Male mdx and control (wt) mice from the same mouse strain (C57BL/10ScSn-D^mdx and C57BL10Sn, Jackson Laboratory, Bar Harbor, ME) were bred in our animal facility. Animals were maintained and treated according to an approved protocol of the Ethics Committee (IACUC) of the Hebrew University Medical School. The Hebrew University is an AAALAC internationally accredited institute. All mice were maintained under a controlled photoperiod (lights on 07:00–19:00). Food and water were provided ad libitum. Body weights were monitored.

Circulating CK values, expressed as units of enzyme activity per liter of serum sample (U/L), were used as an index to identify the genotype of the animals (mdx or wt). CK values in blood samples drawn from orbital sinuses were determined using a standard enzymatic method and an automated clinical analyzer.

Data were collected for samples from individual animals in all experiments. Mean values were obtained by averaging measurements obtained for a group of 4–10 animals for a particular experimental day (as indicated in each experiment). The significance of differences between groups was determined by One-Way ANOVA (parametric with Tukey's multiple comparison post-test) for all groups, or by Kruskal–Wallis (non-parametric) tests, depending whether or not the data showed normal Gaussian distribution. Comparison between two groups was by non-paired t-test with unequal variances (our usual case). All tests were available in the GraphPad Prism4 (GraphPad Inc.), Statistica10 (StatSoft) and Microsoft Office Excel Professional Software. The difference between groups was considered significant when (p < 0.05).

Although the mdx mouse was reproductively competent (thus, T was present and functional), its postnatal growth was hindered compared to that of normal controls. The mdx mice displayed lower and more variable body weight compared to the controls, with the phenomenon being more pronounced in mdx females than in males (not shown). However, the body weight stabilized by 11 weeks of age at levels not significantly different from those of the control mice (Figure 1). The oscillatory shape of the curve was consistent with the finding that mdx mouse muscles undergo cycles of degeneration and regeneration until most muscles stabilize by 10 weeks (Settles et al., 1996). The mice we used were therefore aged 19–22 weeks, at which age their body weights are stable, and the majority of muscle fibers no longer display transient degeneration (Karpati et al., 1988).

Mouse serum contains BChE and AChE, both of which hydrolyze ATCh. Total ChE activities in sera of male mdx mice were at least 15% (p < 0.01) below those in normal mice sera (Figure 2A), indicating reduced levels of AChE and/or BChE in the mdx sera. AChE activity, which was found to be very low in sera of normal mice, was significantly elevated in the mdx sera (Figure 2A, detailed below). In contrast, BChE levels, assayed on BTCh, were found to decrease in male mdx-sera to 64–67% of the levels in wt sera (Figure 2B, p < 0.002). Similar results were observed when BChE was assayed on ATCh, with AChE activity being selectively inhibited by BW284c51 (Figure 2A). Since AChE levels in normal mouse serum are very low, the more sensitive radiometric assay was used to confirm the results obtained using the colorimetric Ellman procedure (Figures 2C and D), showing that AChE activity in mdx-sera indeed increased by 50–80% over the level in control wt sera (p < 0.05), confirming an earlier report (Oliver et al., 1992). BChE activity was shown, also by the radiometric assay, to decrease in mdx-sera to 61–76% of wt values (Figure 2D, p < 0.001). Since BChE is the major ChE form in mouse serum (>70%), the large drop in BChE was responsible for the overall reduction in total ChE activity observed in mdx sera. However, the decrease in BChE levels, taken together with the increase in AChE levels, in mdx sera (as shown in Figures 2C and D), reduced the BChE/AChE ratio from 2.3 ± 0.2 in the wt-sera to 1.3 ± 0.5 in mdx-sera. Similar results were found for adult female mdx-mice and are summarized elsewhere (Anglister et al., 2008).

As an internal control, CK levels in sera were measured to validate the genotype of the animals (mdx compared to wt). Consistent with the 3–14-fold elevation reported by Glesby et al. (1988), the male mdx mice used in our studies showed variable, but consistently increased, CK levels in sera, viz., 9.9 ± 1.4 (× 10³ U/L, mean ± SEM) in mdx mice, compared to 1.3 ± 0.3 in controls (Figure 2E). These differences were highly significant (p < 0.001).

To determine whether the abnormal BChE/AChE ratio in mdx sera is due to hormonal influences, we first investigated regulation by the androgen, T, of ChEs in the sera of adult male wt mice.

Sera from orchidectomized 21 week adult male wt mice were analyzed for ChE activities (Figure 3). Gonadectomy raised BChE activities in adult male mice serum by 20–37% (p < 0.001), whether measured on BTCh, or on ACh in the presence of a selective AChE inhibitor (Figure 3A and B, respectively)

T-replacement in adult wt males, performed concomitantly with castration, abolished the increase caused by castration, restoring BChE activity to the levels observed in the sera of intact males (Figures 3A and B). Thus, a high basal level of BChE activity is found in the sera of agonadal males (in the absence of T), and both endogenous and exogenous androgen down-regulate the enzyme.

Whereas castration of the male wt markedly raised the BChE level in the serum, that of AChE (measured with ACh in the presence of iso-OMPA) did not change significantly (Figure 3C; p = 0.41). The ranges of values recorded radiometrically, with ACh and the respective selective inhibitors of BChE and AChE, in sera of male adult wt mice under the different treatment regimes, are shown in Table 1. The data show clearly that BChE activities were increased from a low level (1830–2350 arbitrary assay units) to a high level (2770–3870 units) after orchidectomy, and reverted to the base level after T-implant. In contrast, the AChE activities observed in adult male mice sera fell in a similar range (530–1460 arbitrary units), whether the mice were intact or castrated, with or without T-implant. These results indicate a specific and selective negative androgen modulation of BChE, but not of AChE. Furthermore, the large increase in BChE after castration resulted in an increased ratio of BChE to AChE activities from 2.3 ± 0.2 in sera of intact wt males to 3.0 ± 0.1 in castrated wt males (Table 1).

We went on to examine whether orchidectomy raises BChE levels also in the sera of mdx mice, and whether, as a consequence, the high agonadal baseline ChE levels seen in wt mice sera can be reached. The data presented in Table 2 and Figure 4 reveal low levels of BChE in the sera of control adult mdx males (32.2–48.6 assay units). Although levels increase substantially after gonadectomy (48.7–61.9 assay units), by an average of 35%, the BChE deficiency observed in untreated mdx mice is sustained (Figure 4): thus, the agonadal “baseline” level in mdx mice was significantly below the baseline level in wt agonadal mice (29%, p < 0.002). It was slightly lower than the level in control wt mice (14%, p = 0.06) or in sham-operated wt mice. As in wt mice, T-replacement reversed the effect of orchidectomy on BChE activity. Thus, after orchidectomy, the BChE in mdx circulation may almost reach the gonadal baseline level of BChE activity in the serum of intact male wt mice, but not the agonadal baseline level in the serum of castrated wt mice.

We went on to examine the effects of orchidectomy and T-replacement on AChE levels in the sera of mdx mice. Specifically, we checked whether orchidectomy of mdx mice, with or without T-implant, affected serum levels of AChE. As documented in Table 3, the mean AChE activity in the sera of intact mdx mice was 1260 ± 120 units, as compared to 790 ± 70 units in the castrated mice. Despite the great variability in AChE levels, castration appears to reduce AChE activity by >30%, p < 0.02). Interestingly, the mean value for agonadal mdx mice was not different from that for gonadal wt mice (p = 0.2).

T-implant into agonadal mdx mice resulted in a mean serum AChE activity of 930 ± 180, not significantly different from the value for agonadal mdx mice lacking the T-implant (p = 0.40). This value is also very similar to the level in the gonadal wt mice (p = 0.89), but lower than the average value for gonadal mdx mice, although a statistically significant difference could not be demonstrated due to the large standard errors within the two groups, resulting in p = 0.11. Furthermore, the values obtained for the small group of “sham” mdx mice analyzed, were intermediate, not being significantly different from either the values for sera of intact mdx and intact wt mice (p = 0.34 and 0.39, respectively). These limitations prevent us from drawing definite conclusions concerning the effect of orchidectomy on serum AChE in mdx mice, although orchidectomy decreases AChE levels. However, the large increase in BChE levels, coupled with the apparent decrease in AChE levels, in the agondal mdx mice, results in an increase in the BChE/AChE activity ratio from 1.2 ± 0.5 in the male mdx-sera to 2.5 ± 0.4 after castration (Table 3), a ratio similar to that in wt-sera (2.3 ± 0.2, Tables 1 and 3).

Fetal calf serum is known to be a good source of AChE (Ralston et al., 1985). It occurred to us that bovine serum might serve as a good system for testing postnatal down-regulation of serum AChE, and for examining, in parallel, the regulation of BChE, assuming BChE to be a serum component common to all species. We therefore compared ChE activities in fetal bovine sera and in post-pubertal (adult) bull sera. Consistent with the literature, we observed a high level of AChE activity, >96% of total ChE, in fetal bovine sera. Interestingly, AChE activity was >10-fold lower in adult bull sera (Table 4). However, whereas in adult mouse serum, BChE was the major ChE component, as in several other species (Ecobichon and Corneau, 1973; Chatonnet and Lockridge, 1989), in bovine sera it was the minor ChE component, with very low activity (Table 4). Although BChE activity in fetal and adult bovine sera was similar, its fraction of the total ChE activity rose from fetal to adult (~4–25%) due to the big drop in AChE activity. Because of the striking difference in ChE composition of mouse and bovine sera, we decided to examine the ChE composition of the sera of rhesus (RHS) monkeys, which are closely related to humans and for which many endocrinological studies have been performed, including examination of the effects of androgens. As seen in Table 4, in adult RHS serum, as in mouse serum, BChE is the major component (89%).

The target of testosterone (or estrogen) that produces modulation of serum BChE levels is not known. It may be in the hypothalamus, in the pituitary, or perhaps in a target organ that produces serum BChE, such as the liver (Leeuwin, 1965), together with pituitary factors. Experiments on hypophysectomized rats have demonstrated that pituitary mediation is required for both androgen and estrogen action (Everett and Sawyer, 1946; Illsley and Lamartiniere, 1981): while sex steroids do modulate ChE activity from a basal level, either due to suppression by androgen in males, or due to stimulation by estrogen in females, their action is abolished by hypophysectomy. It has also been shown in rats that hypophysectomy (Illsley and Lamartiniere, 1981; Edwards and Brimijoin, 1983) or lesion of the arcuate nucleus in the hypothalamus of males (Lamartiniere, 1986) reduces levels of GH and increases levels of ChE in the serum. The effects on serum ChEs were shown to be reversed by exogenous administration of GH after either type of surgery. Thus, both T and GH appear to be negative modulators of rat serum BChE in male rats. Moreover, they can act synergistically, since their levels are inter-related: the hypothalamus regulates the release of pituitary hormones (e.g., GH) and gonadotropins that, in turn, drive the gonads to produce T and estrogen, viz., the H-P-G axis. In addition, T itself influences GH secretion, and GH in turn reduces serum BChE levels; thus, T can also reinforce down-regulation of BChE via GH.

Serum BChE in adults is apparently produced in the liver (Silver, 1974). Indeed, GH has been shown to modulate several sex-differentiated hepatic enzymes, including serum ChE (Lamartiniere, 1981, 1986). Since hypophysectomy abolishes rat liver estrogen receptors and GH restores them, GH may also restore responsiveness of the liver to circulating sex steroids, and result in androgen or estrogen modulation of serum ChE. A similar mechanism could apply to GH regulation of other potential target organ for serum BChE production. In considering the liver as the source for serum BChE, it should be taken into account that, the liver secretes BChE monomers and dimers (Perelman et al., 1990), not the G₄ tetramer which is the major isoform in serum (Lockridge et al., 1979). Furthermore, we have preliminary evidence that T, castration, and T-replacement after castration, alter predominantly the level of G₄ BChE in mouse serum. Thus, it appears that the BChE isoforms produced/secreted by the liver are not the isoform present in serum and modulated by the hormones. This unresolved issue, as well as the influence of T and/or GH on BChE isoforms in serum, liver, and other tissues need to be revisited and further investigated.

It became apparent in this study that the mdx mice, that lacked dystrophin, had less BChE in their sera (both before and after orchidectomy), relative to wt controls. Since T and GH have both been found to be negative modulators of BChE, it is possible that the intact mdx mice secrete higher levels of T and/or GH than the wt mice. Consistent with this suggestion, mdx mice exhibit hypertrophy of somatotroph cells in the pituitary, and slightly elevated pituitary GH in adult females (Anderson et al., 1994). The reduced levels of BChE we show in castrated mdx mice compared to castrated wt are also likely to be due to gender-specific effects of GH at the hypothalamic-pituitary level, and not to a direct effect of T, since castration has eliminated T from the system. Further studies will be needed to determine whether the perturbation is at the pituitary or hypothalamic level.

Further correlation of GH levels with dystrophin deficiency comes from studies on DMD patients that reported mitigation of the severity of the dystrophy in GH-deficient patients (Zatz et al., 1981a,b), or after treatment with a GH inhibitor (Zatz et al., 1986). Another report provided some evidence for an altered hypothalamic-hypophyseal axis (Zaccaria et al., 1989). This is consistent with the observation referred to above, of high GH levels in mdx sera (Anderson et al., 1994). In addition, insulin growth factor-1 (IGF-1), which is secreted in correlation with GH, is elevated in mdx plasma (De Luca et al., 1999). Altogether, dystrophin deficiency is likely to be associated with impaired H-P-G-regulation.

Consistent with Oliver et al. (1992), it became apparent in this study that mdx mice had more AChE in their sera (prior to orchidectomy), than wt controls. Elevated levels of serum AChE were also reported for dystrophic chickens (Lyles et al., 1980) and for other animal dystrophies (Skau, 1985), as well as for fetal calf serum (Ralston et al., 1985). Oliver et al. (1992) proposed that the increased AChE levels in the sera of the dystrophic mice reflect increased release of muscle AChE relative to release in wt controls. Our finding that post-pubertal castration of adult mdx males normalizes their serum ChE levels by elevating BChE and, perhaps reducing AChE, and thus normalizing the BChE:AChE ratio, suggests that the mdx sera BChE levels are not just a reflection of muscle degradation but are under control of the gonads and of the aforementioned higher centers. Moreover, in addition to T, there is likely to be an additional factor involved in the impaired down-regulation of AChE and up-regulation of BChE in mdx sera. This seems likely since although the BChE level increased after castration, it did not reach the agonadal wt level, and T-replacement did not reverse the effect on mdx serum AChE. Before one can extrapolate from our results in mice to human patients, one has to prove that gonadal regulation of BChE occurs in higher primates.

Because, in mdx sera, AChE failed to decrease upon maturation and BChE was low in the adult, we considered the possibility that mdx mice are locked in a pre-adult stage and retain some embryonic regulatory properties. It was shown in mouse sera that in the embryo AChE predominates, while in the adult BChE is the major ChE species (Oliver et al., 1992). Surprisingly, we report here strong postnatal down-regulation of AChE in bovine serum, with only small amounts of BChE in both fetal and adult serum relative to other species (Table 4). However, we found BChE to be the major ChE component in sera of adult normal wt mice and rhesus monkeys (Table 4), as has been reported for many animals (Ecobichon and Corneau, 1973; Chatonnet and Lockridge, 1989). Indeed, while human 12th week fetal serum may contain 40% AChE (Hahn et al., 1993), adult human plasma contains >99% BChE (Brimijoin and Hammond, 1988). Higher primates, such as the rhesus macaque, should provide an appropriate model for further studies on the regulation of both AChE and BChE in human serum.

While in the present study we have shown that the AChE in mouse serum is not regulated by circulating T, it should be noted as mentioned, that AChE decreased ~2-fold in the serum of normal mice, prior to puberty. During that time, a progressive increase in AChE coincident with a reduction in BChE occurred in muscle resulting in inversion in the amounts of the two enzymes in adult muscle (Berman et al., 1987). Thus it may still be that the decrease in serum AChE and increase in muscle AChE are due to one or more postnatal triggers, e.g., humoral factor(s) discharged before the episodic T release at puberty (~5 weeks). In this context, it should be noted that BChE precedes AChE expression in embryonic neural development of birds (Layer, 1991; Layer and Willbold, 1994) and mammals (Koenigsberger et al., 1998).

One needs to bear in mind, considering the various roles attributed to ChEs in development, during normal function and in pathological conditions, that modulation of their levels may both reflect and influence normal processes or existing impairments. In the context of the present study, the abnormal BChE/AChE ratio in the serum of dystrophic mice, as compared to normal, may be developed as a tool for easy detection of dystrophy, provided similar observations are made for DMD or for other human dystrophies.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.