Strain differences in biologic responses to a variety of stimuli have been reported. For example, Lewis rats are quite susceptible to many inflammatory diseases in response to a wide range of stimuli (Sternberg et al. 1989). In addition, we learned recently that mice of the NZBWF₁ strain, routinely used as a model of systemic lupus erythematosus (Hirose et al. 1994, Watanabe et al. 1994), had a diminished affinity for hepatic specific binding for [³H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)⁵ compared with that of C57B/6 mice (R. N. Kurl, Brown University, personal communication). Based upon the latter observation, we utilized this strain (NZBWF₁) along with the Swiss strain of mice as a control, to determine affinities for ³H-tryptophan binding to hepatic nuclei in vitro. Earlier we reported that binding of L-tryptophan to rat hepatic nuclear envelope protein was saturable, stereospecific and of high affinity (Kurl et al. 1987 and 1988). Also, we have reported that the administration of L-tryptophan to CF₁ mice (Sidransky et al. 1968) and rats (Sidransky et al. 1971) stimulated hepatic protein synthesis.

The results of this study indicate that the hepatic nuclei of NZBWF₁ mice have a significantly lower tryptophan receptor binding affinity than that of Swiss mice. The binding affinity of the livers of each strain appears to be related to the degree of enhanced hepatic protein synthesis after the administration of L-tryptophan.

MATERIALS AND METHODS

The mice were fed Purina nonpurified diet (no. 5001; Purina, St. Louis, MO) and maintained in a temperature-controlled room with a 12-h light:dark cycle. Before the experiments were begun, the mice were adapted to their quarters for at least 1 wk and then were deprived of food overnight but had free access to water. Mice were killed by decapitation. The protocol for these studies was reviewed and approved by the institutional animal care and use committee.

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In experiments designed to determine whether hepatic cytosols may affect hepatic nuclear binding to [³H]tryptophan in vitro, hepatic nuclei of Swiss and of NZBWF₁ mice were prepared as described below (Blobel and Potter 1966). Nuclei were resuspended in hepatic cytosols of Swiss or NZBWF₁ mice, and then centrifuged at 11,400 × g. Nuclei of each group were assayed for [³H]tryptophan binding with or without unlabeled L-tryptophan (10⁴ mol/L).

Table 1. Comparison of total and specific 3H-tryptophan binding in vitro to hepatic nuclei of Swiss and NZBWF1 Mice1,2 [View Table]

In one group of experiments, other strains of mice, DBA, SJL and BALB/c, were used in addition to Swiss and NZBWF₁ mice to determine specific binding data for [³H]tryptophan binding to hepatic nuclei.

In experiments designed to determine serum and liver concentrations of free L-tryptophan and liver tryptophan dioxygenase activities, Swiss and NZBWF₁ mice that had been food deprived overnight or had been tube-fed L-tryptophan (20 mg/100 g body weight) 1 h before killing were utilized. In two experiments, groups of mice were also tube-fed other levels (2.5, 5, 10 or 40 mg/100 g body weight) of L-tryptophan for 1 h.

In experiments designed to determine hepatic nuclear RNA efflux, Swiss and NZBWF₁ mice were injected intraperitoneally with [6-¹⁴C]orotic acid (592 kBq/100 g body weight) 30 min before killing. At 10 min before killing, Swiss and NZBWF₁ mice were tube-fed water or L-tryptophan (20 mg/100 g body weight).

In experiments concerned with hepatic protein synthesis, mice of each strain that had been food deprived overnight were tube-fed distilled water (control group) or L-tryptophan (2.5, 5, 10, 20 or 40 mg/100 g body weight) 1 h before killing. In vitro protein synthesis as described below was assayed.

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RESULTS

The results of two experiments concerned with the inhibition of [³H]tryptophan binding in vitro when incubating the hepatic nuclei of NZBWF₁ and of Swiss mice with zero or varying concentrations of unlabeled L-tryptophan (10¹⁰, 10⁸, 10⁶ , 10⁴ and 10² mol/L) are summarized in Figure 1. The percentage of inhibition of [³H]tryptophan binding to hepatic nuclei at all concentrations studied was less for NZBWF₁ than for Swiss mice (comparison at 10⁴ mol/L concentrations, P < 0.01). Using hepatic nuclei of Swiss mice, the 50% inhibition of [³H]tryptophan binding was at 2 × 10⁸ mol/L with unlabeled L-tryptophan (results similar to those obtained with hepatic nuclei of rats, Sidransky and Verney 1994). On the other hand, using hepatic nuclei of NZBWF₁ mice, the percentage of inhibition of [³H]tryptophan binding reached only 65% at 10² mol/L unlabeled L-tryptophan. To evaluate whether the hepatic cytosols could affect the hepatic nuclei during preparation, we determined that the addition of hepatic cytosol from NZBWF₁ mice had no effect on [³H]tryptophan binding of hepatic nuclei of Swiss or NZBWF₁ mice (data not shown).

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The results of experiments to determine whether tube-feeding L-tryptophan affects subsequent [³H] tryptophan binding to hepatic nuclei of Swiss and NZBWF₁ mice in vitro are summarized in Table 2. Control (water-treated) NZBWF₁ mice had 48% less total binding and 62% less specific binding in hepatic nuclei than control Swiss mice. Mice tube-fed 10 mg L-tryptophan 60 min before killing demonstrated 34 and 19% lower total binding and 48 and 14% lower specific binding in hepatic nuclei in Swiss and NZBWF₁ mice, respectively, when compared with controls of each species. Mice tube-fed 20 mg L-tryptophan 60 min before killing demonstrated 45 and 36% lower total binding and 48 and 29% lower specific binding in hepatic nuclei in Swiss and NZBWF₁ mice, respectively, when compared with controls of each species. In vivo administration of L-tryptophan diminished the subsequent in vitro [³H]tryptophan binding to hepatic nuclei (total and specific) of both strains. However, the relative changes (diminishing effects) appeared to be greater in Swiss mice than in NZBWF₁ mice.

Table 2. Influence of L-tryptophan (trp) administration in vivo 60 min before killing on in vitro 3H-tryptophan binding to hepatic nuclei of Swiss and NZBWF1 mice1,2 [View Table]

Hepatic protein synthesis after tube-feeding L-tryptophan. The stimulatory effects of administering L-tryptophan on in vitro [¹⁴C]leucine incorporation into protein using hepatic microsomes tended to be higher in Swiss than in NZBWF₁ mice at all levels of L-tryptophan administered (Fig. 2). At the level of 20 mg L-tryptophan/100 g body weight, the difference was significant (P < 0.05).

Table 3. Serum and hepatic free tryptophan (trp) concentrations and hepatic tryptophan dioxygenase activity of Swiss and NZBWF1 mice1,2 [View Table]

Hepatic enzyme activities in NZBWF₁ and Swiss mice. Hepatic tryptophan dioxygenase activities in the two strains of mice that had been food deprived overnight or following the tube-feeding of L-tryptophan (20 mg/100 g body weight) 60 min before killing are summarized in Table 3. The enzyme activities under both conditions were similar in the two strains of mice.

Table 4. Status of in vitro 14C-orotate-labeled nuclear RNA release, nuclear poly(A)polymerase (PAP) and nucleoside triphosphatase (NTPase) activities of the livers of mice treated with L-tryptophan for 10 or 60 min1 [View Table]

Pretreatment of mice by tube-feeding L-tryptophan (20 mg/100 g body weight) 10 or 60 min before killing revealed higher hepatic nuclear poly(A)polymerase (PAP) (bound and free) activities and hepatic nuclear nucleoside triphosphastase (NTPase) activities compared with controls (water-treated) of each strain of mice (Table 4). These increases were similar to those observed earlier in rats (Kurl et al. 1993, Murty et al. 1980). However, the hepatic free PAP activity at 10 min after L-tryptophan administration was significantly increased in the Swiss mice but not in the NZBWF₁ mice (Table 4). Although the tryptophan-treated Swiss and NZBWF₁ mice (10 or 60 min) had significantly higher hepatic nuclear NTPase activities compared with each control, the tryptophan-treated NZBWF₁ mice showed somewhat less increase than did the Swiss mice (Table 4).

DISCUSSION

Table 5. Effects of L-tryptophan on liver and blood of Swiss and NZBWF1 mice1,2 [View Table]

Furthermore, three other variables, nuclear PAP and NTPase activities and nuclear RNA efflux of liver, which become elevated due to the administration of L-tryptophan (Kurl et al. 1993, Murty et al. 1977 and 1979) and are also probably related to the increased hepatic protein synthesis, were different (lower response to L-tryptophan) in the NZBWF₁ strain of mice compared with the Swiss mice (Table 4). Marked elevations were observed in livers of Swiss mice tube-fed L-tryptophan, but less marked elevations occurred in livers of NZBWF₁ mice treated similarly. These findings support the view that the strains' differences in nuclear specific binding affinity for L-tryptophan were interrelated with or influential in the responses of nuclear PAP and NTPase activities and in nuclear RNA efflux, mechanisms considered to be related to stimulation of hepatic protein synthesis (Sidransky et al. 1984). Thus, our findings indicate an important biochemical interaction between the genotype and the tryptophan-binding phenotype.

The findings that the serum and liver levels of free L-tryptophan and also liver tryptophan dioxygenase activities were similar in Swiss and NZBWF₁ mice (Table 3) is of interest. This occurred in spite of the differences in hepatic nuclear binding affinities for L-tryptophan. These findings are in marked contrast to the differences in the nuclear binding affinities for L-tryptophan of normal liver and hepatoma reported in rats (Sidransky et al. 1995) where the decreased binding of hepatoma has been considered possibly to be associated with the greater free L-tryptophan concentrations in hepatoma [which contain no tryptophan dioxygenase (Feigelson et al. 1977, Rammanarayanan-Murthy et al. 1976)] compared with normal liver. Thus, in hepatoma, like in the liver of NZBWF₁ mice, the nuclear receptor affinity for L-tryptophan was low compared with host rat liver or Swiss liver. However, in hepatoma, the metabolism of free L-tryptophan is unlike that in the liver of NZBWF₁ mice, as reflected by less tryptophan dioxygenase activity and greater free L-tryptophan levels in hepatoma than in livers of NZBWF₁ mice.