTs is observed during gestation, but declines as birth approaches. Changes in GST isoenzyme profile were not observed in the brushtail possum, since a single alpha class GST isoenzyme was detected and purified. Most eutherians studied display adult levels and GST profiles within 2 months of birth (29,35). In the brushtail possum, a similar level of expression was attained after 5 to 6 months of postnatal development. The development of capacity for the metabolism of xeno-
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biotics showed a significant increase between the age of 150 6 15 and 180 6 15 days in the brushtail possum, an age span that correlates with the period following the commencement of weaning in this marsupial. Developmental patterns in which significant increases in xenobiotic metabolism function occur at weaning have been shown in many mammals (6,22,23,27,36,40). The inducibility of the MFO system by plant allelochemicals and other dietary xenobiotics is well characterised (21) and Possum GST 1-1 has been suggested to be important in the detoxication of allelochemicals (and their oxidised metabolites) present in the Eucalyptus spp. diet of the brushtail possum (5). Thus exposure to dietary xenobiotics upon weaning may act as an exogenously derived regulator of expression of these enzymes. Lactational transfer of dietary xenobiotics, as shown by McLennan et al. (18), may explain the low levels of detoxication enzymes expressed by pre-weaning brushtail possums. The features responsible for the differences between eutherian and marsupial mammals in the development of metabolic function are most likely of both physiological and behavioural origin. The rate of development of detoxication enzyme expression varies between eutherian species and the hypothesis of Neims et al. (20) that this is influenced by the relative state of maturity of the young at birth is supported by the present findings. The ontogenic stage at which expression of xenobiotic metabolising capacity is first developed and reaches maximum levels in marsupials is most likely influenced by both the rate of attainment of functional capacity of organs, as well as by exogenous factors such as foodborne xenobiotics and maternal hormones, as well as a myriad of regulatory factors yet to be investigated and defined. This study highlights the different rate of developmental expression of detoxication enzymes in a marsupial in comparison to eutherians. Similarly to many eutherian developmental patterns, the phase I and II enzyme systems studied in the brushtail possum first develop during postpartum differentiation of the liver and then increase in level and activity to adult levels during weaning. However, due to the differences in reproductive strategy and rate of development in the marsupial, physiological development (such as development of functional organs) as well as life history events (such as weaning) occur after a significant period of lactation and postpartum development. As a result of this slower rate of development, marsupials may be considerably more susceptible to environmental chemical exposure during their relatively extended development in comparison to eutherian mammals. Clearly, a better understanding of the mechanisms regulating the developmental expression of detoxication enzymes in marsupials is of great importance to understanding the potential for environmental chemicals to impact developing marsupials. Important future directions include the investigation of enzyme expression at the gene and transcription level, as well as investigation of neonatal and foe-
tal marsupials. This study has provided a much needed fundamental basis for further investigation. RMB is a recipient of a John Storey Memorial Scholarship, RMIT (1991) and an Australian Postgraduate Research Award (1992– 1994). The research was supported by the Australian Research Council.
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