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Free Radical Defenses in the Liver and Kidney of Human Growth Hormone Transgenic Mice

Possible Mechanisms of Early Mortality

^a Department of Physiology, Southern Illinois University School of Medicine, Carbondale

Steven J. Hauck, Department of Physiology, Southern Illinois University School of Medicine, Carbondale, IL 62901-6512 E-mail: shauck{at}siumed.edu.

Decision Editor: John A. Faulkner, PhD

	Abstract

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The long-term effects of growth hormone (GH) administration are unknown. Although limited data on its short-term effects purport health benefits, numerous detrimental effects are the consequence of chronically elevated GH. We used spectrophotometric assay and Western blot to determine the effects of chronic GH excess on hepatic and renal antioxidant enzymes (AOEs) in young and middle-aged PEPCK (phosphoenolpyruvate carboxykinase) hGH (human GH) transgenic mice. In the liver, glutathione peroxidase (GPx) was reduced in transgenics of both age groups, catalase was reduced only in young transgenics, and Cu–Zn superoxide dismutase (SOD) was similar to normal mice, but declined with age. In all groups, hepatic AOE activity correlated significantly with AOE level. In the kidney, AOEs in young transgenics were similar to those of normal mice. However, middle-aged transgenics showed reduced renal SOD and GPx activities when compared with young transgenic or middle-aged normal mice. Similarly, renal SOD and GPx levels in middle-aged transgenics were reduced when compared with those of middle-aged normal mice. AOE activity in the kidney correlated significantly with AOE protein level among middle-aged animals only. These data suggest the following: ⁽¹⁾ GH excess is associated with early declines in SOD and GPx in the kidney and reductions of hepatic GPx at all ages examined, perhaps increasing the risk of free radical-induced damage to these tissues; ⁽²⁾ in the liver of young animals and in the liver and kidney of middle-aged animals, AOE activity reflects the amount of enzyme protein; and ⁽³⁾ age-related reductions in GPx in transgenics may be related to the increased incidence of liver tumors and renal failure in these animals.

DESPITE the essential nature of oxygen for all aerobic organisms, oxidative metabolism is not without consequences. Free radicals, or reactive oxygen species (ROS), the normal products of oxidative metabolism, continuously arise from a variety of cellular reactions ⁽¹⁾⁽²⁾. In addition, the potential consequences of ROS production are as varied as the reactions that produce them. Volumes of literature have been accumulated that implicate free radicals in macromolecular damage, including insults to DNA ⁽³⁾⁽⁴⁾, proteins ⁽⁵⁾⁽⁶⁾, and lipids ⁽⁷⁾⁽⁸⁾. The damage caused by free radicals was first suggested to play a role in the aging process nearly 50 years ago ⁽⁹⁾. The imbalance between pro-oxidants and antioxidant defenses, otherwise known as oxidative stress, increases dramatically with increasing age ⁽¹⁰⁾. Among the observational evidence for this are the measurement of free radicals in biological tissues ^(11)(12)(13), and of antioxidant capacity as a function of age ^(14)(15)(16). Evidence for direct ROS involvement in aging has been demonstrated in transgenic flies ⁽¹⁷⁾, worms ⁽¹⁸⁾, and mice ^(19)(20), which possess extra copies of genes coding for one or more free radical-scavenging antioxidant enzymes (AOEs). These models have an enhanced protection against ROS damage, and under normal or oxygen-stress (mouse model) conditions, they have been shown to live longer than nontransgenic controls. Today, despite the many unanswered questions concerning the extent to which oxidative stress correlates with aging, few would argue that ROS-induced damage to macromolecules does not play a major role in the aging process.

In addition to an expanding list of dietary antioxidants that remove ROS, cells possess several highly efficient enzymes responsible for the bulk of free radical scavenging. The AOEs represent the primary defense against oxygen free radicals and function both in tandem and in parallel to reduce the superoxide radical, the first product of the monoelectric oxygen reduction, to water. Among these are superoxide dismutase (SOD), which converts the superoxide radical to hydrogen peroxide (H₂O₂); catalase (CAT), which quenches the generated radical by conversion of H₂O₂ to water; and glutathione peroxidase (GPx), which, similar to CAT, detoxifies H₂O₂, but primarily scavenges phospholipid and other organic hydroperoxides. These defenses vary widely between species but their existence, in some form, is universal among aerobic organisms, suggesting that protection against the destructive potential of ROS is necessary for survival ⁽²¹⁾.

What has become clear from decades of advances in modern medicine is that despite the great increases in mean life span for humans, maximum life span has remained nearly unchanged ⁽²²⁾. This suggests that the aging process may function independently of age-associated diseases such as cancer and cardiovascular disease. Thus, any method developed for extension of the maximal life span may not translate into a greater period of the functional life span, or the so-called quality life. Indeed, the more relevant challenge for researchers is in how to maximize the number of healthy years of life. One very controversial method for this is the administration of growth hormone (GH).

Growth hormone levels in the human, as well as in experimental animals, decline dramatically with increasing age ^(23)(24). This age-related deficiency in GH is associated with increased adiposity ⁽²⁵⁾, elevated blood lipids ⁽²⁶⁾, and a loss of lean body mass ⁽²⁷⁾. Increased risk of cardiovascular disease and early mortality have also been reported in patients with GH deficiency ⁽²⁸⁾; however, these results may be due to the effects of patient treatments and the previous presence of pituitary tumors rather than GH deficiency per se ⁽²⁹⁾. In contrast, life-long absence of GH signaling in animal models appears to exert anti-aging effects. Hypopituitary Ames dwarf mice have deficiencies in prolactin (PRL), thyroid stimulating hormone (TSH) and GH ⁽³⁰⁾, increased mean and maximal life span by more than 50% ⁽³¹⁾, elevated AOE activities ⁽³²⁾, and greater insulin sensitivity ⁽³³⁾ than normal mice. Similarly, Snell dwarf mice, which are also deficient in TSH, PRL, and GH as a result of a mutation unrelated to Ames dwarfism, live significantly longer than their normal siblings ⁽³⁴⁾. Further evidence of the anti-aging effects of GH deficiency have also been shown in mice with a targeted disruption of the GH receptor gene (GHR-KO) ⁽³⁵⁾. These mice, which produce GH but lack a functional GH receptor, thus eliminating GH signaling, have an increased life span over their normal littermates ⁽³⁶⁾. In addition, a possible link between mitogenic signaling, oxidative stress, and longevity in mammals has been reported in mice with targeted mutation of the p66^shc stress response gene ⁽³⁷⁾. Homozygous "knockout" mice from this line are resistant to growth factor and ROS-induced stresses, and they live significantly longer than normal or heterozygous littermates.

Despite these and other reports, GH has also been shown to exert effects that can be considered beneficial in animal models as well as humans, which has led to its clinical use in elderly patients. In rats, GH administration elevates both CAT activity and glutathione content, which would presumably reduce ROS-mediated damage ⁽³⁸⁾. In humans, GH administration has been shown to reverse the declines associated with GH deficiency and improve overall health. These include increased lean muscle mass and decreased adiposity ⁽³⁹⁾, decreased blood lipids ⁽⁴⁰⁾, and improved cardiac function ⁽⁴¹⁾. However, the effects of long-term GH administration to elderly patients who do not have congenital GH deficiencies have not been fully evaluated, and some findings question the reported benefit of such treatment ^(42)(43).

Consequences of long-term supraphysiological GH elevations have been shown in gigantism, acromegaly ⁽⁴⁴⁾, and in GH transgenic mice. These mice have one or more extra copies of the GH gene and chronic elevations of GH over their entire life span. Although young GH transgenic mice appear to be otherwise healthy ⁽⁴⁵⁾, by as early as 6 months of age they begin to exhibit a number of characteristics that are consistent with accelerated aging processes. These include early loss of reproductive potential ⁽⁴⁶⁾, early development of liver tumors ⁽⁴⁷⁾, increased incidence of renal failure ⁽⁴⁸⁾, higher levels of renal and hepatic superoxide radical and lipid peroxidation ⁽⁴⁹⁾, and severely reduced life span ⁽⁵⁰⁾. Because of this exhibition, GH transgenic mice represent a valuable model for characterizing the possible effects of long-term GH administration.

The objective of this research was to determine the extent to which lifelong GH elevation influences AOE activity and amount of enzyme protein within the liver and kidney. This was approached by using PEPCK (phosphoenolpyruvate carboxykinase) hGH transgenic mice that overexpress human GH (hGH, which has both PRL and GH activities) and have a major reduction in life span. We have previously reported that hGH excess is associated with elevated Cu–Zn SOD activity in the hypothalamus, suggesting that reduced life span is not related to declines in ROS defenses in this critical endocrine regulator ⁽⁵¹⁾. The aims of this study were to further elucidate the impact of hGH expression in organs suspected to be potential contributors to the reduced life span of GH transgenic mice, and to determine the long-term effects of GH excess on antioxidant defense status.

	Methods

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Animals
PEPCK hGH transgenic mice were originally produced by microinjection of a recently fertilized C57BL/6 x C3H F₁ mouse egg with a construct consisting of the rat PEPCK promoter fused with the hGH gene, as previously described ⁽⁵²⁾. The line was designated PEPCK hGH and was derived from a single transgenic founder male and maintained in our colony by mating transgenic male descendants from each generation to C57BL/6 x C3H F₁ hybrid females. This mating system produces approximately equal numbers of hemizygous transgenic and normal (wild-type) animals. Thus normal animals used as controls in these studies differed from transgenics only by the absence of the transgene and otherwise were identical in every respect, including genetic background, maternal effects, and intrauterine environment. Young (2–3 months old) and middle-aged (7–9 months old) transgenic (n = 6–9/age group) and normal females were produced in this manner and maintained in the Southern Illinois University animal facility on a 12:12 light–dark cycle with ad-libitum feed and water. All experiments were approved by the University Animal Care and Utilization Committee and were conducted in accordance with NIH (National Institutes of Health) care guidelines.

Samples
Mice were killed by decapitation, and tissues were rapidly removed, frozen on dry ice, and stored at -60°C until processed. A portion (60 mg) of kidney or liver was thawed and sonicated in ice-cold buffer (1:5 wt/vol; 50 mM of Tris-HC1 plus 1% Triton X-100; pH 7.3) in two 30 second bursts with cooling at 0°C between bursts. Samples were centrifuged at 13,000x g for 30 minutes at 4°C, and the supernatant was collected for determination of AOE status. Total protein was determined by a colorimetric method using BCA protein assay reagent (Pierce, Rockford, IL).

Antioxidant Enzyme Activity
SOD activity was determined as previously described ^(53)(54) and modified for determination of the Cu–Zn isoform by increasing the pH of the reaction mixture to 10.0 and by increasing the concentration of xanthine to 0.1 mM ⁽⁵⁵⁾. One unit of activity was defined as the amount of enzyme necessary to cause 50% inhibition of nitroblue tetrazolium reduction.

CAT activity was determined by ultraviolet spectrophotometry (Spectronic Genesys 5; Milton Roy, Rochester, NY), as previously described ⁽⁵⁶⁾. One unit of activity was defined as micromoles of H₂O₂ disproportionated per minute per milligram of protein.

GPx activity was determined as previously described ⁽⁵⁷⁾. One unit of activity was defined as the amount of enzyme required to oxidize 1 µmol of reduced nicotinamide adenine dinucleotide phosphate to NADP per minute per milligram of protein.

In order to test the hypothesis that an altered ratio of SOD-to-GPx activity is a contributing factor to premature aging ⁽⁵⁸⁾, individual activity ratios were determined by dividing the measured activity of SOD by that of GPx for each sample.

Antioxidant Enzyme Level
As a way to determine whether any of the detected alterations in AOE activity were due to changes in the levels of these enzymes, the relative amount of AOE protein was determined by Western blot analysis. Kidney and liver homogenates (50 µg of protein) were subjected to electrophoresis, in triplicate, on a 10% polyacrylamide gel ⁽⁵⁹⁾. The separated proteins were transferred to nitrocellulose membrane by electroblotting ⁽⁶⁰⁾. The membrane was then blocked and probed with first antibody overnight at 4°C. CAT protein was detected by using antihuman erythrocyte CAT (Calbiochem, La Jolla, CA); Cu–Zn SOD protein was detected by using antibovine erythrocyte Cu–Zn SOD (Chemicon International, Inc., Temecula, CA); or GPx protein was detected by using antibovine erythrocyte GPx (Biogenesis Ltd., England). Following primary incubation, the blots were washed and incubated in secondary antibody conjugated with horseradish peroxidase for 2 hours at room temperature. The blots were washed again, and immunoreactive bands were illuminated by using Enhanced Chemiluminescence (ECL) reagent (Amersham, Arlington Heights, IL) and exposed on Kodak film (Eastman Kodak, Rochester, NY). AOEs were identified by comparison to a commercial molecular weight standard (New England Biolabs Inc., Beverly, MA) and to AOE standards (Sigma, St. Louis, MO) run under the same conditions. Quantification of bands was performed by laser densitometry (Personal Densitometer SI; Molecular Dynamics, Sunnyvale, CA) and is expressed in arbitrary optical density (OD) units per milligram of protein.

Statistical Analysis
Data on AOE activity and level, including the SOD to GPx activity ratios, were analyzed by the use of an analysis of variance; Fisher's Protected Least Significant Difference (PLSD) post hoc test was used to determine specific differences between means. Correlation between enzyme activity and protein level was determined by Spearman's rank correlation coefficient test. A value of p < .05 was considered significant, and data are presented as the mean ± SEM.

	Results

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Cu–Zn Superoxide Dismutase
In young transgenic mice, renal SOD activities and levels were similar to those of normal mice (Fig. 1 and Fig. 1). Despite the similarities between groups, there was no correlation between enzyme activity and its absolute level measured in the kidney (r = .067) as shown in Fig. 2. Liver SOD activities and levels were again similar among transgenic and normal mice (Fig. 3 and Fig. 3). However, SOD activity correlated strongly with protein level (r = .783, p < .0192) as shown in Fig. 4.

View larger version (54K): [in this window] [in a new window]   Figure 1. Renal Cu–Zn superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx): activity, A, and level, B, in female PEPCK hGH transgenic (Tg) and normal (N) mice of young (Yng) and middle (Mid) age. Antioxidant enzyme (AOE) activities were measured by spectrophotometry as described in the text. Immunoreactive AOE levels were measured by Western blot as described in the text. Data reported are mean ± SEM; *: Mean activities that do not share a common superscript are significantly different (p < .05).

View larger version (27K): [in this window] [in a new window]   Figure 2. Renal antioxidant enzyme activity-to-level correlation in young and middle-aged female PEPCK hGH transgenic and normal mice. Correlation comparisons are as follows: Cu–Zn superoxide dismutase (SOD) in young, A, and middle-aged mice, B, catalase (CAT) in young, C, and middle-aged mice, D, and glutathione peroxidase (GPx) in E, young, and F, middle-aged mice. Raw data were plotted on the x- (level) and y- (activity) axes, and the correlation coefficient and statistical significance were determined by Spearman's rank correlation coefficient test; p < .05 was considered a significant correlation.

View larger version (45K): [in this window] [in a new window]   Figure 3. Hepatic Cu–Zn superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx): activity, A, and level, B, in female PEPck hGH transgenic (Tg) and normal (N) mice of young (Yng) and middle (Mid) age. Data reported are mean ± SEM; *: Mean activities that do not share a common superscript are significantly different (p < .05).

View larger version (27K): [in this window] [in a new window]   Figure 4. Hepatic antioxidant enzyme activity-to-level correlation in young and middle-aged female PEPCK hGH transgenic and normal mice. Correlation comparisons are as follows: Cu–Zn superoxide dismutase (SOD) in young, A, and middle-aged mice, B; catalase (CAT) in young, C, and middle-aged mice, D; and glutathione peroxidase (GPx) in young, E, and middle-aged mice, F; p < .05 was considered a significant correlation.

An age-related decline in renal SOD activity was seen in transgenic animals (p < .0281); however, SOD levels in transgenics, though lower than those of normal mice of the middle-aged group, did not decline significantly (p < .1125) from youthful values (Fig. 1 and Fig. 1). In the liver, SOD levels did not change from young to middle-aged mice. However, SOD activity declined with age in both normal and transgenic mice (normal, p < .0402; transgenic, p < .0013), as shown in Fig. 3 and Fig. 3.

Catalase
In young transgenic mice, renal CAT activities and levels were similar to values measured in normal mice (Fig. 1 and Fig. 1). The CAT activities and levels in the kidneys of young mice were not correlated, similarly to what was seen with SOD (r = .069, p < .8166; Fig. 2). In the liver, both CAT activities and levels were lower in transgenic mice compared with those of normal mice (activity, p < .0281; level, p < .0025), as shown in Fig. 3 and Fig. 3. In addition, this group demonstrated a strong activity-to-level correlation (r = .845, p < .0335), as shown in Fig. 4.

In middle-aged transgenics, the activities and levels of renal CAT were nearly identical to those of normal mice and to those of young transgenics (Fig. 1 and Fig. 1). The lack of differences in activity and level of CAT arose from an apparent bimodal distribution in middle-aged mice, which tended to have either high or low measured CAT values. Despite this unusual distribution, the activity-to-level correlation within these mice was highly significant (r = .952, p < .0028), as shown in Fig. 2. In the liver, middle-aged transgenic mice showed no age-associated decline in CAT activity or in CAT protein. However, middle-aged normal animals did appear to lose roughly 33% of CAT activity (p < .1127) and level (p < .1472), though these differences were not significant (Fig. 3 and Fig. 3). As a result of the apparent age-related decline only among normal mice, CAT activities and levels were similar to those of transgenics within the middle-aged group (activity, p < .2823; level, p < .0911). However, the correlation between CAT activity and the amount of CAT protein remained significant from young to middle age (r = .513, p < .0388), as shown in Fig. 4.

Glutathione Peroxidase
In young transgenic mice, renal GPx activities and levels were numerically higher than those in normal mice (activity, p < .0554; level, p < .1548; see Fig. 1 and Fig. 1). Despite these trends, GPx activity in the kidney did not correlate significantly with GPx level (r = .492, p < .1571; Fig. 2). In contrast, hepatic GPx activities and levels were similarly reduced in transgenic mice (activity, p < .0214; level, p < .0268). In addition, the activity-to-level correlation in hepatic GPx was very strong (r = .837, p < .0028; see Fig. 4).

In middle-aged transgenic mice, renal GPx activity was 25% lower (p < .0040) than that measured in normal mice, whereas the amount of GPx protein in transgenics was one-third lower (p < .0081; see Fig. 1 and Fig. 1). The correlation between GPx activity and level in this older group, as with SOD and CAT, was also significant (r = .631, p < .0075; see Fig. 2). Larger differences were seen in the liver of middle-aged mice. The average hepatic GPx activity in middle-aged transgenic mice was roughly half of that measured in controls (p < .0001), and GPx level was similarly reduced (p < .0001), as shown in Fig. 3 and Fig. 3. The consistent reductions in mean GPx activity and level in middle-aged transgenic mice yielded a significant activity-to-level correlation (r = .824, p < .0081; see Fig. 4).

Age-associated declines in GPx activity and level were seen in the kidneys of transgenic animals only. GPx activities and levels were 30% lower in middle-aged transgenics compared with those in young transgenics (activity, p < .0012; level, p < .0170; see Fig. 1 and Fig. 1). In the liver, the reduced activity and level of GPx in transgenics did not decline significantly from young to middle age (Fig. 3 and Fig. 3).

Ratio of Superoxide Dismutase-to-Glutathione Peroxidase Ratio of Activity
In the kidney the SOD-to-GPx ratio was unaffected by GH status or age (Table 1 ). However, in the liver, both age and GH status influenced this ratio. The activity ratio declined significantly with age among normal mice and transgenic mice, and it was 26% and 42% higher in young and middle-aged transgenics, respectively, though these differences were significant only between middle-aged mice (p < .0498).

	Discussion

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Transgenic mice used in this study showed tissue-specific alterations in AOEs, which were, in most cases, age dependent. In the kidney, young transgenic mice exhibited no significant changes in AOE levels or activity although SOD and GPx activities were numerically elevated. This may be expected given that the expression of the hGH transgene, under the control of the PEPCK promoter, which begins shortly after birth, may initially be of benefit through the somatogenic and/or lactogenic effects of this hormone in mice. Indeed, in short-term studies, it has been shown that GH administration elevates CAT activity in normal rats, whereas PRL administration increases Cu–Zn SOD mRNA in normal mice ^(38)(61). However, it could also be argued that the anabolic effects of GH cause a rise in ROS through an increase in the metabolic rate, which may signal an elevation in AOE levels. Given that our findings in the kidneys of young transgenic mice did not show elevated SOD levels or activities, or any effect on CAT, the induction of AOE genes during the first 2–3 months of life, by GH or ROS, appears unlikely.

Although renal GPx activity and level were not altered in young transgenic mice, they were significantly reduced by middle age when normal controls showed no reductions. In view of these findings, the effect of GH and/or free radicals on renal GPx may warrant further investigation. The trend toward higher GPx activity and level in transgenic mice, which have higher levels of superoxide radical in this organ, may be related to the fact that the primary source of GPx in the mouse, and the human, is the proximal tubule of the nephron, suggestive of a protective effect ^(62)(63). Indeed, patients with chronic renal failure on dialysis have low plasma GPx activity, and it has been suggested that GPx activity may serve as a predictive measure of kidney function ⁽⁶⁴⁾. The present data are consistent with this predictive relationship. Transgenic mice expressing human GH, or bovine GH (bGH), which is not lactogenic, die primarily from pathologies associated with the kidney ⁽⁴⁷⁾. Interestingly, mice with GH deficiency do not develop these pathological changes in the kidney, further implicating a GH-mediated mechanism ⁽⁶⁵⁾. Early loss of the protective effects of GPx in the kidneys of transgenic mice presumably can accelerate the accumulation of ROS-induced damage, leading to renal failure and premature mortality. The possibility that the age-related decline of GPx in the kidneys of transgenic mice is caused by renal disease appears unlikely.

The alterations in AOEs shown in the livers of young transgenics are markedly different from those seen in the kidneys. Transgenic mice had lower activities and levels of both hepatic GPx and CAT in the young group, and GPx remained lower than that of normal mice in the middle-aged group. SOD, in contrast, was not affected in these animals. In both young and middle-aged transgenics, SOD activities and levels in the liver were similar to the values measured in normal mice and declined with age in both genotypes. The differences in CAT and GPx between transgenic and normal mice, however, were not altered with age. This would suggest a lower set point for the regulation of AOE gene translation or transcription among transgenic mice. Indeed, the present data showed significant positive correlations among young mice between hepatic AOE activity and relative amount of protein, unlike those seen in the kidney, suggesting a possible mechanism responsible for the maintenance of AOE activity. Thus, GH excess clearly has an early negative impact on hepatic GPx and CAT. This may be related to the fact that the liver is the principal site of action of GH, and to the relationship between excess GH and somatic growth. Grossly elevated GH may disrupt the balance between somatic growth and cellular repair and perhaps interfere with the expression of repair genes, including those for AOEs, thus leading to elevated free radical processes and accelerated aging. Moreover, because the primary site of expression of the PEPCK hGH transgene is the liver, locally high concentrations of hGH may further contribute to alterations in gene expression specifically within this target organ. Indeed, GH treatment of hepatocyte cell cultures suppresses CAT activity (H. M. Brown-Borg, personal communication).

The relationship between gene expression and accelerated aging has been investigated within the context of the SOD-to-GPx activity ratio. Patients with Down's syndrome have an elevated level of expression of the Cu–Zn SOD gene, resulting from the additional copy of chromosome 21 where the Cu–Zn SOD gene is located ⁽⁶⁶⁾. It has been suggested from evidence taken from these patients that the consequent alteration in the SOD-to-GPx ratio plays a role in the premature aging of affected individuals ⁽⁵⁸⁾. More recently, an elevation in this ratio has been shown to lead to elevated H₂O₂ levels and to induce features of cellular senescence ⁽⁶⁷⁾. Data presented here would support the idea that an imbalance in the SOD-to-GPx ratio may be a contributing factor to accelerated aging. The SOD-to-GPx activity ratio in the liver of transgenic mice was numerically higher than those of normal mice of young age and significantly higher than those of middle-aged normal mice. Moreover, the ratio in young transgenic mice resembled that of middle-aged normal mice, which is consistent with premature aging. Taken with previous evidence of an elevated superoxide radical in GH transgenic mice ⁽⁴⁹⁾, these data show that the liver is also a potential target for ROS-induced cellular damage, which could conceivably contribute to the increased incidence of hepatic tumors in these animals ⁽⁴⁷⁾. More recently, it has been shown that transgenic mice have reduced levels of renal inorganic peroxides compared with those of normal animals ⁽⁶⁸⁾. However, these results do not conflict with previous findings of ROS in GH transgenic mice or with the present findings. Rather, they suggest that reductions in GPx and/or CAT without alterations in SOD may favor the production of more destructive radical species.

The profound loss of SOD and GPx in the kidneys of middle-aged transgenic mice in addition to lifelong deficiencies in CAT and GPx in the liver suggest that these tissues may represent weak links in the overall free radical defense systems of these animals. In the kidney, SOD and GPx declined in an age-dependent manner. In the liver, our results suggest that SOD activity is age dependent, but not GH dependent, whereas the expression of CAT and GPx is lower in GH transgenic mice, resulting in lifelong reductions in activity and level of these AOEs. These data are consistent with results of a previous study of renal and hepatic CAT And GPx in the PEPCK bGH transgenic mouse ⁽³²⁾. This study also found that hepatic GPx was lower in transgenic mice. Agreement of these findings with the present results concerning renal GPx and hepatic CAT depends on the age group used for comparisons. Given that the bGH transgenic mice used in the previous study were 4–5 months of age (H. M. Brown-Borg, personal communication), which is between the ages of the young and middle-aged groups examined here, the available data collectively suggest that the reduction in AOE activities in GH transgenic mice is age dependent. This is consistent with the assumption that these mice have accelerated aging. Furthermore, the lactogenic activity of hGH does not appreciably influence the overall effect of the overexpression of GH on AOE activity.

The resulting elevations in ROS in the kidney and the liver could lead to earlier and more extensive damage in both of these organs, thus potentially accelerating the aging process, and ultimately leading to premature mortality. However, the mechanism(s) linking GH excess to reductions in these AOEs is still unknown. An evolutionary theory of aging would propose that the detrimental effects that GH may have on longevity must be outweighed by some benefits. This indeed would appear to be the case. Female bGH transgenic mice have been shown to have accelerated sexual maturation and a greater propensity for ovulation than their nontransgenic littermates ⁽⁶⁹⁾. In addition, young transgenic mice expressing rat GH (rGH) have recently been shown to possess greater learning and memory than normal controls ⁽⁷⁰⁾. These findings would suggest that early in life GH enhances cognition and increases reproductive capacity, two major factors in evolutionary survival strategies.

The present data show that overall free radical defenses in GH transgenic mice are compromised, which is likely to contribute to their reduced longevity. Here we report that AOE activity, particularly among middle-aged mice, was dependent on AOE level, which suggests that lower activity is the result of less protein, and perhaps lower or less efficient expression. However, this does not answer how GH or GH signaling may decrease the expression of AOE genes, or whether GH therapy in the elderly population, which merely restores GH to youthful levels, would have similar detrimental effects. Because GH levels normally decline with age, it is unclear whether GH treatment in the elderly population represents replacement or induction of an excess. The results of the present study would suggest that although GH may improve lean body mass, enhance fertility, and improve psychological well-being, the potential cost could be a reduction in life span.

	Acknowledgments

 
Portions of this work were supported by funds from the Illinois Council on Food and Agricultural Research, C-FAR grant project 99E-035-4. We thank Drs. Jodi Huggenvik and Laura Murphy for their assistance and use of laboratory equipment.

Received April 28, 2000

Accepted October 3, 2000

	References

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1. Hauptmann N, Cadenas E, 1997. Reactive oxygen and apoptosis. Scandalios JG, , ed.Oxidative Stress and the Molecular Biology of Antioxidant Defenses 1-20. Cold Spring Harbor, Plainview, NY.
2. Ames BN, Shigenaga MK, 1992. Oxidants are a major contributor to aging. Ann NY Acad Sci 663:85-96. [Medline]
3. Aust AE, Eveleigh JF, 1999. Mechanisms of DNA oxidation. Proc Soc Exp Biol Med 222:246-252. [Abstract/Free Full Text]
4. Sohal RS, Ku H-H, Agarwal S, Forster MJ, Lal H, 1994. Oxidative damage, mitochondrial oxidant generation, and antioxidant defenses during aging and in response to food restriction in the mouse. Mech Age Dev 74:121-133. [Medline]
5. Agarwal S, Sohal RS, 1994. Aging and protein oxidative damage. Mech Age Dev 75:11-19. [Medline]
6. Sohal RS, Weindruch R, 1996. Oxidative stress, caloric restriction, and aging. Science 273:59-63. [Abstract]
7. Keller JN, Mattson MP, 1998. Roles of lipid peroxidation in modulation of cellular signaling pathways, cell dysfunction, and death in the nervous system. Rev Neurosci 9:105-116. [Medline]
8. Yu BP, Chen JJ, Kang CM, Choe M, Maeng YS, Kristal BS, 1996. Mitochondrial aging and lipoperoxidative products. Ann NY Acad Sci. 786:44-56. [Medline]
9. Harman D, 1956. Aging. A theory based on free radical and radiation chemistry. J Gerontol 11:298-300. [Free Full Text]
10. Nohl H, 1993. Involvement of free radicals in ageing: a consequence or cause of senescence. Br Med Bull 49:653-667. [Abstract/Free Full Text]
11. Sohal RS, Sohal BH, Orr WC, 1995. Mitochondrial superoxide and hydrogen peroxide generation, protein oxidative damage, and longevity in different species of flies. Free Radic Biol Med 19:499-504. [Medline]
12. Sawada M, Carlson JC, 1987. Changes in superoxide radical and lipid peroxide formation in the brain, heart, and liver during the lifetime of the rat. Mech Age Dev 41:125-137. [Medline]
13. Semsei I, Rao G, Richardson A, 1989. Changes in the expression of superoxide dismutase and catalase as a function of age and dietary restriction. Biochem Biophys Res Commun 164:620-625. [Medline]
14. Matsuo M, Gomi F, Dooley MM, 1992. Age-related alterations in antioxidant capacity and lipid peroxidation in brain, liver, and lung homogenates of normal and vitamin E-deficient rats. Mech Age Dev 64:273-292. [Medline]
15. Mo JQ, Hom DG, Anderson JK, 1995. Decreases in protective enzymes correlates with increased oxidative damage in the aging mouse brain. Mech Age Dev 81:73-82. [Medline]
16. Rikans LE, Moore DR, Snowden CD, 1991. Sex-dependent differences in the effects of aging on antioxidant defense mechanisms of rat liver. Biochim Biophys Acta 1074:195-200. [Medline]
17. Orr WC, Sohal RS, 1994. Extension of life-span by overexpression of superoxide dismutase and catalase in Drosophila melanogaster. Science 263:1128-1130. [Abstract/Free Full Text]
18. Taub J, Lau JF, Ma C, et al. 1999. A cytosolic catalase is needed to extend adult lifespan in C. elegans daf-c and clk-1 mutants. Nature 399:162-166. [Medline]
19. Cardozo-Pelaez F, Song S, Parthasarathy A, Epstein CJ, Sanchez-Ramos J, 1998. Attenuation of age-dependent oxidative damage to DNA and protein in the brainstem of Tg Cu/Zn SOD mice. Neurobiol Aging 19:311-316. [Medline]
20. White CW, Avraham KB, Shanley PF, Groner Y, 1991. Transgenic mice with expression of elevated levels of copper-zinc superoxide dismutase are resistant to pulmonary toxicity. J Clin Invest 87:2162-2168.
21. Beckman KB, Ames BN, 1998. The free radical theory of aging matures. Physiol Rev 78:547-581. [Abstract/Free Full Text]
22. Johnson FB, Sinclair DA, Guarente L, 1999. Molecular biology of aging. Cell 96:291-302. [Medline]
23. Iranmanesh A, Lizarralde GB, Veldhuis JD, 1991. Age and relative obesity are specific negative determinants of the frequency and amplitude of growth hormone (GH) secretion secretory bursts and the half-life of endogenous GH in healthy men. J Clin Endocrinol Metab 73:1081-1088. [Abstract/Free Full Text]
24. Muller EE, Cella SG, de Gennaro Colonna V, Parenti M, Cocchi D, Locatelli V, 1993. Aspects of the neuroendocrine control of growth hormone secretion in ageing animals. J Reprod Fertil 46: (suppl) 99-114.
25. Hoffman DM, O'Sullivan AJ, Freund J, Ho KK, 1995. Adults with growth hormone deficiency have abnormal body composition but normal energy metabolism. J Clin Endocrinol Metab 80:72-77. [Abstract]
26. de Boer H, Blok G-J, van der Veen EA, 1995. Clinical aspects of growth hormone deficiency in adults. Endocr Rev 66:63-86.
27. Corpas E, Harman SM, Blackman MB, 1993. Human growth hormone and human aging. Endocrine Rev 14:20-38. [Abstract/Free Full Text]
28. Rosen T, Bengsson B, 1990. Premature mortality due to cardiovascular disease in hypopituitarism. Lancet 336:285-288. [Medline]
29. Bates AS, Hoff WV, Jones PJ, Clayton RN, 1996. The effect of hypopituitarism on life expectancy. J Clin Endocrinol Metab 81:1169-1172. [Abstract]
30. Sornson MW, Wu W, Dasen JS, et al. 1996. Pituitary lineage determination by the prophet of pit-1 homeodomain factor defective in Ames dwarfism. Nature 384:327-333. [Medline]
31. Brown-Borg H, Borg KE, Meliska CJ, Bartke A, 1996. Dwarf mice and the ageing process. Nature 384:33[Medline]
32. Brown-Borg HM, Bode AM, Borg KE, Carlson J, Bartke A, 1999. Growth hormone and oxidation state: possible role in the aging process. Endocrine 11:41-48. [Medline]
33. Borg KE, Brown-Borg HM, Bartke A, 1995. Assessment of the primary adrenal cortical and pancreatic hormone basal levels in relation to plasma glucose and age in the unstressed Ames dwarf mouse. Proc Soc Exp Biol Med 210:126-133. [Medline]
34. Miller RA, 1999. Kleemeier award lecture: Are there genes for aging?. J Gerontol Biol Sci 54A:B297-B307. [Abstract]
35. Zhou Y, Xu BC, Maheshwari HG, et al. 1997. A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse). Proc Natl Acad Sci USA 94:13215-13220. [Abstract/Free Full Text]
36. Kopchick JJ, Laron Z, 1999. Is the Laron mouse an accurate model of Laron syndrome?. Mol Genet Metab 68:232-236. [Medline]
37. Migliaccio E, Giorgio M, Mele S, et al. 1999. The p66^shc adaptor protein controls oxidative stress and life span in mammals. Nature 402:309-313. [Medline]
38. Youn YK, Suh GJ, Jung SE, Oh SK, Demling R, 1998. Recombinant human growth hormone decreases lung and liver lipid peroxidation and increases antioxidant activity after thermal injury in rats. J Burn Care Rehab 19:542-548. [Medline]
39. Rudman D, Feller AG, Nagraj HS, et al. 1990. Effects of human growth hormone in men over 60 years old. N Engl J Med 323:1-6. [Abstract/Free Full Text]
40. Beshyah SA, Henderson A, Niththyanathan R, et al. 1995. The effects of short and long term growth hormone replacement therapy in hypopituitary adults on lipid metabolism and carbohydrate metabolism. J Clin Endocrinol Metab 80:356-363. [Abstract]
41. Pfeifer M, Verhovec R, Zizec B, Prezelj J, Poredos P, Clayton RN, 1999. Growth hormone (GH) treatment reverses early atherosclerotic changes in GH-deficient adults. J Clin Endocrinol Metab 84:453-457. [Abstract/Free Full Text]
42. Holloway L, Butterfield G, Hintz RL, Gesundheit N, Marcus R, 1994. Effects of recombinant human growth hormone on metabolic indices, body composition, and bone turnover in healthy elderly women. J Clin Endocrinol Metab 79:470-479. [Abstract]
43. Papadakis MA, Grady D, Black D, et al. 1996. Growth hormone replacement in healthy older men improves body composition but not functional ability. Ann Intern Med 124:708-716. [Abstract/Free Full Text]
44. Sacca L, Cittadini A, Fazio S, 1994. Growth hormone and the heart. Endocr Rev 15:555-573. [Abstract/Free Full Text]
45. Bartke A, Borwn-Borg HM, Bode AM, Carlson J, Hunter WS, Bronson RT, 1998. Does growth hormone prevent or accelerate aging?. Exp Gerontol 33:675-687. [Medline]
46. Cecim M, Kerr J, Bartke A, 1995. Effects of bovine growth hormone (bGH) transgene expression or bGH treatment on reproductive functions in female mice. Biol Reprod 52:1144-1148. [Abstract]
47. Wolf E, Kahnt E, Ehrlein J, Hermanns W, Brem G, Wanke R, 1993. Effects of long-term elevated serum levels of growth hormone on life expectancy of mice: lessons from transgenic animal models. Mech Age Dev 68:71-87. [Medline]
48. Wanke R, Wolf E, Hermanns W, Folger S, Buchmüller T, Brem G, 1992. The GH-transgenic mouse as an experimental model for growth research: clinical and pathological studies. Hormone Res 37:74-87.
49. Rollo CD, Carlson J, Sawada M, 1996. Accelerated aging of giant transgenic mice is associated with elevated free radical processes. Can J Zool 74:606-620.
50. Cecim M, Bartke A, Yun JS, Wagner TE, 1994. Expression of human, but not bovine growth hormone genes promotes development of mammary tumors in transgenic mice. Transgenics 1:431-437.
51. Hauck S, Bartke A, 2000. Effects of growth hormone on hypothalamic catalase and Cu/Zn superoxide dismutase. Free Radic Biol Med 28:970-978. [Medline]
52. Milton S, Cecim M, Li YS, Yun JS, Wagner TE, Bartke A, 1992. Transgenic female mice with high human growth hormone levels are fertile and capable of normal lactation without having been pregnant. Endocrinology 131:536-538. [Abstract/Free Full Text]
53. Sun Y, Oberley LW, Li Y, 1988. A simple method for clinical assay of superoxide dismutase. Clin Chem 34:497-500. [Abstract/Free Full Text]
54. Sun Y, Oberley LW, 1994. Suitability of copper chloride as a reaction terminator for superoxide dismutase activity assay. Clin Chim Acta 226:101-103. [Medline]
55. Crapo JD, McCord JM, Fridovich I. Preparation and assay of superoxide dismutases. In: Fleischer S, Packer L, eds. Methods in Enzymology. New York: Academic Press; 1978;53:382–393.
56. Aebi HE. Catalase. In: Bergmeyer HU, ed. Methods in Enzymatic Analysis. New York: Academic Press; 1974;2:673–686.
57. Tappel AL. Glutathione peroxidase and hydroperoxides. In: Fleisher S, Packer L, eds. Methods in Enzymology. New York: Academic Press; 1978;52:506–513.
58. Groner Y, Elroy-Stein O, Avraham KB, et al. 1990. Down syndrome clinical symptoms are manifested in transfected cells and transgenic mice overexpressing the Cu/Zn–superoxide dismutase gene. J Physiol Paris 84:53-77. [Medline]
59. Laemmli UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. [Medline]
60. Towbin H, Staehelin T, Gordon J, 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350-4354. [Abstract/Free Full Text]
61. Sugino N, Takamori MH, Zhong L, Telleria CM, Shiota K, Gibori G, 1998. Hormonal regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase messenger ribonucleic acid in the rat corpus luteum: induction by prolactin and placental lactogens. Biol Reprod 59:599-605. [Abstract/Free Full Text]
62. Maser RL, Magenheimer BS, Calvet JP, 1994. Mouse plasma glutathione peroxidase: cDNA sequence analysis and renal proximal tubular expression and secretion. J Biol Chem 269:27066-27073. [Abstract/Free Full Text]
63. Avissar N, Ornt DB, Yagil Y, et al. 1994. Human kidney proximal tubules are the main source of plasma glutathione peroxidase. Am J Physiol 35:C367-C375.
64. Whitin JC, Tham DM, Bhamre S, et al. 1998. Plasma glutathione peroxidase and its relationship to renal proximal tubule function. Mol Genet Metab 65:238-245. [Medline]
65. Bellush LL, Doublier S, Holland AN, Striker LJ, Striker GE, Kopchick JJ, 2000. Protection against diabetes-induced nephropathy in growth hormone/binding protein gene-disrupted mice. Endocrinology 141:163-168. [Abstract/Free Full Text]
66. Tan YH, Tischfield J, Ruddle FH, 1973. The linkage of genes for the human interferon-induced antiviral protein and indophenol oxidase B traits to chromosome 21. J Exp Med 137:317-330. [Abstract]
67. de Haan JB, Cristiano F, Iannello R, Bladier C, Kelner MJ, Kola I, 1996. Elevation in the ratio of Cu/Zn-superoxide dismutase to glutathione peroxidase activity induces features of cellular senescence and this effect is mediated by hydrogen peroxide. Human Mol Genet 5:283-292. [Abstract/Free Full Text]
68. Carlson JC, Bharadwaj R, Bartke A, 1999. Oxidative stress in hypopituitary dwarf mice and transgenic mice overexpressing human and bovine GH. Age 22:181-186.
69. Danilovich NA, Bartke A, Winters TA, 2000. Ovarian follicle apoptosis in bovine growth hormone transgenic mice. Biol Reprod 62:103-107. [Abstract/Free Full Text]
70. Rollo CD, Ko CV, Tyerman JGA, Kajiura LJ, 1999. The growth hormone axis and cognition: empirical results and integrated theory derived from giant transgenic mice. Can J Zool 77:1874-1890.

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